Chapter 2

Chaperones: General Characteristics

and Classifications

Abstract This chapter presents the classification of chaperones, their molecular

properties among which that of forming functional complexes involving various
molecules, and their distribution inside and outside the cell. The chaperone genes
in the human genome are listed and briefly described, focusing on the small heat-
shock proteins (sHsp), Hsp60, Hsp70, and Hsp90, and mentioning all others
known. The chapter also introduces the concept of chaperoning system, i.e., the
physiological system of an organism which is composed of all its chaperones,
co-chaperones, and chaperone co-factors.

 
Keywords Chaperone genes, human genome sHsp Hsp40/DnaJ Hsp60  
  
Hsp70 Hsp90 Chaperoning teams Chaperoning networks Chaperoning 
 
system Ubiquitin-proteasome system (UPS) Apoptosis

2.1 Classification of Chaperones

Hsp-chaperones are classified in various ways. For example, according to molecular

weight they fall into several groups, Table 2.1. In some of these groups are clas-
sified well known families of phylogenetically related proteins, such as the Hsp90,
Hsp70, Hsp40, and sHsp-crystallin families. Other members of these groups are not
members of those families although they have molecular weights within the stip-
ulated ranges. Chaperonopathies involve chaperones pertaining to all these groups.
The classification of chaperones according to their apparent molecular weight
(MW) as determined, for instance, by gel electrophoresis is quite useful. If one
knows the MW of the chaperone under scrutiny and wants to check if it is present in
a sample (serum or another biologic fluid, secretion, or tissue extract), a simple gel
electrophoresis with protein staining might give the first piece of information
desired. Subsequent steps applying complementary methods such as Western
blotting (using specific anti-chaperone antibodies; see for example Fig. 9.1), 2D
electrophoresis and mass spectrometry will definitely clarify the situation. This is
very encouraging because the need to determine presence, levels, and distribution of

A. J. L. Macario et al., The Chaperonopathies, SpringerBriefs in Biochemistry 15

and Molecular Biology, DOI: 10.1007/978-94-007-4667-1_2, Ó The Author(s) 2013
16 2 Chaperones: General Characteristics and Classifications

Table 2.1 Subpopulations of Hsp-chaperones classified according to apparent molecular weight

MW Classical family in this Other members in this range implicated in the causation
(kDa) MW range of disease (chaperonopathies)a
range
200 or None Sacsin
higher
100–199 Hsp100–110
81–99 Hsp90 Paraplegin [SPG7]
65–80 Hsp70/DnaK Spastin [SPG4]; LARP7
55–64 Hsp60 (chaperonins groups Myocilin; protein disulphide-isomerases (PDI)
I and II, e.g., Cpn60 and
CCT, respectively)
35–54 Hsp40/DnaJ AIP; AIPL1; torsin A; clusterin; DNAJC19 (TIM14)
34 or less sHsp (crystallins) Hsp10 (Cpn10); Alpha hemoglobin-stabilizing protein
(AHSP); cyclophilin type peptidyl-prolyl cis–trans
isomerase (PPI); alpha-synuclein; HSPB11
a
Others are named in the tables, for example Table 4.3, in which the pertinent chaperonopathies
are listed
Source Macario AJL (1995) Heat-shock proteins and molecular chaperones: Implications for
pathogenesis, diagnostics, and therapeutics. Intl J Clin Lab Res 25:59–70; and Macario AJL,
Conway de Macario E (2009) The chaperoning system: Physiology and pathology. Exp Med Rev
Vol. 2–3: Years 2008/09, pp. 9–21 (http://www.unipa.it/giovanni.zummo)
 2.1 Classification of Chaperones 17

chaperones in clinical and other biological samples in research is quite common. It

has to be borne in mind, though, that the electrophoretic characteristics of a chap-
erone may vary due to structural changes caused by post-translational modifications
or mutations. In the case of deletions, the length of the protein may be considerably
altered. So, the results have to be examined very critically, particularly when a
chaperonopathy is suspected and the chaperone molecule might by abnormal.

2.2 Chaperones can Form Functional Complexes

that Work as Multimolecular Machines

Chaperones usually do not work alone but form teams, sometimes called chap-
eroning machines. Teams can be formed by varios members of the same family,
i.e., oligomers, in which all members are identical, homo-oligomers, or not,

b Fig. 2.1 Examples of chaperoning teams. CM. The chaperoning machine (CM) is a team of
essentially three proteins, Hsp70 (70), Hsp40 (40), and nucleotide (N; e.g., adenosine triphosphate,
abbreviated ATP) exchange factor (NEF). Hsp70 binds a client (e.g., unfolded or misfolded)
polypeptide (Cp) and, in collaboration with the other members of the team, assists the polypeptide
to fold correctly to achieve its native conformation. This chaperoning process involves ATP
hydrolysis mediated by the ATPase domain of Hsp70 and stimulated by Hsp40, and exchange of
adenosine diphosphate (ADP) for ATP, an exchange that is induced by the nucleotide exchange
factor (e.g., BAG-1, which means Bcl-2-associated athanogene 1, where Bcl-2 stands for B cell
lymphoma 2). See Fig. 2.2. PFN. Prefoldin (PFN) is composed of distinct subunits (1 through 6 in
humans) arranged in a medusa type of structure. MTC. The mitochondrial chaperonin (MTC) is a
barrel shaped complex of two multimeric assemblages. The larger assemblage is formed of two
superimposed rings, each with seven identical subunits (Hsp60 also named Cpn60, where Cpn
stands for chaperonin), delimiting a central cavity, the folding chamber. The smaller assemblage is
also ring shaped, being formed by seven identical subunits (Hsp10, also named Cpn10) that are
considerably smaller than those around the folding chamber. The smaller ring serves as a lid to
close the folding chamber while the polypeptide folding process is taking place inside. CCT. The
chaperonin-containing TCP-1 poypeptide (CCT), where TCP-1 means tailless complex polypep-
tide 1, is similar in overall structure to the mitochondrial chaperonin since it is also formed of two
superimposed multisubunit rings and it has a central cavity. However, the CCT rings are formed of
eight distinct subunits (A through H, also named alpha through theta) rather than seven identical
subunits, and there is no third ring or lid. sHsp. The small heat-shock proteins (sHsp) are
chaperones, such as the alpha-crystallins, that are usually 34 kDa or less in molecular weight (see
Table 2.1) and are normally present as monomers, or multimers of varios degrees of complexity,
depending on which sHsp, cell type, and conditions (e.g., stress vs. basal, constitutive) is
considered. Source Macario AJL, Conway de Macario E (2005) Sick chaperones, cellular stress
and disease. New Eng J Med 353:1489–1501; Macario AJL, Conway de Macario E (2001) The
molecular chaperone system and other anti-stress mechanisms in archaea. Front Biosci 6:
d262–283. To see Table of Contents, Abstract, Figures, and Tables go to http://www.bioscience.
org/2001/v6/d/macario/fulltext.htm; Macario AJL, Malz M, Conway de Macario E (2004) Evo-
lution of assisted protein folding: The distribution of the main chaperoning systems within the
phylogenetic domain Archaea. Front Biosci 9: 1318–1332. To see Table of Contents and Abstract
go to http://www.bioscience.org/2004/v9/af/1328/fulltext.htm; and Macario, AJL, Conway de
Macario E (2007) Molecular chaperones: Multiple functions, pathologies, and potential applica-
tions. Front Biosci 12: 2588–2600. To see Table of Contents, Abstract and Figures go to http://
www.bioscience.org/2007/v12/af/2257/fulltext.htm
18 2 Chaperones: General Characteristics and Classifications

