Group Title: BMC Evolutionary Biology
Title: hsp70 genes in the human genome: Conservation and differentiation patterns predict a wide array of overlapping and specialized functions
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Title: hsp70 genes in the human genome: Conservation and differentiation patterns predict a wide array of overlapping and specialized functions
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Creator: Brocchieri, Luciano
Conway de Macario, Everly
Macario, Alberto
Publisher: BMC Evolutionary Biology
Publication Date: 2008
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Abstract: BACKGROUND:Hsp70 chaperones are required for key cellular processes and response to environmental changes and survival but they have not been fully characterized yet. The human hsp70-gene family has an unknown number of members (eleven counted over ten years ago); some have been described but the information is incomplete and inconsistent. A coherent body of knowledge encompassing all family components that would facilitate their study individually and as a group is lacking. Nowadays, the study of chaperone genes benefits from the availability of genome sequences and a new protocol, chaperonomics, which we applied to elucidate the human hsp70 family.RESULTS:We identified 47 hsp70 sequences, 17 genes and 30 pseudogenes. The genes distributed into seven evolutionarily distinct groups with distinguishable subgroups according to phylogenetic and other data, such as exon-intron and protein features. The N-terminal ATP-binding domain (ABD) was conserved at least partially in the majority of the proteins but the C-terminal substrate-binding domain (SBD) was not. Nine proteins were typical Hsp70s (65–80 kDa) with ABD and SBD, two were lighter lacking partly or totally the SBD, and six were heavier (>80 kDa) with divergent C-terminal domains. We also analyzed exon-intron features, transcriptional variants and protein structure and isoforms, and modality and patterns of expression in various tissues and developmental stages. Evolutionary analyses, including human hsp70 genes and pseudogenes, and other eukaryotic hsp70 genes, showed that six human genes encoding cytosolic Hsp70s and 27 pseudogenes originated from retro-transposition of HSPA8, a gene highly expressed in most tissues and developmental stages.CONCLUSION:The human hsp70-gene family is characterized by a remarkable evolutionary diversity that mainly resulted from multiple duplications and retrotranspositions of a highly expressed gene, HSPA8. Human Hsp70 proteins are clustered into seven evolutionary Groups, with divergent C-terminal domains likely defining their distinctive functions. These functions may also be further defined by the observed differences in the N-terminal domain.
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Research article

hsp70 genes in the human genome: Conservation and
differentiation patterns predict a wide array of overlapping and
specialized functions
Luciano Brocchieril, Everly Conway de Macario2 and Alberto JL Macario*2


Address: 'University of Florida, Department of Molecular Genetics and Microbiology and UF Genetics Institute, Gainesville, FL 32610, USA and
2Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, MD 21202, USA
Email: Luciano Brocchieri lucianob@ufl.edu; Everly Conway de Macario macarioe@umbi.umd.edu;
Alberto JL Macario* macario@umbi.umd.edu
* Corresponding author


Published: 23 January 2008
BMC Evolutionary Biology 2008, 8:19 doi: 10.1 186/1471-2148-8-19


Received: 5 November 2007
Accepted: 23 January 2008


This article is available from: http://www.biomedcentral.com/1471-2148/8/19
2008 Brocchieri et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: Hsp70 chaperones are required for key cellular processes and response to
environmental changes and survival but they have not been fully characterized yet. The human
hsp70-gene family has an unknown number of members (eleven counted over ten years ago); some
have been described but the information is incomplete and inconsistent. A coherent body of
knowledge encompassing all family components that would facilitate their study individually and as
a group is lacking. Nowadays, the study of chaperone genes benefits from the availability of genome
sequences and a new protocol, chaperonomics, which we applied to elucidate the human hsp70
family.
Results: We identified 47 hsp70 sequences, 17 genes and 30 pseudogenes. The genes distributed
into seven evolutionarily distinct groups with distinguishable subgroups according to phylogenetic
and other data, such as exon-intron and protein features. The N-terminal ATP-binding domain
(ABD) was conserved at least partially in the majority of the proteins but the C-terminal substrate-
binding domain (SBD) was not. Nine proteins were typical Hsp70s (65-80 kDa) with ABD and SBD,
two were lighter lacking partly or totally the SBD, and six were heavier (>80 kDa) with divergent
C-terminal domains. We also analyzed exon-intron features, transcriptional variants and protein
structure and isoforms, and modality and patterns of expression in various tissues and
developmental stages. Evolutionary analyses, including human hsp70 genes and pseudogenes, and
other eukaryotic hsp70 genes, showed that six human genes encoding cytosolic Hsp70s and 27
pseudogenes originated from retro-transposition of HSPA8, a gene highly expressed in most tissues
and developmental stages.
Conclusion: The human hsp70-gene family is characterized by a remarkable evolutionary diversity
that mainly resulted from multiple duplications and retrotranspositions of a highly expressed gene,
HSPA8. Human Hsp70 proteins are clustered into seven evolutionary Groups, with divergent C-
terminal domains likely defining their distinctive functions. These functions may also be further
defined by the observed differences in the N-terminal domain.


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Background
Molecular chaperones encompass several families, play
important physiological roles, some of which essential for
life, and are key components of anti-stress mechanisms in
all organisms, tissues and cells [1]. The Hsp70 family is
thought to be required for various cellular processes and
for the response to environmental changes and survival
[2,3]. Although there is no doubt about the biological and
medical importance to the human Hsp70 chaperone fam-
ily, a coherent, unifying informational framework encom-
passing all members, that would facilitate their
understanding individually and as a group is lacking. A
framework of multiple data pertaining to all family mem-
bers is necessary for planning future experiments to fur-
ther elucidate the evolution, synthesis and maturation,
structure, functions, trafficking and pathology, of these
genes and proteins. Likewise, this framework would pro-
vide an essential context for interpreting the results and
for inferring new properties that can be submitted to
experimental testing.

Hsp70 proteins and their parent genes have been studied
in prokaryotic and eukaryotic organisms chiefly because
of their participation in protein folding under normal and
stress conditions, and of their potential role in ageing and
pathology, e.g., cancer, and apoptosis [4-7]. Many hsp70
genes have been reported, generating a confusing array of
data, variants, and nomenclatures in databases and
printed literature. The use of different names for the same
hsp70 gene and/or protein and the use of the same name
for various hsp70 genes and/or proteins make interpreta-
tion of the reported results very difficult if not impossible.
Furthermore, in many instances it is not entirely clear if
reference is made to the gene or its protein product, and
in other instances, it is impossible to know exactly what
gene or protein in the family is being considered, since
only the word Hsp70 is used without further clarification.

Analyses of hsp70 genes at the genome level have been car-
ried out for a few organisms, such as Caenorhabditis [8],
Drosophila [9,10], and Ciona intestinalis [11]. An earlier
attempt, more than ten years ago, at defining the human
Hsp70 family was based on printed literature searches and
yielded 11 genes [12]. Since then, no systematic, compre-
hensive analysis of human hsp70 genes has to our knowl-
edge been published.

In this report, we provide a comprehensive characteriza-
tion of the extended family of human hsp70 genes and
their proteins based on genomic, structural, and evolu-
tionary analyses. The overall picture is that of a diversified
family of proteins that achieved diversity at various levels:
genetic, transcriptional, and post-transcriptional. The
implications of this diversity for function, cell and tissue
localization, and pathology can be predicted to be wide


and important for life and to have a significant impact on
biomedical sciences. The unified and coherent matrix of
data we generated pertaining to evolution, genetics, tran-
scription, structure, anatomic distribution, and regulation
of the entire human Hsp70 family should provide a solid
context for future studies and for the development of ther-
apeutic means specifically targeted to well-defined family
members [13].

Results
Human hsp70 genes
We carried out extensive literature and database searches
and found 130 human Hsp70 proteins with correspond-
ing names and identifiers. Similarity analysis of these pro-
teins revealed 86 groups of non-identical Hsp70
sequences, which in turn could be clustered by further
analysis into 13 groups. From each of these 13 groups, we
chose a representative sequence to query the human
genome NCBI Build 36.1 for conserved and diverged
sequences, as described in Methods, and we found 47 loci
encoding hsp70-like sequences. Of these sequences, 16
corresponded to genes recognized in the genome annota-
tion. Another sequence was identified as an hsp70 gene,
named HSPA7, which is transcribed under certain condi-
tions although it is considered by some as a pseudogene
(see later). The main characteristics of the 17 hsp70 genes
are shown in Table 1. We identified thirty other sequences
as belonging to the hsp70-gene family, but they had fea-
tures such as frame-shifts and in-frame stop codons indi-
cating that they are pseudogenes. The main characteristics
of these 30 hsp70-related pseudogenes are shown in Table
2. The chromosomal distribution of all hsp70 genes and
pseudogenes is displayed in Table 3. The genes and pseu-
dogenes were distributed over 18 chromosomes, some of
which had both protein-coding genes and pseudogenes,
while chromosomes 15-17, 19 and 22 had neither.
Hsp70 genes and pseudogenes were distributed among
chromosomes irrespective of their evolutionary relations,
with the exception of the triad of genes HSPA1A, HSPA1B,
HSPA1L, all encoded in close proximity on chromosome
6, and of the pair of genes HSPA6, HSPA7, encoded near
each other on chromosome 1.

Variants and isoforms
The protein isoforms shown in Table 1 pertain in the
majority of cases to known mRNA variants. In the case of
HSPA7, a transcription product was terminated by a stop
codon after 367 amino acids. However, a complete con-
served Hsp70 protein of 641 amino acids could be
encoded bypassing a frame shift at codon position 340.
For the HYOU1 gene, we found four mRNA variants two
of which using the same translation initiation site, corre-
sponding to three protein isoforms (Table 1).