hetero-oligomers. An example of the former is the mitochondrial chaperonin

Hsp60 complex, which is formed by seven identical subunits, Fig. 2.1, MTC,
Hsp60. An example of hetero-oligomer is the chaperonin of Group II present in the
cytosol, named chaperonin-containing TCP-1 polypeptide (CCT), Fig. 2.1.
Also shown in Fig. 2.1 are prefoldin (PFN), which comprises six different but
very similar subunits in humans; Hsp10 (that builds heptamers; MTC, Hsp10), and
other small heat-shock proteins, indicated as sHsp; and Hsp70 (ATPase 70).
Hsp70 forms a chaperoning machine (CM) by interacting with a co-chaperone,
Hsp40/Dnaj (40) and with a co-factor, nucleotide exchange factor (NEF)
and, thus, with participation of ATP (N) folds nascent polypeptides (client
polypeptide, Cp), as illustrated in Fig. 2.2.
It is believed that the sHsp, or at least some of them, undergo dynamic assembly
into mono- and poly-disperse oligomers (Fig. 2.1) with variable chaperoning
ability. For example, the alpha-A crystallin (HspB4 in Table 2.3), the largest being
composed of 30–40 identical subunits; this results in a highly dynamic quaternary
structure with the subunits exchanging between the oligomers of various multi-
plicities. The chaperoning ability seems to depend on the rate of oligomer disas-
sembly. Another sHsp, Hsp27, may occur as multimers under basal conditions but
the complexes disaggregate upon cellular stress and the free monomers become
involved in counteracting the consequences of stress such as protein aggregation.
It becomes clear from these considerations and from the illustrations in
Figs. 2.1, 2.2 that each single molecule of a chaperone must have different
functional domains to carry out the various functions pertaining to team building
and polypeptide folding. In addition, teams interact between them to build
 2.2 Chaperones can Form Functional Complexes 19

b Fig. 2.2 Examples of chaperoning teams forming a network. The Hsp70–Hsp40-nucleotide-

exchange factor (NEF) chaperone machine (CM) is a dynamic complex or team involved in protein
folding that also interacts with other teams. Top left Hsp70 (70) binds the unfolded client
polypeptide (Cp; represented as a slightly ondulating line), via its peptide-binding domain near the
middle of the molecule, when ADP is bound to its ATPase domain and Hsp40 (40) is bound to its
C-terminal domain. Top middle the client polypeptide, still unfolded (Pu) but already bound to
Hsp70, enters the folding cycle. The nucleotide-exchange factor (e.g., BAG-1, or GrpE in
prokaryotes, shown as NEF) promotes exchange of ADP for ATP. When Hsp70 is bound to ATP, its
affinity for the folding polypeptide decreases, and the folded polypeptide (Pf; represented as ?) is
released. The nucleotide-exchange factor is replaced by Hsp40, ATP hydrolysis occurs with release
of pyrophosphate (Pi), and a new cycle of peptide binding, folding and release begins. N,
nucleotide, e.g., ATP or ADP. The bottom half of the figure shows other alternatives. For example,
the partially folded polypeptide (represented as S) is handed over by the CM to the CCT complex,
directly or with participation of Hsp90 (90; to the left) and/or prefoldin (PFN; to the right). The
polypeptide is then folded inside the CCT-folding chamber and released. CM and Hsp90 can also be
involved in a pathway leading to protein degradation (Pd) in the proteasome, for instance. While
CM occurs in the three life Domains (i.e., Bacteria, Archaea, and Eukarya), CCT and PFN exist in
eukaryotes (typically the cytosol) and archaea but not in bacteria. CM also exists in the eukaryotic
cell compartments: nucleus, mitochondria, endoplasmic reticulum, and chloroplasts. Red arrows
and blue discontinuous arrow indicate possible interactions between chaperoning teams to form a
functional network. The violet arrow indicate the pathway to degradation by intracellular proteases,
including the ubiquitin–proteasome system. The green vertical arc near each team indicate that the
team members interact with each other and that the whole complex is dynamic, i.e., it changes
conformation while performing its function. See also Sect. 7.2 for a brief discussion on the inter-
action of the chaperonin system with the ubiquitin–proteasome system (UPS) involved in protein
degradation, and with autophagy, particularly chaperone-mediated autophagy (CMA), and with
apoptosis (programmed cell death). Source Macario AJL, Conway de Macario E (2001) The
molecular chaperone system and other anti-stress mechanisms in archaea. Front Biosci 6:
d262–283. To see Table of Contents, Abstract and Figures go to http://www.bioscience.org/2001/
v6/d/macario/fulltext.htm; Macario AJL, Malz M, Conway de Macario E (2004) Evolution of
assisted protein folding: The distribution of the main chaperoning systems within the phylogenetic
domain Archaea. Front Biosci 9: 1318–1332. To see Table of Contents and Abstract go to http://
www.bioscience.org/2004/v9/af/1328/fulltext.htm; and Macario, AJL, Conway de Macario E
(2007) Molecular chaperones: Multiple functions, pathologies, and potential applications. Front
Biosci 12: 2588–2600. To see Table of Contents, Abstract, and Figures go to http://
www.bioscience.org/2007/v12/af/2257/fulltext.htm

functional networks. Therefore, individual molecules must have domains dedi-

cated to form networks. It is then evident that there are many sites in the chap-
erone molecules in which a change due to mutation, or to post-translational
modification, may abolish or alter one or more of their interactions and functions
with the potential of causing a chaperonopathy.

2.3 Distribution of Chaperones

Chaperones are found in all cells, cell compartments, tissues, and outside cells in
the intercellular space, in circulation, and in secretions (Table 2.2). Consequently,
it is to be expected that a chaperonopathy might have an impact on various
locations, generating pleiotropic and variable pathological and clinical pictures.
20 2 Chaperones: General Characteristics and Classifications

Table 2.2 Residences of chaperones in eukaryotes

Location Compartment
Intracellular Nucleus; cytosol; mitochondria; endoplasmic reticulum (ER); lysosomes; vesicles;
plasma membrane; chloroplasts (in plants)
Pericellular Cell membrane on the outside
Extracellular Intercellular space; blood (plasma, serum): soluble or in vesicles (e.g., exosomes);
lymph; cerebrospinal fluid; intrasynovial space (joint cavity); secretions (e.g.,
saliva, urine)
Source Macario AJL, Conway de Macario E (2009) The chaperoning system: physiology and
pathology. Exp Med Rev Vol. 2–3: Years 2008/09, pp. 9–21, (http://www.unipa.it/
giovanni.zummo); and Cappello F, Conway de Macario E, Marasa L, Zummo G, Macario AJL
(2008) Hsp60: new locations, functions, and perspectives for cancer diagnosis and therapy.
Cancer Biol Ther 7:801–809

It has become clear from recent studies that chaperones thought to be confined
to a single cell compartment, e.g., the mitochondria, can also be found in other
locations. It is of particular interest that several chaperones are now known to be
on the plasma-cell membrane, exposed to the outside of the cell, especially in
tumors. This knowledge opens new avenues for research and for developing
diagnostic means centered on detecting tumors by targeting chaperones on their
cells using antibodies. Even more exciting is the possibility of utilizing these
antibodies to deliver anticancer drugs to the cancer cells, or to kill them immu-
nologically (see also Chap. 9).
As a direct consequence of the re-assessment of the distribution of chaperones
intracellularly and extracellularly and its alterations in disease, morphological
methods that detect chaperones in situ have been re-evaluated and given consid-
erable importance. Immunocytochemistry and immunohistochemistry with spe-
cific antibodies, and complementary morphological and quantitative techniques
(e.g., confocal microscopy), are instrumental to estimate chaperone levels in cells
and tissues and their changes with disease progression and response to treatment,
for instance. These morphological methods are also very useful to map the dis-
tribution of chaperones in cells and tissues, and its variations in pathological
situations such as the chaperonopathies. If electron microscopy is added, the
immunogold technique with specific antibodies can provide crucial information on
the chaperone location, for instance plasma-cell membrane, mitochondria, Golgi
apparatus, or cytosol (see also Sect. 7.4 and Chap. 9).

2.4 How Many Chaperones in the Human Species?

Given that molecular chaperones can be affected by abnormalities, genetic or

acquired, structural and/or functional, it is pertinent to ask how many chaperones
are there in humans that might cause problems. How many chaperonopathies are
possible? The answer to this question has become accessible since the development
of strategies and methods for genome sequencing and analysis. We developed a
2.4 How Many Chaperones in the Human Species? 21

protocol, chaperonomics, for identifying chaperone genes and, applying this

protocol, we elucidated the complement of Hsp60/CCT and Hsp70 genes in the
human genome. Likewise, others have identified other members of the chaperoning
system in humans. It is likely that more members will be discovered as the
understanding of the genome progresses and the boundaries of genes and the extent
of regulatory regions are better defined. In any case, the information already
available provides an excellent basis to study chaperones and their abnormalities or
chaperonopathies. In addition, the diversity of chaperones stemming from the set
of genes in the genome is most likely enlarged by, for instance, variations in the
transcription initiation site, differential (alternative) splicing, and post-translational
modifications. This diversity of mechanisms involved in the synthesis and
production of chaperone molecules could be the basis of the diverse functions and
locations that are constantly being unveiled for the chaperone proteins (see
Table 2.2, and Sect. 2.5). These transcriptional and post-translational variations
will, no doubt, be the focus of research in the immediate future because they have
the potential of uncovering fundamental principles of biology in what regards
molecular migration and diversification of function.
Unfortunately, many chaperones have been given various names, which can be
very confusing and frustrating. To assist the reader, we have included in some of