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Table I: hsp70 genes in the human genome: Main characteristics (NCBI Build 36.1)a


Location S Start/End


nt aa Ex Is


Hsp70 kDa 6 (HSP70B')/HSPA6
Hsp70 kDa 7 (HSP70B)/HSPA7
Hsp70 kDa 4-like/HSPA4L
Hsp70 kDa 9B/HSPA9B
Hsp70 kDa 4/HSPA4


Hsp70 kDa I-like/HSPAI L (HSP70-Hom)
Hsp70 kDa IA/HSPAIA (HSP70-1)
Hsp70 kDa I B/HSPAI B (HSP70-2)
Hsp70 kDa 5 (Grp78)/HSPA5
Hsp70 kDa 12A/HSPAI2A
Hsp70 kDa 14/HSPAI4


Hsp70 kDa 8/HSPA8


150 kDa oxygen-regulated protein/HYOU I


Hsp 105 kDa/HSPH I


Hsp70 kDa 2/HSPA2
Hsp70 kDa 12B/HSPAI2B
Stress 70 protein chaperone/STCH


Iq23.3
Iq23.3
4q28.1
5q31.2
5q31.1

6p21.33
6p21.33
6p21.32
9q33.3
10q25.3
10pl3

I iq24.1

I Iq23.3



13ql2.3

14q23.3
20pl3
21qI 1.2


+ 159,761,073/159,763,001
+ 159,842,705/159,844,628b
+ 128,923,156/128,973,476
- 137,919,628/137,938,906
+ 132,415,842/132,468,024
132,415,842/132,468,024
- 31,885,806/31,887,728
+ 31,891,513/31,893,435
+ 31,903,707/31,905,629
127,038,695/127,043,226
I 18,424,285/118,456,787
+ 14,920,408/14,953,608
14,920,408/14,924,181
122,433,655/122,437,242
122,433,655/122,437,242
18,421,518/118,432,082
I 18,421,518/I 18,432,082
118,421,518/1 18,431,751
I 18,424,940/I 18,432,082
30,609,458/30,633,719
30,609,458/30,633,719
+ 64,077,321/64,079,237
+ 3,667,322/3,680,810
- 14,667,812/14,677,311


aAbbreviations and symbols: ID, identifying designation used in this work; S, DNA strand; Start/End, chromosome positions comprising the coding
region (see Table S I, Additional File I for start and end positions of the primary transcript); nt, number of nucleotides between Start and End; aa,
length in amino acid of the protein productss; Ex, exons (non coding exons excluded); Is, isoform names (for the 150 kDa oxygen-regulated
protein/HYOU I there are four mRNA variants but only three protein isoforms because two variants have the same translation initiation site).
bBypassing an internal frame-shift (see text). The putative coding region would otherwise end at position 159,843,805 and encode for a protein 367
aa long.


Besides the described mRNA variants indicated in Table 1,
many more processed variants for the majority of the
genes were predicted from the analysis of EST (Expressed
Sequence Tag) and SAGE (Serial Analysis of Gene Expres-
sion) data. Prediction of transcript variants based on these
data (ECgene database [14,15] and NCBI AceView data-
base, D. Thierry-Mieg, J. Thierry-Mieg, M. Potdevin and M.
Sienkiewicz, unpublished) are displayed and compared in
Table 4, which also shows that ECgene and AceView pre-
dictions differ considerably. For example, for HSPA8 at
least 19 protein variants are predicted by ECgene vs. 14
predicted by AceView, 13 vs. 10 variants are predicted for
HSPA1A or HSPA1B, 3 vs. 15 are predicted for HYOU1, 1
vs. 12 are predicted for HSPA6, and similar differences
were observed for most of the other genes. These incon-
sistencies could result from the use of somewhat different
datasets, or from different quality-control procedures
applied to the EST data. In any case, EST analysis sug-
gested that there is a significantly greater number of tran-
script variants and isoforms for hsp70 genes than
previously suspected.


Evolutionary history of the human hsp70 genes
We computed phylogenetic trees of the human hsp70
genes based on the alignment of their protein products
using both distance and maximum-likelihood procedures
(see Methods) obtaining with both methods highly simi-
lar results. We excluded from the overall alignment the
sequences of HSPA12A and HSPA12B, which would have
significantly reduced the useful size of the alignment. A
phylogenetic tree of the proteins encoded by the 17 hsp70
genes (Figure 1) distinguished seven major evolutionarily
related groups, which we defined by a bootstrap-support
values over 85%. These Groups are indicated in Figure 1
with brackets. Group I was composed of the most
diverged sequences, HSPA12A and HSPA12B, of uncer-
tain relation with other eukaryotic and prokaryotic
Hsp70s. It had previously been noticed that HSPA12A
and HSPA12B branch in an intermediate position
between eukaryotic and prokaryotic hsp70 sequences [16]
but the origin of these two genes was not investigated fur-
ther. Although conservation of a few sequence motifs
clearly identifies these two genes as members of the
extended hsp70 gene family, their sequence conservation
was insufficient to allow for a reliable, accurate determi-


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Name/ID


1,929
1,924
50,321
19,279
52,183
52,183
1,923
1,923
1,923
4532
32,503
33,201
3,774
3,588
3,588
10,565
10,565
10,234
7,143
24,262
24,262
1,917
13,489
9,500


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Table 2: hsp70-related pseudogenes in the human genome: Main characteristics (NCBI Build 36.1)


Chra S Start/End


Name

P I
PI1.2
P2.1
P2.2
P2.3
P3.1
P3.2
P4
P5
F5
F6.1
F6.2
F6.3
F6.4
P7.1
P7.2
F7
P8
F8.1
F8.2
P9
PIO
P12.1
PI 12.2
F13
FI 18.1
FI18.2
PX. I
PX.2
PX.3


aa F Stops


+ 38,947,258/38,949,148
+ 245,459,865/245,461,803
- 209,645,569/209,647,064
+ 222,534,878/222,536,560
- 235,317,093/235,318,989
+ 139,081,827/139,083,766
- 19,956,765/19,959,824
+ 189,871,681/189,873,662
+ 129,503,623/129,505,555
63,644,128/63,644,314
151,776,456/151,776,854
+ 147,708,650/147,708,925
+ 47,769,360/147,769,647
- 80,187,101/80,187,848
+ 10,457,465/10,459,400
+ 84,495,080/84,496,823
+ 97,272,880/97,273,101
+ 30,214,440/30,218,052
+ 47,581,377/47,581,649
- 47,778,468/47,778,785
+ 30,977,881/30,980,601
+ 27,043,495/27,044,962
+ 4,076,878/4,078,947
+ 1 10,304,007/1 10,307,864
88,996,627/88,997,451
25,090,760/25,090,992
62,025,362/62,025,705
120,164,760/120,166,561
123,166,030/123,167,337
113,915,646/113,917,590


aAbbreviations and symbols: Chr, chromosome; S, DNA strand; Start/End, positions at which the homology region begins and ends, respectively; nt,
number of nucleotides between start and end, i.e., length of the recognized homology region; aa, number of amino acids, namely codons in the
predicted pseudocoding region; F, frame shifts; Stops, in-frame stop codons; I, apparent pseudointrons. In parentheses signifies that the inserted
element is short.


nation of their evolutionary position compared to other
human Hsp70s.

Group II was composed of the mitochondrial protein
HSPA9B, considered of alphaproteobacterial origin
[17,18], which, accordingly, clustered with the DnaK
sequence from E. coli (Gammaproteobacteria) with very
high bootstrap support.

Group III encompassed the 105/110 kDa proteins HSPA4,
HSPA4L, HSPH1 (clustered with 100% bootstrap sup-
port) and the more distantly related sequence HYOU1,
coding for the 170 kDa protein Grp170. HSPA14 was also
related to proteins in Group III but with lower (81.7%)
bootstrap support than that (85% or higher) we adopted
to identify closely related sequences as members of a dis-
tinct Group. Therefore, we classified HSPA14 separately in
Group IV. Also joined to the sequences of Groups III and
IV, was the sequence STCH, but with considerably lower
bootstrap support (55.6%) than that required to differen-


tiate the Groups, and thus we assigned STCH to another
group, i.e., Group V.

Group VI was composed of sequences clustered with
100% bootstrap support and, among them, we distin-
guish three subgroups, one including HSPA1A, HSPA1B
and HSPA1L, a second including HSPA8 and HSPA2, and
a third including HSPA6 and HSPA7.

Group VII included sequence HSPA5, expressed in the
endoplasmic-reticulum (ER), which was joined to Group
VI with lower bootstrap support (78.9%) than the mini-
mum adopted for distinguishing the main groups.

We considered the sequences from Groups II (mitochon-
drial), VI (cytosolic/nuclear) and VII (ER resident) typical
Hsp70 proteins because they had conserved ATP-binding
and substrate-binding domains (ABD and SBD, respec-
tively; see later). In contrast, we considered the sequences
in all the other Groups atypical because they had in what



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1,891
1,939
1,496
1,683
1,897
1,940
3,060
1,982
1,933
187
399
276
288
748
1,936
1,744
222
3,613
273
318
2,721
1,468
2,070
3,858
825
233
344
1,802
1,308
1,945


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Table 3: Human chromosomes with hsp70 genes and hsp70-
related pseudogenes


Chra Protein-coding gene

I HSPA6, HSPA7
2
3
4 HSPA4L
5 HSPA9B, HSPA4
6 HSPAIL, HSPAIA, HSPAIB
7
8
9 HSPA5
10 HSPA12A,HSPAI4
II HSPA8, HYOUI
12
13 HSPHI
14 HSPA2
18
20 HSPA12B
21 STCH


Pseudogene

PI.1, P1.2
P2.1, P2.2, P2.3
P3.1, P3.2
P4
P5, F5
F6.1, F6.2, F6.3, F6.4
P7.1, P7.2, F7
P8, 8.1, F8.2
P9
PIO

P12.1, P12.2
F13

F 18.1, F18.2


PX. I, PX.2, PX.3


aChr, chromosome


would be the SBD of the typical Hsp70 molecules no
equivalent of this domain, or incomplete or highly
diverged equivalents.