Table 2.3 Human sHsp genes and proteins

Alpha- New nomenclature Other names Gene aa
crystallin ID
Gene Protein
family
HspB1 HSPB1 HSPB1 CMT2F; HMN2B; HSP27; HSP28; HSP25; 3315 205
HS.76067; DKFZp586P1322
HspB2 HSPB2 HSPB2 MKBP; HSP27; Hs.78846; LOH11CR1K; 3316 182
MGC133245
HspB3 HSPB3 HSPB3 HSPL27 8988 150
HspB4 HSPB4 HSPB4 Crystallin alpha A; CRYAA, CRYA1 1409 173
HspB5 HSPB5 HSPB5 Crystallin alpha B, CRYAB; CRYA2 1410 175
HspB6i HSPB6 HSPB6 HSP20; FLJ32389 126393 160
HspB7 HSPB7 HSPB7 cvHSP; FLJ32733; DKFZp779D0968 27129 170
HspB8 HSPB8 HSPB8 H11; HMN2; CMT2L; DHMN2; E2IG1; 26353 196
HMN2A; HSP22; CRYAC
HspB9 HSPB9 HSPB9 FLJ27437 94086 159
HspB10 HSPB10a HSPB10 ODF1; ODF; RT7; ODF2; ODFP; SODF; 4956 250
ODF27; ODFPG; ODFPGA; ODFPGB;
MGC129928; MGC129929
HSPB11 HSPB11 HSP16.2; C1orf41; PP25; IFT25; HSPCO34 51668 144
Source Kappe G, Franck E, Verschuure P, Boelens WC, Leunissen JAM, de Jong WW (2003)
The human genome encodes 10 alpha-crystallin–related small heat shock proteins: HspB1–10.
Cell Stress Chaperones 8:53–61; Kampinga HH, Hageman J, Vos MJ, Kubota H, Tanguay RM,
Bruford EA, Cheetham ME, Chen B, Hightower LE (2009) Guidelines for the nomenclature of
the human heat shock proteins. Cell Stress Chaperones 14: 105–111; doi: 10.1007/s12192-008-
0068-7; and Hu Z, Yang B, Lu W, Zhou W, Zeng L, Li T, Wang X (2008) HSPB2/MKBP, a
novel and unique member of the small heat-shock protein family. J Neurosci Res 86:2125–2133
22 2 Chaperones: General Characteristics and Classifications

the Tables describing chaperones not only the preferred name but also some other
names that are, or have been, used in data bases and publications.

2.4.1 Human Small Size (34 kDa or Less) Chaperones

Thus far 10 genes encoding small heat shock proteins (sHsp) with the crystallin
domain have been identified in the human genome (Table 2.3). However, there are
several other chaperones with a MW similar to those of the sHsp (e.g., HspB11 in
Table 2.3, and many others some of which are listed in Table 2.1), which can be
affected by pathologic changes and, thus, can be the basis of a chaperonopathy.

Table 2.4 Human hsp60/CCT genes

Name Alternative names Start End Str Chr Loc Is Ex aa
CCT1 TCP1, CCTa, TCP-1 160,119,520 160,130,731 - 6 q25.3 2 12, 7 556, 401
alpha
CCT2 TCP1 beta 68,266,317 68,280,052 + 12 q15 1 14 535
CCT3 TCP1 gamma 154,545,617 154,572,307 - 1 q23.1 3 13, 13, 545, 544,
12 507
CCT4 TCPD, TCP-1 delta 61,950,076 61,969,146 - 2 p15 1 13 539
CCT5 TCP1E, TCP1 epsilon, 10,303,453 10,317,892 + 5 p15.2 1 11 541
KIAA0098
CCT6A TCP1 zeta, CCT6, Cctz, 56,087,036 56,098,269 + 7 p11.2 2 14, 13 531, 486
HTR3, TCP20,
TCPZ, TTCP20
CCT6B Cctz2, TSA303, Tcp20 30,279,183 30,312,525 - 17 q12 1 14 530
CCT7 TCP1 eta 73,320,279 73,333,494 + 2 p13.2 2 12, 7 543, 339
CCT8 TCP1 theta 29,350,670 29,367,782 - 21 q21.3 1 15 548
CCT8L1 LOC155100 151,773,495 151,775,165 + 7 q36.1 1 1 557
CCT8L2 GROL, CESK1 15,451,770 15,453,440 - 22 q11.1 1 1 557
MKKS BBS6 10,333,898 10,342,162 - 20 p12.2 2 4, 4 570, 570
BBS10 C12orf58, FLJ23560 75,263,727 75,266,269 – 12 q21.2 1 2 723
BBS12 C4orf24, FLJ35630, 123,882,498 123,884,627 + 4 q27 1 1 710
FLJ41559
HSPD1 GROEL, HSP60, SPG13, 198,060,018 198,071,817 - 2 q33.1 2 11, 11 573, 573
CPN60, HuCHA60
PIKFYVE CFD, FAB1, PIP5 K, 209,182,591 209,190,094 + 2 q34 1 5 224
PIP5K3
Key Name, official NCBI Entrez gene database name. For CCT1, the official Entrez name is TCP1 but we
chose CCT1 for consistency with other subunit gene names; Start and End, start and end of coding region;
Str, DNA strand with positive or negative signs indicating sequenced or complementary strand, respectively;
Chr, chromosome; Loc, chromosomal location; Is, number of isoforms or mRNA variants; Ex, number of
exons-multiple numbers indicate the number of exons in each isoform; aa, amino acids encoded; (PIKFYVE),
Fab1_TCP sequence domain of the PIKFYVE kinase, most similar to the apical domain of CCT3, in which
features refer to the domain portion of the gene/protein
Source Brocchieri L, Conway de Macario E, Macario AJL (2007) Chaperonomics, a new tool to study ageing
and associated diseases. Mechan Ageing Develop. 128:125–136; and Mukherjee K, Conway de Macario E,
Macario AJL, Brocchieri L (2010) Chaperonin genes on the rise: new divergent classes and intense duplication
in human and other vertebrate genomes. BMC Evolutionary Biology 2010, 10:64; doi:10.1186/1471-2148-10-
64. http://www.biomedcentral.com/1471-2148/10/64
2.4 How Many Chaperones in the Human Species? 23

Fig. 2.3 The branching patterns of the canonical (CCT1–CCT8) subunits in the maximum
likelihood tree indicate that distinct clades are formed by the subunits CCT4 and CCT5, by the
subunits CCT3 and CCT6, and, with strong bootstrap support, by the subunits CCT1, CCT2 and
CCT7. The data also indicate that the BBS clade and CCT8L share a common ancestor with CCT8
but are not necessarily derived from CCT8. The tree was rooted by the archaeal thermosome alpha
subunit of Sulfolobus solfataricus (SULSO2). Source Mukherjee K, Conway de Macario E,
Macario AJL, Brocchieri L (2010) Chaperonin genes on the rise: new divergent classes and intense
duplication in human and other vertebrate genomes. BMC Evolutionary Biology 2010, 10:64;
doi:10.1186/1471-2148-10-64. http://www.biomedcentral.com/1471-2148/10/64

2.4.2 Human Hsp60/CCT Genes and Proteins

The human genome harbors 16 hsp60/CCT genes, including the mitochondrial

Hsp60 or Cpn60 (Table 2.4) and many related pseudogenes. The CCT genes form
4 (or 5, depending on the criteria applied) evolutionarily related groups (Fig. 2.3).