Origin and evolution of the human hsp70O-related
pseudogenes
An evolutionary tree of the proteins that would be
encoded by the pseudogenes identified in this work in

Table 4: Predicted mRNA variants of human hsp70 genes


relation to the proteins encoded in the 17 hsp70 genes is
shown in Figure 2. We included in the tree the 16 less cor-
rupted sequences out of the 30 pseudogenes. The evolu-
tionary tree indicates that the vast majority of the hsp70
pseudogenes originated from HSPA8. We also identified
the Hsp70 sequence most similar to each of the 30 pseu-
dogenes. The results are shown in Table 5 also in compar-
ison to sequence characterizations for the pseudogenes
available at the Pseudogene Database [19]. Twenty-three
of the 30 pseudogenes were predicted in the Pseudogene
Database, while seven pseudogenes were not and are
newly reported here.

Phylogenetic analysis of the most conserved pseudogenes
and detailed comparison of the most corrupted ones with
the closest sequence among the 17 hsp70 genes, revealed
that 27 of the 30 pseudogenes originated from the HSPA8,
two from HSPA9B, and one from HSPA5 (Figure 2 and
Table 5).

Exonlintron structure of the human hsp70 genes
The number of exons for each gene are shown in Table 1,
and a detailed characterization of the genome position
and length of each exon is shown in Table S1, Additional
File 1. An analysis of the exon/intron structure of these
genes provided important insights into their evolutionary
relations. With the exception of all but one of the genes in
group VI (see below), all hsp70 genes are multiexonic,
ranging from a minimum of five exons in STCH up to 25
exons in HYOU 1. Details on the intron-exon structure of


ECgene variant predictions


AceView variant predictions


mRNAs ESTs


HSPA8
HSPA12A
HSPA12B
HSPA9B
HSPA4
HSPA4L
HSPH I
HYOU I
HSPA14
STCH
HSPA2
HSPAIA/B
HSPAI L
HSPA6
HSPA7
HSPA5


9 (11)
2(2)
I (I)
5 (5)
5 (5)
n.a.d
8 (8)
4 (4)
6 (6)
3 (3)
3 (5)
8 (8)
2 (2)
2 (4)
n.a.
5 (6)


3156 (4729)
108 (112)
37 (38)
906 (1010)
441 (464)
n.a.
405 (444)
469 (482)
209 (226)
209 (210)
187(261)
14 11 (14 11)
36 (36)
2 (2)
n.a.
S1174 (1305)


Transcripts

21 (95)
4(6)
1 (2)
10(267)
12(30)
n.a.
9(55)
3 (13)
10 (17)
3 (5)
6(7)
21 (21)
2 (2)
1 (2)
n.a.
10 (18)


Proteins

19(52)
4(6)
I (2)
3 (42)
5 (11)
n.a.
7 (25)
3 (7)
5 (9)
2(3)
6(7)
13 (13)
1 (1)
1 (1)
n.a.
6 (10)


Transcripts

14


Proteins

14


aWithin parentheses are the total number of predicted transcripts and proteins including those of lower confidence level.
bData from [14,15].
cNCBI AceView Database [41].
dNot available.


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Gene


ECgene datab


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HSPA12B
H.RPA12A


III





]v





VI





] VII


Figure I
Phylogenetic tree of the 17 human Hsp70 proteins encoded in the genes studied in this work. The seven distinct
evolutionary groups supported by bootstrap values (1000 samples) equal or greater than 85% are indicated by brackets to the
right. Alignments were obtained with Clustal W [42] and the evolutionary tree was obtained using the neighbor-joining algo-
rithm [38] with the distance transformation method of Kimura [39], as implemented in Clustal W. A similar tree (not shown)
was obtained using the maximum likelihood approach implemented in the program PHYML [40].


0.2


individual gene sequences and their relations within and
between the hsp70 Groups are provided below.

Group I
The two similar sequences HSPA12A and HSPA12B pos-
sessed an identical exon-intron structure with 12 exons
within the coding region (Table SI, Additional File 1).
HSPA12B also possessed an intron in its 5' UTR region.
The HSPA12A and HSPA12B proteins were quite dissimi-
lar from the other Hsp70 sequences and, within the few
regions of homology we found no splice site in common
with sequences from other groups.

Groups II, VI and VII (Figure S I, Additional File I)
Sequences from groups II, VI, and VII included typical
Hsp70 sequences expressed, respectively, in the mito-
chondrion, cytosol/nucleus, and ER. The genes HSPA1A,
HSPA1B and HSPA1L (frequently called Hsp70-1, Hsp70-
2 and Hsp70-Hom, respectively) from Group VI are posi-


tioned within the MHC-III region on the short arm of
chromosome 6 [7,20], and are intronless. In fact, among
the genes in Group VI, only HSPA8 possessed multiple
exons (8 and 7 in the two isoforms) within the coding
region. We compared the splice positions in HSPA8
(Group VI), HSPA5 (Group VII) and HSPA9B (Group II),
and found a splice site conserved within one codon posi-
tion in HSPA8 (between codons 188 and 189) and HSPA5
(between codons 164 and 165), and we also found one
splice site conserved within two codon positions between
HSPA8-2 (within codon 463) and HSPA9B (between
codons 505 and 506).

Group III
Within Group III (Figure S2, Additional File 1), the closely
related HSPA4, HSPA4L and HSPH 1 sequences conserved
a common exon-intron structure of 18-19 exons, further
supporting the cohesiveness of this group. Among these
sequences, HSPH1 was distinguished by fusion of the


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HSPA12B
SHSPA 1I2A
1000 -HYOU I
1000 0-HSPA4
430 HSPA4L
603 4721 HSPH I
IHSPA14
STCH
F---PX.1
12 2 PS
J----A -0.P3.2

d1n i E_ PI Fu2.2 P2. I


312 7P7.2

8T4 5( ieP3 1
51 1P.2
-HSPA8*
1000 -HSPA2
1IHSPAlB
748 HSPAIA
55 LHSPA I L 0.2
557 HSPA.
HSPA7
1000 HSPA5
-- Pl.I

6 1000 r-HSPA9B *
DnaK E. coli

Figure 2
Phylogenetic tree of the 17 human Hsp70 proteins and the proteins that would be encoded in the related pseu-
dogenes identified in this work. Same as tree shown in Figure I but including the most conserved pseudogenes (see text).
Shaded areas highlight each group of pseudogenes with the gene from which they originated, indicated by an asterisk. A similar
tree (not shown) was obtained using the maximum-likelihood approach implemented in the program PHYML [40]. See legend
for Figure I and text for details on Methods.




Table 5: hsp70-related pseudogenes in the human genome: Type and evolutionary origin


Namea Type PD Ori PD Ori


Sim Name Type PD Ori PD Ori


HSPA5 HSPA51
HSPA81
HSPA8 HSPA81
HSPA9BI
HSPA82
HSPA81
HSPA6 HSPA81
HSPA82
HSPA8 HSPA81
HSPA82
HSPA82
HSPA82
HSPA82
HSPA82
HSPA8 HSPA81


P7.2
F7
P8
F8.1
F8.2
P9
PIO
P 12.1
PI 2.2
F13
F 18.1
FI18.2
PX. I
PX.2
PX.3


HSPA8 HSPA81
HSPA82
HSPA81
HSPA82
HSPA82
HSPA82
HSPA82
HSPA8 HSPA81
HSPA8 HSPA81
HSPA82
HSPA9B2
HSPA82
HSPA81
HSPA6 HSPA82
HSPA8 HSPA81


aAbbreviations and symbols: pseudogenes with a predicted pseudocoding region > 300 aa are called P, and those shorter than 300 aa are called F
(fragment); PD, Pseudogene Database [19]; Type PD, predicted pseudogene type as per the Pseudogene Database; Ori PD, pseudogene origin as
per the Pseudogene Database; Ori, pseudogene origin determined in this work, based on phylogenetic-tree branching (I superscript) and based on
similarity with the closest sequence (2 superscript); Sim, percent similarity (identities plus conservative substitutions of the predicted
pseudoprotein); P, processed (retrotranscribed); A, ambiguous; D, duplicated.


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exons labeled VII and VIII of HSPA4 and HSPA4L into a
single exon named VII (Table SI, Additional File 1), with
an apparent intron-loss/intron-insertion event in the sub-
group. A remarkable feature of the exon-intron structure
of this subgroup was that most splice sites and all of the
last seven splice sites (all but two of the splice sites within
the substrate-binding domain) were between codons (i.e.,
the splice sites did not disrupt codons). Within Group III,
HYOU1 was the most distantly related. It included 27
splice sites, of which 25 (24 or 16 in alternatively spliced
variants) were within the coding region. Ten of these sites
were within the ABD (8-9 splice sites were within this
region in the other sequences of Group III). One splice site
was conserved throughout all sequences in Group III (the
third splice site between codon positions 102 and 103 of
the HSPA4, HSPH1 subgroup and between codon posi-
tions 88 and 89 of HYOU1-1). Six other splice sites were
either in close proximity (within 5 codons) or bordering
diverged inserted elements. Besides the ABD region,
HYOU 1 shared similarity with other sequences of Group
III in a short segment immediately following the ABD and
mostly within a region of about 200 codons between posi-
tions 710 and 917. This relatively conserved region
included five splice sites in HYOU1 as well as in HSPA4,
HSPA4L, and HSPH1. One of these five splice sites was
conserved among all sequences (between codons 604 and
605 in HSPA4L, and between nucleotides 2 and 3 of
codon 722 in HYOU1). In conclusion, by comparing
HYOU 1 splice sites with those of the other members of
Group III, we recognized two conserved splice sites (one
in the ABD and one in the SBD) in this Group. We also
found a few other splice sites in close proximity within
conserved sequence elements or in correspondence to
inserted/deleted elements.