2.4.3 Human Hsp70 Genes and Proteins

The human genome harbors 17 hsp70 genes (Table 2.5, parts 1 and 2) and many
related pseudogenes. The genes form seven evolutionarily related groups (Fig. 2.4).
Table 2.5 hsp70 genes in the human genome
24

Name Location Str Start/End nt Ex Is aa

Part 1
Hsp70kDa 6 (HSP70B’)/HSPA6 1q23.3 + 159,761,073/ 1,929 1 1 643
159,763,001 N
Hsp70kDa 7 (HSP70B)/HSPA7 1q23.1 + 159,842,705/159,844,628 1,924 1 1 641
Hsp70kDa 4-like/HSPA4L 4q28.1 + 128,923,156/ 50,321 19 1 839
128,973,476 N
Hsp70kDa 9B/HSPA9B 5q31.2 – 137,919,628/137,938,906 19,279 17 1 679
Hsp70kDa 4/HSPA4 5q31.1 + 132,415,842/132,468,024 52,183 19 2; a 840
132,415,842/132,468,024 52,183 5 b 148
Hsp70kDa 1-like/HSPA1L/HSP70-Hom 6p21.33 – 31,885,806/31,887,728 1,923 1 1 641
Hsp70kDa 1A/HSPA1A/HSP70-1 6p21.33 + 31,891,513/31,893,435 1,923 1 1 641
Hsp70 kDa 1B/HSPA1B/HSP70-2 6p21.32 + 31,903,707/31,905,629 1,923 1 1 641
Hsp70 kDa 5 (Grp78; BiP)/HSPA5 9q33.3 – 127,038,695/127,043,226 4532 8 1 654
Part 2
Hsp70kDa 12A/HSPA12A 10q25.3 – 118,424,285/118,456,787 32,503 12 1 675
Hsp70kDa 14/HSPA14 10p13 + 14,920,408/14,953,608 33,201 14 2 509
14,920,408/14,924,181 3,774 4 88
Hsp70kDa 8/HSPA8 11q24.1 – 122,433,655/122,437,242 3,588 8 2 646
122,433,655/122,437,242 3,588 7 493
150kDa oxygen-regulated protein/ 11q23.3 – 118,421,518/118,432,082 10,565 25 4 999
HYOU1 118,421,518 / 118,432,082 10,565 25 [3] 999
118,421,518/118,432,082 10,234 24 964
\118,424,940/118,432,082 [7,143 16 687
(continued)
2 Chaperones: General Characteristics and Classifications
Table 2.5 (continued)
Name Location Str Start/End nt Ex Is aa
Hsp105kD/HSPH1 13q12.3 – 30,609,458/30,633,719 24,262 18 2; a 858
30,609,458/30,633,719 24,262 17 b 814
Hsp70kDa 2/HSPA2 14q23.3 + 64,077,321/64,079,237 1,917 1 1 639
Hsp70kD 12B/HSPA12B 20p13 + 3,667,322/3,680,810 13,489 12 1 686
Stress 70 protein chaperone/STCH 21q11.2 – 14,667,812/14,677,311 9,500 5 1 471
Parts 1 and 2. Human hsp70 genes. Key Name, official NCBI Entrez gene database name; Str, DNA strand with positive or negative signs indicating
sequenced or complementary strand, respectively; Location, chromosomal location as per UCSC genomic browser; Start/End, genomic positions at the 50
and 30 ends of the mRNA, including the 50 -UTR and 30 -UTR, except for HSPA7, for which the coding-region length is shown since there is no mRNA
sequence available for this gene. The data are as per the UCSC genomic browser website; nt, gene length in nucleotides (base pairs); Ex, exons, excluding
non-coding exons when present; Is, protein isoforms and mRNA variants, as pertinent. For the HYOU1 gene four mRNA variants and three protein isoforms
(shown within brackets) have been determined; aa, amino acids encoded. HSPA7 was found to be transcribed under heat-shock in fibroblasts and it was
concluded that the gene is functional, although this is still under scrutiny. It is possible that HSPA7 could be a sometimes transcribed pseudogene. There is
no conclusive evidence on whether its mRNA produces or not a protein with a defined function
Source Brocchieri L, Conway de Macario E, Macario AJL (2007) Chaperonomics, a new tool to study ageing and associated diseases. Mechan Ageing
Develop. 128:125–136; and Brocchieri L, Conway de Macario E, Macario AJL (2008) hsp-70 genes in the human genome: conservation and differentiation
2.4 How Many Chaperones in the Human Species?

patterns predict a wide array of overlapping and specialized functions. BMC Evolutionary Biology 2008, 8:19; doi:10.1186/1471-2148-8-19. http://
www.biomedcentral.com/1471-2148/8/19
25
26 2 Chaperones: General Characteristics and Classifications

Fig. 2.4 A phylogenetic tree of the proteins encoded by the 17 hsp70 genes distinguishing seven
major evolutionarily related groups (shown by the vertical red lines), which were defined by
bootstrap-support values over 85 %. The asterisks indicate the genes for which related
pseudogenes were found. Group I was composed of the most diverged sequences, HSPA12A and
HSPA12B, of uncertain relation with other eukaryotic and prokaryotic Hsp70s. 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 determination of their evolutionary position compared to other human Hsp70s. Group II
was composed of the mitochondrial protein HSPA9B, considered of alpha-proteobacterial origin,
which accordingly, clustered with the DnaK sequence from E. coli (Gamma-proteobacteria) with
very high bootstrap support. Group III encompassed the 105/110 kDa proteins HSPA4,
HSPA4L, HSPH1 (clustered with 100 % bootstrap support) 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) adopted to
identify closely related sequences as members of a distinct Group. Therefore, HSPA14 was
classified 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
differentiate the Groups, and thus STCH was assigned to another group, i.e., Group V. Group VI
was composed of sequences clustered with 100 % bootstrap support and, among them, three
subgroups were distinguish, 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 minimum adopted for distinguishing the main groups. Source
Brocchieri L, Conway de Macario E, Macario AJL (2008) hsp-70 genes in the human genome:
conservation and differentiation patterns predict a wide array of overlapping and specialized
functions. BMC Evolutionary Biology 2008, 8:19; doi:10.1186/1471-2148-8-19 http://
www.biomedcentral.com/1471-2148/8/19