We compared Group III with other sequences (Figure S3,
Additional File 1) and showed a splice site in homologous
positions in HSPA4, HSPA4L, and HSPH1 (positioned
between nucleotides 1 and 2 of codon 329 in HSPA4L)
and in the mitochondrial gene HSPA9B (positioned
between codons 394 and 395). Also, we found a splice site
between codons 379 and 380 in HSPA4L within two
homologous codon positions (within position 374) of a
corresponding splice site in HSPA8 isoform 1.

Groups IV and V
Group IV (Figures S2 and S3, Additional File 1) was com-
posed of one sequence, HSPA14, which in the phyloge-
netic tree shown in Figure 1 was associated to Group III
with bootstrap support 81%, marginally below the thresh-
old of bootstrap support (85%) that we chose to define
evolutionary Groups. Within the coding regions of
HSPA14-a there were 14 splice sites (in HSPA14-b there
were 4 splice sites within the coding region and one in the
3' UTR). In HSPA14-a, we found a splice site within codon


191 in a position homologous to a splice site in HYOU1
(in which, however, the splice site was between codons
226 and 227). A second splice site of HSPA14-a, within
codon 297, was homologous to a corresponding splice
site in HSPA4L (within codon 303) and closely related
sequences. A third splice site, positioned after codon 90 of
HSPA14-a, coincided with a splice site in a homologous
position in HSPA5 (after codon 118) and in STCH (after
codon 122), although weak conservation in this region
made the alignment less reliable. A splice site of HSPA14-
a at codon 191 was within a short variable region in which
splice sites occurred also in HYOU1 (between codons 226
and 227 of isoforms 1 and 2) and in HSPA9B (within
codon 239). Finally, a splice site within codon 245
(between nucleotides 2 and 3) of HSPA14-a was within
one homologous codon of another splice site in HSPA9B
(between nucleotides 2 and 3 of codon 293).

The STCH (Group V) gene had 5 splice sites in its coding
region. Since STCH was weakly associated (with 55.6%
bootstrap support) in the phylogenetic tree to the
sequences of Group III and to HSPA14 (Group IV), it was
interesting to see whether some similarities in the exon-
intron patterns of these sequences could be detected but
we did not detect any common splice site associating
STCH (Group V) to Group III. However, we did find a
splice site in common among STCH (between codons 122
and 123), HSPA14-a (between codons 90 and 91), and
HSPA5 (between codon 118 and 119).

Evolution of eukaryotic Hsp70 sequences
We reconstructed the phylogenetic tree of eukaryotic typi-
cal Hsp70s, including the human ones (Groups II, VI and
VII), and 137 other typical sequences from completely
sequenced eukaryotic genomes. The tree is shown in Fig-
ure S4, Additional File 1, in which all branches with boot-
strap support < 40% have been collapsed into multi-
partitions. The tree constructed using all the information
available, did not have sufficient resolution to discern
most of the ancient phylogenetic relations. However, the
tree did provide several important pieces of information.
For example, thirty-three eukaryotic mitochondrial
sequences clustered with the E. coli DnaK in a separate
group, which roots the tree of other eukaryotic sequences.
Apart from a few protist sequences of uncertain origin and
a sequence from Microsporidia (Fungi), the cluster of ER
proteins, including Protists, Plants, Fungi, and Animals
(including human HSPA5) separated early, as in the trees
of human sequences (e.g., Figure 1), indicating that the
separation of ER and cytosolic sequences happened early
in the evolution of eukaryotes. The cytosolic sequences
presented several lineages of uncertain relations (Figure
S4, Additional File 1), many involving sequences from
Protists, two from Fungi and a third set from Plants. A
major cluster of sequences with weak bootstrap support


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(47.3%) included all human cytosolic sequences. Within
this cluster, two subclusters with relatively high support
(77.7% and 72.0%) included, respectively, the human
HSPA8 and HSPA2. A third cluster with lower support
(51.8%) included HSPA1A, HSPA1B and HSPA1L.

Figure 3 displays an evolutionary tree of eukaryotic atypi-
cal Hsp70 proteins from completely sequenced genomes,
including representatives of Protists, Plants, Fungi, and
Animals encompassing Nematodes, Insects, Fish,
Amphibians, and Mammals. In these genomes, we found
sequences most closely related to human Group III,
Group IV, and Group V. The tree is rooted by a group
encompassing DnaK from E. coli and human mitochon-
drial HSPA9B and include, for comparison, other repre-
sentatives of the human typical Hsp70s such as the ER-
residing HSPA5 and the cytosolic HSPA8 proteins. As
mentioned earlier, we excluded from the tree sequences of
Group I (i.e., HSPA12A and B), which were too diverged
from the other sequences to be used in the alignment. The
tree in Figure 3 shows, similarly to those in preceding fig-
ures, that the relation between Groups III, IV, and V are
uncertain, a likely consequence of their ancient diver-
gence. Also noteworthy is that contrary to the trees in the
previous figures, in the tree shown in Figure 3 Groups IV
and V (represented in humans by HSPA14 and STCH,
respectively) appear to have diverged from a common
ancestor, with weak bootstrap support (32.5%) but con-
sistent nonetheless with their similarity in molecular
weight (about 60 kDa) and sequence domain composi-
tion (see later). Interestingly, the cluster including STCH
(Group V) contained sequences from animals such as C.
elegans (but not Drosophila) and from Protists
(Enthamoeba) but not from Plants or Fungi, whereas the
cluster containing HSPA14 (Group IV) included only
sequences from Vertebrates. Apart from a single sequence
(SCHPO4) from S. pombe of uncertain classification, the
sequences of the high molecular weight proteins, corre-
sponding to the human Group III, were distinguished into
the two subgroups of 105/110 kDa and 170 kDa as in the
human tree shown in Figure 1, with high bootstrap sup-
port (91.7% and 97.7%, respectively). Sequences of the
105/110 kDa subcluster (including the human HSPH1,
HSPA4L and HSPA4) were present in various Protists and
in higher eukaryotes, suggesting that they originated from
an ancient gene duplication event. A duplication of the
gene into two branches, corresponding respectively to the
human sequences HSPA4 and HSPH1 (representing two
of the three subgroups of Group III; Figure 1) appears to
have occurred early in the evolution of the Vertebrate lin-
eage, before the appearance of Tetrapods (amphibians,
reptiles, birds, and mammals). A subsequent duplication
of HSPA4 into two separate genes (corresponding to
HSPA4 and HSPA4L) seems also to have occurred before
the appearance of Tetrapods (as indicated by the position


in the tree of the sequence DANRE20). Interestingly, no
representatives of HSPA4L from fish or amphibian were
found. Multiple paralogs from the HSPH1 and HSPA4
gene-lines seem to have been recently generated in the
chicken genome. Unexpectedly, the cluster containing the
170 kDa subgroup of the human Group III (HYOU1)
included only sequences from animals and plants, sug-
gesting gene loss or divergence in other lineages, includ-
ing Fungi.

We also studied the evolutionary relations of all human
Hsp70 proteins (excluding HSPA12A and HSPA12B) with
prokaryotic DnaK(Hsp70) proteins, resulting in the evo-
lutionary tree displayed in Figure 4 that shows the close
relation of HSPA9B (mitochondrial) with alpha-proteo-
bacterial sequences. The tree also suggests that all other
human hsp70 genes originated and differentiated from a
unique prokaryotic ancestor of uncertain identity.

Evolution of HSPA 12
We also searched for the genes encoding proteins like
HSPA12A and HSPA12B in the sequenced genomes exam-
ined in Figure 3. We found homologs of these genes only
in fish (Danio), Amphibian (Xenopus), Bird (chicken), and
Mammal (human and mouse), a finding that together
with their evolutionary relations shown in Figure 5, sug-
gests that the various HSPA12 genes evolved at an early
stage in the evolution of vertebrates (or that versions of
the gene that diverged earlier do not conserve significant
similarity to current human homologs). We found in
Danio rerio (fish) three recently diverged copies of the
gene (similarities 97-99%) that were quite diverged from
the human and amphibian sequences (similarities in the
range 25-32%). Chicken had only one copy, which was
most similar to the human HSPA12B (92% similarity),
whereas we found two copies of this gene in mammals
and amphibians. The evolutionary tree of the gene sug-
gests that the amphibian and mammalian copies resulted
from a unique duplication event that occurred in the tetra-
pod lineage before the radiation of the amphibian and
mammalian lineages. Within each of the one amphibian
and two mammals examined, the two gene copies had
similarities in the range 61-65%. Each paralog was quite
conserved among the three groups, with similarities
between mammals and amphibian of 82-83% for the
HSPA12A paralog, and of 71-73% for the HSPA12B par-
alog, whereas between human and mouse similarities
were 97% and 94%, respectively. The phylogenetic rela-
tions of the vertebrate lineages imply that one of the two
copies of the gene has been lost from the chicken genome.
Interestingly, we identified in the chicken genome a non-
annotated sequence of 186 nt (positions
45,897,943-45,898,128 of chromosome 1) in the com-
plementary strand, encoding a protein with 56.5% iden-



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917


Group III
A6W


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607 CHICK12
7il CHICKII
CHICK9
94 992 CHICKIO
XI ---- NTR7
736 s77 MOU0SF6
I 71 IIPllHI
DANREI4
987 IMxx) DANREI3
S --CHICK8
MOUSE
999 IISPA4L
959 i DANRE20
U ---XENTRI
775 8721 9 I, .i | M L I'll k6
S.slt ilU k0


ANO-A-CAEEL7
744 ANOGA4
SCIHP06
99177-- NEUCR4
24087 5-A I"
8 00 o-CANGL8
994___ TRCR6
I,, [--.TRICRI
432 I00-- LREIMA4
*---------------------MT 114
760 ARATH8
15 1000 ARATH7
ARATH6
968 95, A R %Ili
ARATH2
CYAME3