Other genes related functionally with Hsp70. Information on another impor-

tant group of Hsp-chaperones, Hsp40/DnaJ, can be found in Qiu XB, Shao YM,
Miao S, Wang L (2006) The diversity of the DnaJ/Hsp40 family, the crucial partners
for Hsp70 chaperones. Cell Mol Life Sci 63:2560–2570; and Kakkar V, Prins LC,
Table 2.6 hsp90 genes
Group Species Total HTPG TRAP HSP90C HSP90B HSP90A HSP90AA HSP90AB
studied (bacterial (mito.) (chlor.) (ER) (cytosolic) (cytosolic (cytosolic
Hsp90) [HSPC5]a [HSPC4] [HSPC1] alpha) beta)
[HSPC2] [HSPC3]
Animalia 9 3–16 None (only Yes (9 of 9 None (only Yes (all 9 Yes (6 of 9 Yes (3 of 9 Yes (3 of 9
in bacteria) species) in plants) species) species) species) species
Homo 5 (16) None 1 (1) None 1 (3) None 2 (6) 1 (6)
sapiens
All studied (6) 32
Coding region 1–19 1 (1)–1 (1) 3 (3)-19–19 10 (?)–21 (21) 1 (?)–18 (18) 1 (1)-11 (12) 8 (?)–12 (12) 7 (7)–11 (12)
exons
(mRNA)
AA mature 588–854 588 (588)–681 644 (688)–687 756 (810)–785 695 (711)–800 689 (689)–745 728 (728)–854 724 (724)–737
protein (588–854) (681) (719) (811) (823) (745) (854) (737)
2.4 How Many Chaperones in the Human Species?

(precursor)
kDa mature 66.7–98.1 66.7 (66.7)–78.0 74.8 (78.1)–77.9 84.2 (89.3)–89.0 79.5 (81.2)–91.5 78.3 (78.3)–86.2 84.1 (84.1)–98.1 83.3 (83.3)–84.8
protein (66.7–98.1) (78.0) (81.5) (91.5) (94.2) (86.2) (98.1) (84.8)
(precursor)
Homo Ref. n.a Ref. n.a. Ref. n.a. Ref. Ref.
sapiens
a
Abbreviations mito., mitochondria; chlor., chloroplast; ER, endoplasmic reticulum; Ref., see reference below; n.a., not applicable. Within brackets, new nomenclature for the
human molecules
Source Chen B, Piel WH, Gui L, Bruford E,Monteiro A (2005) The HSP90 family of genes in the human genome: Insights into their divergence and evolution. Genomics
86:627–637; Chen B, Zhong D, Monteiro A (2006) Comparative genomics and evolution of the HSP90 family of genes across all kingdoms of organisms. BMC Genomics 7:156
doi:10.1186/1471-2164-7-156; and Kampinga HH, Hageman J, Vos MJ, Kubota H, Tanguay RM, Bruford EA, Cheetham ME, Chen B, Hightower LE (2009) Guidelines for the
nomenclature of the human heat shock proteins. Cell Stress Chaperones 14: 105–111; doi:10.1007/s12192-008-0068-7
27
28 2 Chaperones: General Characteristics and Classifications

Kampinga HH (2012) DNAJ proteins and protein aggregation diseases. Curr Top
Med Chem 12:2479–2490 (see also Sect. 4.3).

2.4.4 The Hsp90 Chaperone Genes and Proteins

The extended family of Hsp90 genes in the three life Domains comprises at least 17
genes, of which five are present in humans. These are: Hsp90AA1 (the cytosolic
Hsp89, or Hsp90, or HSPC1), Hsp90 alpha (or Hsp90AA2, or HSPC2), Hsp90 beta
(or HSP90B1, or HSPC3), TRAP (the mitochondrial Hsp90L, or Hsp75, or HSPC5),
and Hsp90B1 (the ER gp96, or grp94, or endoplasmin, or HSPC4). In addition, the
human genome harbors several related pseudogenes. The Hsp90 genes identified in
eukaryotes along with HTPG, which is present in bacteria, are listed in the Table 2.6.
These genes form four evolutionarily related groups (not shown).

2.5 The Chaperoning System

As a working hypothesis it may be assumed that all Hsp-chaperones in an

organism form a physiologic system, akin to the immune system. Thus, the
chaperoning system is composed of all Hsp-chaperones, co-chaperones and
chaperone co-factors of an organism. This is illustrated by the distribution and
migrations of certain chaperones, e.g., Hsp60, Fig. 2.5.
The chaperone molecules can be classified into subpopulations according to their
origin with regard to the cell in which they function, precise location inside or
outside the cell, and to their mobility, and also considering whether or not they are
associated to other molecules or molecular assemblies. For example, according to its
origin a chaperone can be autochthonous if residing in the cell in which it originated
or imported if it comes from another cell; sessile if attached to a structure or mobile if
free-moving in the cytosol, extracellular environment or body fluids (blood, lymph,
cerebrospinal fluid), as illustrated in Fig. 2.5. In addition, chaperones can be
single or member of a chaperoning team with other chaperones, co-chaperones, and
co-factors, also named chaperoning machine (See Fig. 2.1). A chaperoning team or
machine can be a member of a chaperoning network, which is formed by various
chaperoning teams and, possibly, other molecules or molecular assemblies (See
Fig. 2.2). A chaperone can also form a complex with another molecule or structure
(e.g., tumor antigen, cell-surface receptor, cytosolic glucocorticoid-hormone
receptor, chemical compound, members of the caspase cascade), but in this case the
complex is not a chaperoning machine; it has other functions unrelated to its
canonical role in the maintenance of protein homeostasis. Examples of these non-
chaperoning complexes are: (1) Hsp70 forms complexes with tumor antigens
(peptides) and cell-surface receptors; it binds mRNAs containing AU-rich elements
and acts as posttranscriptional regulator of gene expression; HspA2 associates with a
2.5 The Chaperoning System 29

Fig. 2.5 The chaperoning system. Circled C, molecular chaperone; 1, mobile chaperone in the
cytosol; 2, chaperone inside an organelle, such as the nucleus or a mitochondrion; 3, sessile
chaperone anchored to a particle (e.g., ribosome) in the cytosol; 4 and 5, sessile chaperone
anchored to the cell membrane on the cytosolic side (4) or on the outside in the extracellular
space (5)–chaperones can also be located, at least transitorily, in the plasma membrane (i.e.,
transmembrane location); 6, mobile chaperone in the intercellular space; 7, mobile chaperone in
circulation inside a vessel (blood or lymph) in suspension or, 7a, on the surface of circulating
erythrocytes, lymphocytes, granulocytes, or platelets; 8, sessile chaperone anchored to the vessel
wall on the inside; 8a, chaperone inside a biological space, such as the intrasynovial space in the
cavity of many joints, and the space between the pia and the arachnoid maters in the central
nervous system (cerebrum ventricles, cisterns, and sulci, and spinal cord central canal); 9, mobile
chaperone in the cytosol like that shown in 1, but imported from another cell. Molecular
chaperones can be found also in other locations such as cerebrospinal fluid (8a) and secretions
(e.g., saliva and urine), the latter two not shown in this figure (see Table 2.2); 10, mobile or
sessile chaperone that originated in the blood or on a nearby cell (same as 6 if mobile) and is now
in the intercellular space. Arrows indicate the various directions of movement of a chaperone
molecule from inside a cell to the extracellular space or vessel lumen and vice versa. A chaperone
can gain the extracellular space from inside a cell or from inside a vessel and it can go into a
vessel directly from a cell or from the extracellular space. Source Macario AJL, Conway de
Macario E (2009) The chaperoning system: Physiology and pathology. Exp Med Rev Vol. 2–3:
Years 2008/09, pp. 9–21 (http://www.unipa.it/giovanni.zummo); and Macario AJL, Cappello F,
Zummo G, Conway de Macario E (2010) Chaperonopathies of senescence and the scrambling of
the interactions between the chaperoning and the immune systems. Ann New York Acad Sci
1197: 85–93

complex including arylsulfatase A (ARSA) and sperm adhesion molecule 1

(SPAM1) in the process of spermatozoa capacitation; (2) Hsp90 binds glucocorti-
coid-hormone receptor (a protein that is a transcription factor); (3) Hsp90 binds
some anti-tumor compounds like the antibiotic geldanamycin; and (4) Hsp60 forms
a complex with procaspase-3.
30 2 Chaperones: General Characteristics and Classifications

The eukaryotic cell, for example a cell in the human body, has organelles, such
as mitochondria and the endoplasmic reticulum (ER). These organelles have their
own subsets of chaperones, i.e., chaperoning subsystems, with defined functions in
health and roles in pathology. For instance, the mitochondrial chaperoning sub-
system is implicated in the pathogenesis of various conditions such as ageing, and
some types of cancer and neurological disorders (e.g., mitochondrial encepha-
lopathy). In these conditions, mitochondrial functions are abnormal. Likewise, the
ER chaperoning subsystem plays a pathogenic role in some malignant tumors, and
genetic (i.e., the Marinesco-Sjogren syndrome) and inflammatory disorders (e.g.,
synovial inflammation in some forms of arthritis), just to mention a few examples.
See Table 4.9, part 1; Table 7.2; and Table 7.4.
The concept of chaperoning system offers a unified view of a set of molecules
that interact directly or indirectly with one another to achieve a particular objec-
tive, i.e., to carry out a physiological process usually essential for life and in the
response to stress. It may be assumed that the chaperoning system, or very
primitive forms of it, appeared very early in evolution and played a defensive role
against stressors. With time, the system became multifacetic and it assumed also a
regulatory role as the living forms became more complex, multicellular and
multiorgan, therefore needing internal coordination of parts. This role could nat-
urally be played by a population of circulating molecules such as the Hsp-
chaperones able to interact widely, including with the immune system. The latter
probably appeared later in evolution to assume also defensive roles against a
widening range of stressors, including microbes and eukaryotic parasites. The
result is that today, the chaperoning and the immune systems interact widely.
From the practical standpoint the concept of chaperoning system is a working
hypothesis that provides a scaffolding to add pertinent data as they become
available and a blueprint for future research to obtain new data. It also serves as a
guide for diagnosis and differential diagnosis, and for assessing the impact
throughout the body that any given chaperonopathy might have. All of these are
essential requisites for adequate treatment and prevention.

Further Reading

See also Chap. 1 for Further Reading

Sections 2.1–2.4

Clusterin
Carver JA, Rekas A, Thorn DC, Wilson MR (2003) Small heat-shock proteins and clusterings:
intra- and extracellular molecular chaperones with a common mechanism of action and
function? IUBMB Life 55:661–668
Wyatt AR, Yerbury JJ, Berghofer P, Greguric I, Katsifis A, Dobson CM, Wilson MR (2011)
Clusterin facilitates in vivo clearance of extracellular misfolded proteins. Cell Mol Life Sci
68:3919–3931. doi:10.1007/s00018-011-0684-8
Further Reading 31

Alpha Synuclein and Myocilin

Anderssohn AM, Cox K, O’Malley K, Dees S, Hosseini M, Boren L, Wagner A, Bradley JM,
Kelley MJ, Acott TS (2011) Molecular chaperone function for myocilin. Invest Ophthalmol
Vis Sci 52:7548–7555
Rekas A, Ahn KJ, Kim J, Carver JA (2012) The chaperone activity of a-synuclein: Utilizing
deletion mutants to map its interaction with target proteins. Proteins 80:1316–1325

AIPL1 Part of Chaperone Complex

AIPL1 (aryl hydrocarbon-interacting receptor protein-like 1)
Hidalgo-de-Quintana J, Evans RJ, Cheetham ME, van der Spuy J (2008) The Leber congenital
amaurosis protein AIPL1 functions as part of a chaperone heterocomplex. Invest Ophthalmol
Vis Sci 49:2878–2887