ARATHI I
553 DROMEI
97 CAELI3
964 CAEU2
966 548 XENTR6
9 ------ 789 CHICK16
MUS"~
-Ii IyOI I
SCHP04


Group V .-1000mT .
-- -- -- -----------------CAKEE 4
[ 606 -- XENTR5
r) IDANRE22
901 70J3 .i IIAN-- \RI21
I i |1.0l|).NRI i'1
25 ,x- --- CHICK 17
88$ ____iMOUSE12
999 -'STC
I- 7i9.i-----XF R12
I000 I --CHICK 15
944 I --MOUSE3
9S0 -HSPA14
___.--HSPA8
60--- HSPA5 0.2
ISPA91 I
993 -- DnaK (E. Cni)



Figure 3
Phylogenetic tree of atypical eukaryotic hsp70 genes. Included are the proteins encoded in the atypical human hsp70
genes (Groups Ill, IV, and V in Figure I) and in the hsp70 genes of eighteen other completely sequenced eukaryotic genomes.
Sequences from the latter eighteen non-human genomes were identified using as queries human proteins from Groups II, VI,
and VII and the SSPA procedure [36]. The tree is rooted by the DnaK sequence (AAC73125.1) from E. coli (DnaK E coli) and
by representatives of typical human Hsp70s (from Groups II, VI, and VII). See legend to Figure I and text for details on meth-
ods and calculation of bootstrap values. Human proteins are in red. Acronyms in black indicate the eukaryotic genomes in
which other hsp70 genes were found, as follows: ANOGA: Anopheles gambiae (Insects); ARATH: Arabidopsis thaliana (Plants);
CANGL: Candida glabrata (Fungi); CAEEL: Caenorhabditis elegans (Nematoda); CHICK: Gallus gallus (Birds); CYAME: Cyanidio-
schyzon merolae (Red alga); DANRE: Danio rerio (Fish); DROME: Drosophila melanogaster (Insects); ENTHI: Entamoeba histolytica
(Protists); LEIMA: Leishmania major (Protists); MOUSE: Mus musculus (Mammals); NEUCR: Neurospora crassa (Fungi); SCHPO:
Schizosaccharomyces pombe (Fungi); TRYCR: Trypanosoma cruzi (Protists); XENTR: Xenopus tropicalis (Amphibians); YEAST: Sac-
charomyces caerevisiae (Fungi).





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621
576-
690

S968 HSPA5
HSPA8


-HYOUI


HSPA14
STCH


294[ Epsilon-proteobacteria
I Actinobacteria
83 43 Chlorobi
8 0 4- Bacteroidetes
5,8 Spirochaetales
528 Chlamydiales

978 163 396999 Beta-proteobacteria
Gamma-proteobacteria
967 "59--2 Alpha-proteobacteria
92I HSPA9B
176 1170 Chloroflexi
1558 Firmicutes
Methanosarcinales
999 Methanobacteriales
441 100 Thermoplasmales
556- Thermotogales
S i5 Halobacteria
4-68 Deinococcus-Thermus
8- Cyanobacteria
Aquificales

0.1

Figure 4
Phylogenetic tree of human Hsp70 proteins and prokaryotic DnaK. Included are all human Hsp70 proteins except the
highly diverged HSPAI2A and HSPAI2B. One sequence was chosen for each prokaryotic (bacterial in black, and archaeal in
blue) group. Human proteins are in red.


tity to a segment of the annotated chicken hspal2 gene.
This short gene is possibly a remnant of HSPA12A.

Hsp70 proteins: Structural domain characteristics of the
groups and subgroups
Conserved and unique sequence elements of the different
members of the human Hsp70 protein family were iden-
tified and are schematically displayed in Figure 6. Hsp70
proteins typically comprise two major domains: an ATP-
binding domain (ABD) and a substrate-binding domain
(SBD). Most of the ABD of the human HSPA1A (positions
1-382) has been resolved by crystallography [21], PDB
identifier 1s3x, and most of the SBD of the rat HSPA8
(corresponding to human HSPA8 and homologous to
human HSPA1A positions 385-543) has been elucidated
by NMR [22], PDB identifier ickr. The positions of the
sequences corresponding to the solved structures of the
ABD and SBD domains are shown in Figure 6 aligned to
the corresponding sequence domains conserved among
the human Hsp70 proteins, drawn as white boxes. Other
domains conserved among groups or subgroups of
sequences are shown as aligned boxes of matching colors.


The ABD was conserved in most proteins; segments of it
were present in all of them, thus providing one parameter
for recognizing them as members of the hsp70 family. We
found variants of ABD in the two subgroups of Group III
distinguished by high bootstrap support, namely the
HSPA4-HSPA4L-HSPH1 subgroup, and the HYOU1 sub-
group, with regions of differentiation and inserted ele-
ments (shown in different color and as loops with length
indicated, respectively) in correspondence to exposed
loops in the crystal structure of HSPA1A (see below). In
HSPA12A and HSPA12B (Group I), most of the ABD was
highly divergent, with scattered conserved regions that
allowed their identification as members of the extended
Hsp70 protein family. An incomplete ABD was present in
HSPA14-b (Group IV), and in HSPA4-b (Group III).

The SBD was considerably less conserved than the ABD.
For example, the typical SBD as observed in HSPA8 was
entirely conserved only in the sequences of Groups II, VI,
and VII. A C-terminal portion of proteins in Groups VI
and VII of about 70 amino acids, comprising the 35 C-ter-
minal residues included in the crystal structure and 66 res-


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---a,


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5951 XENTR3
99 -CHICK 14
MOUSE18
943
HSPA12A


S0.1


Figure 5
Phylogenetic tree of eukaryotic Hsp70 protein 12.
The tree includes the human HSPAI 2A and HSPAI 2B pro-
teins and the homologs found in eighteen sequenced eukary-
otic genomes. See legend for Figure 3 and text for methods
and other details. Homologs of human HSPAI2A and
HSPAI2B were found only in vertebrates. The evolutionary
tree suggests that the human genes originated from a unique
progenitor also appearing in fish possibly due to a duplication
event that occurred in Tetrapoda before the divergence of
Amphibia. Only one HSPAI2 copy was found in reptiles and
birds, suggesting gene loss (or extreme divergence) of one
HSPAI2 copy from this lineage.



idues not included in the crystal structure, shown in
yellow in Figure 6, was found in the proteins of Group III
(HSPA4L, HSPA4-a, and HSPH1-a and -b, light green seg-
ments; and HYOU1-1, 2, and 3, orange segments). In all
of these proteins, this sequence segment was interrupted
by a long insertion characteristic of Group III, somewhat
differentiated in the HSPA4L and HSPA4-a sequences in
which it is shown in greenish yellow, compared to
HYOU 1 in which it is shown in brownish. In contrast, we
did not find in the proteins of Group I any sequence sim-
ilarity to the SBD of Group VI sequences.

Other noteworthy findings are listed below (see Figure 6):

1) HSPA8-2 and HSPA14-a had only a partial, N-terminal
sequence of the SBD;

2) The HSPA4 and HSPH 1 subgroups were characterized
by distinctive domains (dark green) different from the
ABD and SBD. HYOU1 had inserted long, unique ele-
ments (reddish) that separated shorter regions of homol-
ogy to the SBD of Group VI;

3) The sequence C-terminal to the part of SBD resolved in
the rat HSPA8 structure (1ckr) was not conserved in


HSPA9B, in which it was instead more similar to the cor-
responding prokaryotic sequence of DnaK (shown by a
straight line);

4) HSPA12A and HSPA12B (Group I) had, as mentioned
earlier, portions (white segments) of the canonical ABD
typical of the other groups (markers of the Hsp70 family),
but did not share any similarity with their SBD;

5) The ABD of STCH was characterized by a long insertion
of 50 aa (positions 311-360) that substituted residues
285-292 of HSPA1A, corresponding to a loop connecting
two beta-strands in the apical part of one of the two ATP-
binding jaws of the ABD. This inserted element was not
predicted to have an ordered secondary structure (as
determined by the secondary-structure prediction pro-
gram PHD). An unrelated inserted element in the same
position was present in HSPA9B (22 aa at positions
323-344, substituting 18 aa at positions 278-295 in
HSPA1A). A more extended region of divergence occurred
in the same region also in HSPA12A and B, in which posi-
tions 278-307 (30 aa) of HSPA1A were substituted by a
diverged element of 53 aa (positions 402-454 in
HSPA12B). These changes may reflect functional differen-
tiation endowing the HSPA12 proteins with specific capa-
bilities not shared with other Hsp70s.

6) Another major region of differentiation corresponded
to positions 211-224 of HSPA1A (ls3x), which were sub-
stituted by a diverged segment of 21 aa in HYOU1 (posi-
tions 245-265 in HYOU1 isoforms 1 and 2). A shorter
overlapping peptide within the same region (correspond-
ing to positions 210-217 in ls3x) was diverged in other
Group III sequences (shown in green and corresponding
to positions 215-222 in HSPA4 and positions 213-220 in
HSPA4L and HSPH1). An insertion of four aa (sequence
LRLR) was present in HYOU1 (positions 277-280 in iso-
forms 1-2) inserted in correspondence to positions 234
and 235 of HSPA1A. These positions are part of an alpha-
helix whose structure most likely influences the ATP-bind-
ing site's architecture and thereby its functionality.

7) Still another region that was modified in all atypical
Hsp70s (typical proteins are those in Groups II, VI, and
VII, as mentioned earlier) corresponded to positions
189-192 of HSPA1A (ls3x), in which this short segment
of four aa was substituted by longer loops (nine aa) in the
HSPA4 and HSPH1 subgroups and in the HYOU1 sub-
group (seven aa insertion). The same region represents the
C-terminal part of a longer segment of 25 aa (positions
168-192) substituted by a long inserted element of 86 aa
in HSPA12A and HSPA12B (positions 229-314 of
HSPA12B). While positions 189-192 formed an exposed
loop in HSPA1A, the longer peptide of 25 aa involved



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BMC Evolutionary Biology 2008, 8:19





1 s3x UHP
168-192 211-224 278-307 343-378


http://www.biomedcentral.com/1471-2148/8/19


1 ckr 509
544-560


VI HPA 2 6.7.8j-1 1(
I 115PAS-2 4


VIIi HSPA5

II HSPA9B -

V STCH __ I-- ,I


DQDTG


581-6W9


C'I-I


254


IVHSPA 14-a
HSPA14-b

IAISPA4L
HSPA4-a
HSPA4-b
HSPH 1-a
IIHSPH I-b

HYOU1 -1,2
HYOU 1-3
H-YOU 1-4


n 8

47= 1Ll C
M)


S91 02&661 760-78B

.......-......


710 j45-81i


7 F;.


88094M


5-


H-SPA12AI51

H SPA 1213B-)5


I TPRi-binding site

I ER retention signal
n ER Import signal


100aa


Figure 6
Structural features of human Hsp70 proteins. Typical Hsp70 structures comprise two major domains: ATP-binding
domain (ABD) and substrate-binding domain (SBD). The regions comprising the crystal structure of the ABD (I s3x [21],
human HSPAIA, positions 1-382) and the NMR structure of the SBD (lckr [22], rat HSPA8, corresponding to human HSPA8
and HSPAIA homologous positions 385-543) are shown as lines at the top of the figure. Domains conserved in human Hsp70
sequences within these regions are shown as aligned white boxes. Other domains conserved among groups of sequences are
shown as aligned boxes of matching colors. Roman numerals to the left indicate the Hsp70 evolutionary groups. UBP, ubiqui-
tin-binding peptide; TPRI, tetratrico peptide repeat I; ER, endoplasmic reticulum. See text for other details.


more substantial structural elements, including an
extended loop followed by a mostly-buried alpha-helix.

8) The interaction of Hsp70 proteins with Hsp90 proteins
is mediated by the adaptor protein Hop, whose TPR1
domain specifically recognizes an Hsp70 C-terminal TPR1
domain-binding site [23]. This site was conserved in the
sequences of Group VI, in HSPA4L, in HSPA4 (both iso-
forms), and in HSPH1 (both isoforms), ending with the
acidic-aliphatic-acidic triad EVD, EVD, DID and DLD,
respectively. This finding suggests that the functions of the
Hsp70s just mentioned include interacting with Hsp90.
In contrast, we did not find the TPR1 domain-binding site


in HSPA5, HSPA9B, STCH, HSPA14, HYOUI1, HSPA12A,
or HSPA12B.

9) In STCH, a sequence segment in close proximity to the
C-terminus of the ABD (shown as UBP for ubiquitin-
binding peptide in Figure 6) was shown to be involved in
interactions with the ubiquitin-system proteins Chapl1
and Chap2 [241. This peptide corresponds to positions
411-445 of the STCH sequence. Its homologous posi-
tions 343-378 in the HSPA1A structure [211 correspond
to an exposed extended loop (425-DPNTSVDPD-433)
anchored by two surrounding alpha-helices. The UBP
(with alpha-helical regions shaded in Figure 6) was con-
served in all human Hsp70s with the exceptions of



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HSPA12A and B, in which only the N-terminal alpha-
helix sequence was somewhat recognizable. This conser-
vation was particularly evident in the typical Hsp70s
(Groups II, VI, and VII), in which the UBPs had similari-
ties of 54-64% with the STCH counterpart, suggesting
that functions associated with the ubiquitin proteasome
system (and hence with protein-degradation pathways)
are shared by the majority of Hsp70 proteins, including
atypical ones.

10) Consistent with their localization in the ER, we iden-
tified ER-import signal peptides in HSPA5, STCH and
HYOU 1 isoforms 1, 2 and 4 (i.e., excluding isoform 3). An
ER-retention signal was identified in HSPA5 and in
HYOU1 isoforms 1, 2, and 3, but not in the microsome-
associated protein STCH.

Patterns of hsp70 gene expression in tissues,
developmental stages, and cellular compartments
The expression levels of hsp70 genes in various tissues can
be estimated by assessing the relative number of ESTs per
tissue type. Summary results on the frequency of human
hsp70 gene ESTs per tissue type obtained from the NCBI
UniGene database [25] are displayed in Table 6. Notewor-
thy were the relatively very high expression levels of
HSPA8 and HSPA1A in vascular tissue and in the spleen,
respectively. At the opposite end of the spectrum, the
expression levels of HSPA1L and HSPA12B were very low,
or undetectable in the majority of tissues examined. All
genes were expressed each in only one tissue at much
higher levels than in the other tissues, with one exception,
HSPA12A, which was preferentially expressed in two tis-
sues, parathyroid and nerve. These findings suggest that
accurate determination of gene regulation and protein
function for different hsp70 paralogs should be experi-
mentally investigated for each pertinent tissue and that
findings pertaining to a tissue must not be assumed to
apply to other tissues.

We also obtained data on expression of hsp70 genes at suc-
cessive developmental stages (embryo, and less than 4
weeks, 4 weeks to 17 years, and over 17 years of age)
(Table 7). The results indicated that most hsp70 paralogs
are preferentially expressed at specific developmental
stages. Seven genes (HSPA8, HSPA1A, HSPA9B, HSPA1B,
HSPH1, HYOU1, HSPA12A) were expressed at their high-
est levels in juvenile tissues (4 weeks to 17 years), three
genes (HSPA1L, STCH, HSPA14) were expressed at their
highest levels in the embryo, three genes (HSPA2, HSPA6,
HSPA12B) were most expressed in the neonate (less than
4 weeks old), only two genes (HSPA5, HSPA4) were most
expressed in adult tissue (17 years old and older), and one
gene (HSPA4L) was equally expressed, at very low levels,
in the embryo and in the adult and was undetectable in
the neonate and juvenile tissues.


We obtained data on the mode of expression of hsp70
genes and the locale of residence of their proteins as
shown in Table 8. Ten of the 14 genes studied were
expressed constitutively, and out of these 10 genes five
were expressed only constitutively (induction by stress
was either untested or when tested the results indicated
that the gene was not stress inducible). Only one gene was
not constitutively expressed, and nine genes were stress
inducible.

Discussion
Multiple genes and mRNA variants: Hsp70 diversity
We identified in the human genome 17 genes (Table 1)
encoding proteins of the extended Hsp70 family. This
count includes one putative gene, HSPA7, transcribed
after a 45 C heat shock in fibroblasts [26] but also pro-
posed to be a pseudogene transcribed only under particu-
lar conditions [27]. This list considerably expands the
count of 11 genes resulting from a "pre-genomic" attempt
at a comprehensive classification of human hsp70 genes
[12] and resizes a recent list of 21 putative genes [11] col-
lected from public databases, which included four pseu-
dogene sequences. At least five genes are characterized by
multiple mRNA variants and protein isoforms. We also
identified in the human genome 30 hsp70 pseudogenes
(seven for the first time), most of which (27 pseudogenes)
we determined originated from the gene HSPA8.

Our sequence and domain comparisons show that the
Hsp70 family is defined by its unique ATP-binding
domain (ABD), conserved in all of its members with the
exception of the two HSPA12 genes, where the ABD is
more divergent but still recognizable. Within the ABD we
have identified sequence insertions, most of which
uniquely characterize sequences from Group III (HSPA4,
HSPH and HYOU1, see Figure 6). These regions are inter-
esting targets for future functional characterization stud-
ies. In contrast to the substantial conservation of the ABD,
the substrate-binding domain (SBD) has greatly diversi-
fied among the various Hsp70 Groups, including com-
plete or partial loss respectively in STCH and HSPA14,
significant sequence divergence and insertion of novel
sequence elements in Group III genes, and complete
domain substitution in the Group I sequences (HSPA12A
and HSPA12B). The extent of sequence divergence, exem-
plified by substitution or loss within the SBD, suggests
that the Hsp70-like proteins belonging to Groups I, III, IV,
and V are likely to have evolved structures and functions
in the SBD domain that are not necessarily associated to
the classic hydrophobic peptide-recognition mechanism
of the typical Hsp70 sequences of Groups II, VI and VII.

We also characterized presence or absence of protein-pro-
tein interaction sequence motifs in different family mem-
bers, indicating which members can be predicted to


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Table 6: Expression levels of hsp70 genes in various human tissues

TISSUEIGENE HSPA HSP HSPA HSPA HSPA HSPA HSPA HSPA STCH HSPA HSPA HSPA HSPH HYOU HSPA HSPA
9B AS IA IB IL 8 2 6 14 4 4L I I 12A 12B


Blood
Bone
Bone marrow
Brain
Connective
Adipose
Ear
Liver
Pancreas
Adrenal gland
Parathyroid
Thyroid
Pituitary gland
Placenta
Eye
Embryonic
Abdominal cav.
Cervix
Ovary
Uterus
Prostate
Testis
Bladder
Kidney
Tongue
Larynx
Pharynx
Salivary gl.
Heart
Lymph node
Lymph
Tonsil
Spleen
Thymus
Mammary gl.
Muscle
Lung
Trachea
Skin
Umbilical cord
Vascular
Small intestine
Colon
Stomach
Esophagus
Nerve tissue


336 172 546 72
341 505 150 0
448 529 61 81
364 99 805 235
289 373 476 56
73 591 2885 591
177 118 769 0
306 316 397 62
150 388 480 155
538 348 1805 411
616 47 0 0
523 1571 776 271
223 503 167 0
219 293 169 13
129 177 220 57
306 701 98 41
964 642 98 49
537 247 123 103
93 409 74 83
287 608 721 266
153 262 1410 237
383 252 197 44
423 195 782 1108
349 130 931 214
590 132 1388 118
132 498 365 66
69 139 69 0
96 0 0 0
246 190 1692 347
165 176 10 10
334 44 0 0
0 0 115 0
302 113 7292 1416
279 39 1982 399
524 647 647 104
393 113 262 52
247 213 1467 153
224 40 2595 797
432 173 358 42
148 148 0 0
411 176 1038 58
357 447 3914 380
251 374 320 103
126 466 641 145
578 1261 2732 1839
156 509 3725 470


aEach number represents the number of Gene ESTs times 106 over total number of Tissue ESTs, as reported in the NCBI UniGene database (2/2007). Boldface highlights the maximum
for each Gene (two very close maxima are highlighted for HSPA12A). Abbreviations: cav., cavity; gl., gland.