AIP
AIP (aryl-hydrocarbon receptor-interacting protein; or aryl hydrocarbon receptor-associated
protein 9, ARA9)
Cain JW, Miljic D, Popovic V, Korbonits M (2010) Role of the aryl hydrocarbon receptor-
interacting protein in familial isolated pituitary adenoma. Experts Rev Endocrinol Metabolism
5:681–695

Torsin
Burdette AJ, Churchill PF, Caldwell GA, Caldwell KA (2010) The early-onset torsion dystonia-
associated protein, torsinA, displays molecular chaperone activity in vitro. Cell Stress
Chaperones 15:605–617
Chen P, Burdette AJ, Porter JC, Ricketts JC, Fox SA, Nery FC, Hewett JW, Berkowitz LA,
Breakefield XO, Caldwell KA, Caldwell GA (2010) The early-onset torsion dystonia-
associated protein, torsinA, is a homeostatic regulator of endoplasmic reticulum stress
response. Hum Mol Genet 19:3502–3515
Ozelius LJ, Page CE, Klein C, Hewett JW, Mineta M, Leung J, Shalish C, Bressman SB, de Leon
D, Brin MF, Fahn S, Corey DP, Breakefield XO (1999) The TOR1A (DYT1) gene family and
its role in early onset torsion dystonia. Genomics 62:377–384

AHSP (Alpha-Hemoglobin-Stabilizing Protein)

Bank A (2007) AHSP: a novel hemoglobin helper. J Clin Invest 117:1746–1749
Favero ME (2011) Costa FF (2011) alpha-hemoglobin-stabilizing protein: an erythroid molecular
chaperone. Biochem Res Int 2011:373859

Hsp10
Corrao S, Campanella C, Anzalone R, Farina F, Zummo G, Conway de Macario E, Macario AJL,
Cappello F, La Rocca G (2010) Human Hsp10 and early pregnancy factor (EPF) and their
relationship and involvement in cancer and immunity: current knowledge and perspectives.
Life Sci 86:145–152
Czarnecka AM, Campanella C, Zummo G, Cappello F (2006) Heat shock protein 10 and signal
transduction: a ‘‘capsula eburnea’’ of carcinogenesis? Cell Stress Chaperones 11:287–294
32 2 Chaperones: General Characteristics and Classifications

Chaperoning Networks: Balance Between Protein Folding and Degradation

Muller P, Ruckova E, Halada P, Coates PJ, Hrstka R, Lane DP, Vojtesek B (2012) C-terminal
phosphorylation of Hsp70 and Hsp90 regulates alternate binding to co-chaperones CHIP and
HOP to determine cellular protein folding/degradation balances. Oncogene. doi:10.1038/
onc.2012.314

CHAPERONES: REALM
CCT, The Cytosolic Chaperonin is also Present in the Nucleus and Perform
Functions Other than the Canonical Protein Folding
Pejanovic N, Hochrainer K, Liu T, Aerne BL, Soares MP, Anrather J (2012) Regulation of
nuclear factor jB (NF–jB) transcriptional activity via p65 acetylation by the chaperonin
containing TCP1 (CCT). PLoS ONE 7(7):e42020
Huang R, Yu M, Li CY, Zhan YQ, Xu WX, Xu F, Ge CH, Li W, Yang XM (2012) New insights
into the functions and localization of nuclear CCT protein complex in K562 leukemia cells.
Proteomics Clin Appl 6:467–475

Chaperones on the Surface of Tumor Cells

Stangl S, Gehrmann M, Riegger J, Kuhs K, Riederer I, Sievert W, Hube K, Mocikat R, Dressel R,
Kremmer E, Pockley AG, Friedrich L, Vigh L, Skerra A, Multhoff G (2011) Targeting
membrane heat-shock protein 70 (Hsp70) on tumors by cmHsp70.1 antibody. Proc Natl Acad
Sci USA 108:733–738
Shipp C, Derhovanessian E, Pawelec G (2012) Effect of culture at low oxygen tension on the
expression of heat shock proteins in a panel of melanoma cell lines. PLoS ONE 7(6):e37475

Saliva and Serum Hsp60

Yuan J, Dunn P, Martinus RD (2011) Detection of Hsp60 in saliva and serum from type 2
diabetic and non-diabetic control subjects. Cell Stress Chaperones 16:689–693

ER chaperones are Also Outside the Organelle

Gold LI, Eggleton P, Sweetwyne MT, Van Duyn LB, Greives MR, Naylor SM, Michalak M,
Murphy-Ullrich JE (2010) Calreticulin: non-endoplasmic reticulum functions in physiology
and disease. FASEB J 24:665–683

How Many Chaperones in the Human Species?

Brocchieri L, Conway de Macario E, Macario AJL (2007) Chaperonomics, a new tool to study
ageing and associated diseases. Mechan Ageing Develop 128:125–136

Section 2.5

The Chaperonin System and Non-chaperoning Complexes Formed by

Chaperones (see also Further Reading in Sects. 7.3–7.4)
Csermely P, Korcsmáros T, Kovács IA, Szalay MS, Soti C (2008) Systems biology of molecular
chaperone networks. Novartis Found Symp 291:45–54; discussion 54–8, 137–40
Further Reading 33

Echeverría PC, Bernthaler A, Dupuis P, Mayer B, Picard D (2011) An interaction network

predicted from public data as a discovery tool: application to the Hsp90 molecular chaperone
machine. PLoS ONE. 2011; 6(10): e2604410.1371/journal.pone.0026044
Kishor A, Tandukar B, Ly YV, Toth EA, Suarez Y, Brewer G, Wilson GM (2013) Hsp70 is a
novel posttranscriptional regulator of gene expression that binds and stabilizes selected
mRNAs containing AU-rich elements. Mol Cell Biol 33:71–84. doi:10.1128/MCB.01275-12
Redgrove KA, Anderson AL, McLaughlin EA, O’Bryan MK, Aitken RJ, Nixon B (2013)
Investigation of the mechanisms by which the molecular chaperone HSPA2 regulates the
expression of sperm surface receptors involved in human sperm-oocyte recognition. Mol Hum
Reprod 19:120–135. doi:10.1093/molehr/gas064