participate in certain interaction networks. For example, only in STCH [241, suggesting that all typical and the
we found that the TPR1 domain-binding motif [23] majority of the atypical Hsp70 proteins are involved in
(shown as TPR in Figure 6), mediating interaction of the ubiquitin system and protein-degradation pathways.
Hsp70 with Hsp90, was conserved in all the sequences of
Group VI and in HSPA4L, HSPA4 and HSPH1, strongly Analyses of EST data suggest that some hsp70 genes may
suggesting a functional relation of these proteins through express an impressively high number of mRNA variants
their interaction with Hsp90. In contrast, the motif was and protein isoforms (see for example Table 4). To what
absent in HSPA5, HSPA9B, STCH, HSPA14, HYOU1, extent these variants correspond to proteins with distinct
HSPA12A, or HSPA12B, suggesting in this respect a differ- functions and/or disparate mechanisms of action, or to
ent role for these proteins in the human interactome. In a transcriptional errors, remains to be established. In any
similar vein, we identified in most Hsp70 sequences case, the results indicate that many more hsp70 gene tran-
(excepting only HSPA12A and B) a ubiquitin-binding scripts and protein variants than previously suspected
peptide (shown as UBP in Figure 6) previously described may be expressed in human cells. This suggests that the




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0 2412 9 118 36 36 18 0 109 54
0 1326 0 41 54 54 164 13 123 109
0 1691 40 20 40 40 61 0 142 448
3 3355 753 14 127 58 45 43 308 120
18 2943 37 18 84 37 74 0 93 130
73 1035 0 73 0 73 73 0 147 73
0 591 118 0 0 0 59 59 177 0
0 1198 4 0 23 28 52 19 47 306
0 818 4 18 27 36 82 4 77 128
0 3959 63 95 95 31 158 0 190 221
0 568 0 0 0 331 0 47 47 0
0 1318 0 0 0 72 36 18 144 198
55 1734 55 55 0 55 167 55 559 55
0 856 219 53 79 66 89 13 86 86
14 989 43 28 96 43 124 28 67 91
0 1304 15 0 67 77 171 5 124 213
0 2743 0 0 24 24 247 24 98 123
0 1177 20 0 103 82 123 20 82 103
0 1581 9 18 46 37 102 0 37 130
0 2334 125 8 79 41 133 16 95 125
6 1653 32 19 44 38 115 32 44 102
67 2222 411 3 140 73 118 220 389 130
0 1858 0 130 228 97 0 65 195 32
9 4470 204 9 97 23 69 55 158 274
0 1905 162 14 29 103 236 29 280 44
0 565 0 0 0 66 0 0 66 66
0 765 69 0 0 0 0 139 0 69
0 387 145 0 48 96 145 48 0 48
II 2129 67 44 33 22 33 II 56 44
0 590 0 0 82 217 103 0 176 10
0 1381 0 0 0 0 89 0 III Ill
0 463 57 0 0 231 115 0 57 57
0 5516 94 75 18 18 18 0 37 56
0 4444 13 172 53 53 39 13 172 0
5 2145 17 17 64 52 169 II 81 1119
0 2963 26 8 26 34 87 52 139 157
8 1166 17 76 48 25 102 22 125 201
0 3310 61 265 265 40 20 163 408 143
0 2861 179 26 21 47 226 0 131 158
0 1782 816 0 0 0 0 0 0 297
0 8247 19 0 117 0 0 19 176 842
22 4853 357 0 67 22 134 0 134 201
0 2986 9 29 24 39 123 9 118 226
0 1856 38 29 0 19 456 9 204 58
0 1839 262 157 52 105 105 52 262 683
0 1215 39 39 0 39 39 0 196 196








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Table 7: Expression levels of human hsp70 genes at various stages of development


Developmental stage


Neonate (<4 weeks)

597
74
523
149
0
7023
410
74
0
0
0
0
186
0
37
37


juvenile (< 17 years)

798
272
1124
235
0
7384
36
0
0
36
72
0
362
526
108
0


Adult (>17 years)

276
418
337
106
2
2006
69
30
55
47
144
18
133
142
41
13


aEach number represents the number of Gene ESTs times 106 over total number of Tissue ESTs as reported in the NCBI UniGene database (2/
2007). Boldface highlights the maximum for each Gene.


use of alternative forms of gene expression, either through
different transcription initiation and/or translation initia-
tion sites or alternative splicing, may be a phenomenon
significantly more important than previously recognized
in generating the many functionalities of the Hsp70 fam-


ily in the various tissues and cell compartments. The dif-
ferent functionalities that may be generated through these
mechanisms are exemplified by the HYOU1 gene. Two
transcript variants of this gene are generated from two
alternative transcription-initiation sites corresponding to


Table 8: Human hsp70 genes: Expression modality and location of their proteins


Gene

HSPA8


HSPA2


HSPAIA and HSPAIB

HSPAI L

HSPA6

HSPA5
HSPA9B

STCH
HSPA14
HSPA4

HSPA4L

HSPH I

HYOU I

HSPA12A
HSPA12B


Cell locale


Tissue


Cytosol. Translocate to nucleus and
nucleoli upon heat shock. Surface of
embryonic stem cells
Nucleus

Cytosol. Nucleus and nucleoli upon heat
shock
Mostly cytosol under basal conditions.
Nucleus but not nucleoli upon heat shock
Cytosol. Nucleus

Endoplasmic reticulum (ER)
Mitochondria. ER. Cytosol. Cytosolic
vesicles. Membrane surface
Microsomas
Cytosol. Associated to ribosomes
Cytosolic (?)

Cytosol. Nucleus upon heat shock

Cytosol. Nucleus upon heat shock, possibly
nucleoli
ER. Cytosol (isoform 3)

Undetermined yet
Undetermined yet


Modality of expression


Highly expressed in all tisuses


Mostly in testis, skeletal and heart
muscles, esophagous, brain
All tissues

Spermatides

Most tissues

All tissue
Many tissues

All tissues
Many tissues (low expression)
Most tissues

Mostly testis

Most tissues

Liver, pancreas. Highly expressed in
cells with well developed ER
Brain. Kidney. Muscle
Skeletal and heart muscles


Constitutive. Moderately induced by
heat shock

Constitutive

Strongly induced by heat shock

Constitutive. Not induced by heat
shock
Induced by heat shock. No basal
expression
Induced by ER stressors
Constitutive

Constitutive
Undetermined
Constitutive. Not induced by heat
shock
Constitutive. Induced by heat shock
in somatic cells but not in germ cells
Constitutive. Induced by heat shock

Induced by ER stressors

Constitutive. Induced by stress
Constitutive. Induced by stress




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Gene


Embryo


HSPA9B
HSPA5
HSPAIA
HSPAI B
HSPAIL
HASPA8
HSPA2
HSPA6
STCH
HSPA14
HSPA4
HSPA4L
HSPH I
HYOU I
HSPAI2A
HSPAI2B


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alternative first exons 1A and 1B of different lengths but
with the same translation-initiation site. A 21 bp cis-acting
element overlaps the 5' end of exon 1A and is involved in
hypoxic stress-dependent induction of the alternative
transcript that begins with exon lB, resulting in increased
accumulation of the protein in the ER [28]. In a third tran-
scription variant, starting within what is exon 2 of the
transcripts mentioned above, a different protein isoform
is produced with translation-initiation within exon 3,
lacking the ER-import signal peptide. This variant is
thought to have housekeeping functions in the cytosol
[28]. The elucidation of the significance of the known and
predicted mRNA variants of hsp70 genes would be of great
importance for understanding the various functions of
Hsp70 proteins in all cellular compartments, tissues, and
developmental stages under diverse environmental condi-
tions.

Evolution, groups, and subgroups of human Hsp70
Their great diversity notwithstanding, human Hsp70s can
be sorted into evolutionarily related clusters. Early analy-
ses based on sequences of eight typical Hsp70 proteins
from yeast and other typical Hsp70 proteins from plants
and animals, including one from humans, showed that
typical hsp70 genes cluster in four evolutionary groups
that are respectively characterized by expression in cyto-
plasm, ER, mitochondria or plastids [29]. Our phyloge-
netic analysis revealed that the 17 human Hsp70s
characterized in this work belong to seven major Groups
(Figure 1), confirmed by other sequence features, such as
"signature sequences" (sequence elements present in one
Group but not in others) and exon-intron gene structure,
and correlated with other data (e.g., molecular mass and
locale of residence). The phylogenetic tree of the human
Hsp70s of about 70 kDa molecular weight unveiled three
major evolutionary events: firstly, two genes with prod-
ucts of molecular weight around 70 kDa converged into a
primordial eukaryotic organism, one derived from an Alp-
haproteobacterium, from which HSPA9B of mitochon-
drial origin derived, and one, of unrecognized prokaryotic
origin, corresponding to the nuclear gene progenitor of
HSPA8 and HSPA5; secondly, the nuclear gene most likely
first duplicated into two genes, one specialized to the
cytosol/nucleus (orthologous to HSPA8), and one special-
ized to the ER (orthologous to HSPA5); thirdly, the gene
specialized to the cytosol/nucleus later multiplied into a
multigene family (Group VI) represented in humans by
six or seven genes (HSPA8, HSPA2, HSPA1A, HSPA1B,
HSPA1L, HSPA6 and possibly HSPA7) and at least 27
pseudogenes (Figures 1 and 2, and Tables 2 and 6).

Within Group VI, only the coding region of HSPA8 was
encoded by multiple exons (two isoforms encoded by
eight and seven exons, respectively), whereas the coding
regions of all other genes are encoded within a single exon


(although HSPA2 and HSPA1L each possesses a 5'
untranslated exon). Similarly, most pseudogenes associ-
ated to HSPA8 did not show signs of exon-intron struc-
tures. This suggests that the sequences of group VI and
related pseudogenes were all derived from HSPA8 by ret-
rotransposition. The impressive retrotransposition activ-
ity (perhaps LI associated) involving HSPA8 is also
consistent with the very high level of expression of this
gene in comparison with the other members of the hsp70
family. The multi-exon structure of all other genes sug-
gests instead that sets of similar sequences (e.g., the
HSPA4 and HSPA12 subgroups) were generated by dupli-
cation events.

The evolutionary history of HSPA1A, HSPA1B and
HSPA1L has been analyzed in detail in the past [30] and
revisited more recently [31]. The three genes are clustered
with the same organizational structure in the human and
mouse genomes, strongly suggesting that their duplica-
tion happened before the separation of the rodent and
primate lineages. The phylogenetic trees, however, suggest
that HSPA1A and HSPA1B resulted from independent
duplications in human and mouse. This apparent contra-
diction can be interpreted to be the result of gene transfor-
mation between HSPA1A and HSPA1B occurring
independently in human and mouse.

The human HSPA6 and HSPA7 sequences do not signifi-
cantly cluster with any other animal protein. This result is
consistent with an early separation of HSPA6/7 and the
common progenitor of HSPA8, HSPA2 and HSPA1, prob-
ably predating the evolution of Vertebrates. The presence
of sequences from Fish and Amphibians in the lineage of
HSPA8 and the absence of a sequence from Fish in the lin-
eages of HSPA1 and HSPA2 suggests that HSPA1 and
HSPA2 originated from HSPA8 early in the evolution of
the tetrapod lineage, with the exclusion of Fish, although
homologs of these genes might be found in Danio rerio
among the many hsp70 genes of unresolved origin.

Less well defined is the origin of the atypical human
Hsp70-like proteins, comprising two lighter sequences
(about 60 kDa), HSPA14 and STCH, the first associated to
ribosomes and the second expressed in microsomes, and
of the heavier 105/110 kDa HSPA4, HSPA4L, HSPH1 pro-
teins and 150 kDa HYOU1 protein (the latter also local-
ized to the ER). However, the greater similarity of these
sequences to the typical human Hsp70 product compared
to the prokaryotic DnaK proteins suggests that these genes
also originated from a common progenitor of the typical
nuclear eukaryotic genes from a gene duplication that pre-
dated the proliferation of the hsp7O-typical gene
sequences. Also uncertain is the evolutionary position of
HSPA12A and HSPA12B, which conserve only sparse sim-
ilarity to the ATP-binding domain of typical human


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Hsp70 proteins, and are marginally more similar to
prokaryotic DnaK [16]. We found that the evolutionary
relations of these multiexon sequences are not clarified by
their gene structure. In fact, whereas the positions of the
splice sites within each Group were generally conserved,
we found very few conserved splice sites when comparing
sequences from different Groups.

Conclusion
The human hsp70-gene family is in the main the result of
multiple duplications facilitated by frequent retrotranspo-
sition events of a single highly expressed gene, HSPA8.
Human Hsp70 proteins can be clustered into seven dis-
tinct evolutionary Groups, many of which are character-
ized by highly divergent C-terminal (substrate-binding)
domains. Hence, functional specificities of the various
Hsp70 proteins are likely to be associated to the func-
tional properties of their C-terminal domains. In addi-
tion, other noticeable differences in the nucleotide-
binding N-terminal domain of the various Hsp70s may
also affect the functional role of these proteins.

Overall, the results revealed an evolutionary diversity at
genetic, transcriptional, and post-transcriptional levels
that generated a multiplicity of structures, functions, reg-
ulation modes, and anatomic locations of Hsp70s, pre-
dicting a wide array of roles in cell physiology and
pathogenesis.

A thorough classification of all the members of the hsp70
gene family encoded in the genome is an essential step
towards their unambiguous functional characterization.
The identification of the chaperonopathies [5-7,32,33]
has made more pressing the need for a better characteriza-
tion of these genes in order to pinpoint with accuracy the
gene-proteins involved, understand pathogenetic mecha-
nisms, and devise diagnostic and therapeutic means. Bio-
informatics analyses of evolutionary relations and
sequence features of the numerous hsp70 paralogs found
in the human genome suggest many interesting avenues
of experimental studies (e.g., to determine specific func-
tions in specific tissues of any given Hsp70) and provide
the basis for selecting the most efficient molecule for use
in chaperonotherapy or the most appropriate target
(Hsp70 molecule and/or segment therein) for anti-chap-
erone agents.

Methods
Chaperonomics
The strategies and methods described previously for chap-
eronomics [31] were applied throughout. Chaperonomics
encompasses a series of concatenated, complementary
methods that are applied in sequence to obtain layer after
layer of results that progress toward the building of a


detailed picture of genes and their proteins, including
structural and evolutionary information at various levels.

Gene identification
hsp70-like DNA sequences were identified as matches to
13 distinct reference-Hsp70 query-sequences (proteins)
selected from a much larger pool of sequences available in
public databases, mining the NCBI reference human
genome sequence Build 36.1 (2006) using the search
engines BLAT [34], at the University of California at Santa
Cruz (UCSC), and BLAST [35], at NCBI (TBLASTN for
Protein to DNA sequence). The DNA strand and chromo-
somal location were recorded as per the UCSC genomic
browser.

Exon and intron identification
Initially, intron, exons, and alternative spliced isoforms
were recorded as reported in the gene description pages
from the NCBI "Entrez Gene" annotation and from the
UCSC Genome Browser annotation links. We also carried
out literature searches to identify some of the variants
(e.g., for HYOU1). As a last step, we verified all intron/
exon structures by aligning the protein and processed
mRNA sequences to the genomic sequence, using the
BLAT [34] search engine, identifying introns as genome
sequences interspersed between aligned regions (exons).

Identification and characterization of diverged or
degenerate sequences
Diverged or degenerate sequences were identified with the
BLAST search engine, enabling us to collect and evaluate
sequence regions that displayed similarity to hsp70 genes
of lower significance. For each BLAST hit (aligned seg-
ment), we extracted the flanking genome sequence up to
several thousand nucleotides 5' and 3' of the hit. We then
translated the sequences encoded in the three frames of
the strand where the hit was identified and, subsequently,
we aligned the three translations to the query Hsp70 pro-
teins using ITERALIGN [36] and, by visual inspection of
the alignments, we identified all conserved Hsp70-like
sequence regions linked by short intervening sequences.
When regions proximal to the 5' end of the query genes
were conserved, the sequence was extended up to the
proximal Start codon (ATG) in the approximately
expected position, when present; otherwise, the sequence
was initiated at the first conserved region. Similarly, the
sequence was extended 3' of the last conserved segment
up to the fist stop codon, when present, or else terminated
at the last conserved segment. The predicted sequence was
matched to the human genome using BLAT. Long genome
sequences interspersed between the aligned portions (pre-
dicted as exons) of the query sequence were considered
potential introns.




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The sequence assembly process described above enabled
us to obtain a complete characterization of the patterns of
conservation for these sequences, and to identify indica-
tors of degeneracy in the coding region, such as frame-
shifts (locations where the alignment in one frame of
translation switched to an alignment in a different frame
of translation) and in-frame stop codons (i.e., stop
codons within a conserved hsp70-like sequences).

Identification of other eukaryotic, nonhuman hsp70 genes
We collected hsp70 genes and Hsp70 proteins from non-
human eukaryotic organisms for which complete genome
sequences are available, by querying the respective
genome sequences with the protein similarity search pro-
gram SSPA [36].

Protein similarity
All similarities reported are SSPA (Significant Segment
Pair Alignment) similarities [36] determined by using the
Blosum62 [37] amino acid similarity matrix.

Protein isoform identification
Many protein/mRNA isoforms were recorded as listed in
the annotations from the UCSC BLAT server Browser
pages and in the NCBI Entrez Gene pages. In addition, we
identified other variants by comparing sequences and
through literature searches.

Phylogenetic analysis
Evolutionary analyses were performed using protein
sequences from the hsp70 genes collected from the human
genome and other eukaryotic genomes and pseudo-trans-
lations of human hsp70 pseudogenes. We excluded all
sequences of length less than 300 aa (labeled as "frag-
ment") or too diverged to produce reliable alignments.
We performed the alignments with Clustal W and con-
structed evolutionary trees using the neighbor-joining
algorithm [38] with the Kimura transformation for pro-
tein sequences [39], and with bootstrap analyses based on
1000 samples. For evolutionary trees of human sequences
we repeated the analyses using the maximum-likelihood
method implemented in PHYML [40].

Sources of gene structure and expression data based on
EST and SAGE analyses
Predictions of gene structure variants were collected and
organized from the ECgene web-site [43] and database
[14,15] and from the NCBI AceView database [41], based
on EST and SAGE expression data. Expression data based
on the number of tissue-specific and developmental-
stage-specific ESTs were collected from the NCBI UniGene
database [25].


Abbreviations
ABD, ATP-binding domain; SBD, substrate-binding
domain; UBP, ubiquitin-binding peptide; TPR1, tetratrico
peptide repeat 1; ER, endoplasmic reticulum

Authors' contributions
All authors contributed equally to this work in discussing
research strategy and development, in data collection and
classification, and in writing the manuscript. LB per-
formed the bioinformatics analyses. All authors read and
approved the final manuscript.

Additional material


Additional file 1
Figures S1 -S4 and Table S1. Alignments of Hsp70 sequences with splice-
site positions (Figures S1-S3), evolutionary tree of eukaryotic typical
hsp70 genes (Figure S4), and genome position and length of each exon
of hsp70-like genes (Table Sl).
Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-
2148-8-19-S1.pdf]


Acknowledgements
We thank Craig Stropkay and Kristen Washburn for assistance with data-
base searches.

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