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Small Heat Shock Proteins: Countering Protein Aggregates

The small heat shock proteins (sHsp) are a very diverse group of chaperones that play an important role in maintaining the protein quality control in the cell. Their diversity extends to molecular weights, which vary from 16 kD in C. Elegans to 40 kD in a protozoan S. Mansoni. They have similar hydropathy profiles and small region’s amino acid identity (1, 11). Recent report support the presence of an I-X-I, I/V-X-I/V or an I/V/L-X-I/V/L motif in the C-terminal part of sHsp sequences (7).

Most of the sHsps share some common features such as; (i) presence of a conserved α-crystallin domain of ~90 residues, (ii) a small molecular mass of 12–43 kDa, (iii) formation of large oligomers, (iv) a dynamic quaternary structure and (v) induction by stress conditions and chaperone activity in suppressing protein aggregation (2).

Structure and Organization

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Domain organisation of sHsps

One of the most characteristic features of sHsps is their ability to organize as large oligomeric structures. The structures of the members of five sHsp family have been determined by X-ray crystallography or electron microscopy. The outside diameters range from 100 Å to 180 Å. Hsp16.5 from Methanocaldococcus jannaschii, Hsp16.3 (Acr1) from Mycobacterium tuberculosis and Hsp26 from Saccharomyces cerevisiae form hollow, ball-like structures. However, the number of subunits in the oligomers varies. For example, M. Jannaschii Hsp16.5 and yeast Hsp26 complexes have 24 subunits, whereas M. Tuberculosis Hsp16.3 has 12. In contrast, Hsp16.9 from wheat is a barrel-shaped structure assembled from two hexameric double disks with a total of 12 subunits (7). Distinct from these two defined oligomeric structures, the quaternary structure of mammalian α-crystallin is variable. It forms polydisperse oligomeric assemblies with up to 50 subunits per complex (1). Despite their sequence diversity and differences in oligomeric assemblies, the sHsp proteins have a conserved structural organization. They consist of an N-terminal region followed by the conserved α-crystallin domain and a C-terminal region (1, 5). Alpha crystallin domain forms the signature motif of sHsps. The amino acid sequence of the domain is variable, barring a few positions (8). However, the compact β-sheet sandwich structures similar to the immunoglobulin-like folds, is conserved throughout the sHsp family. The sheet is composed of two layers of three and five antiparallel strands respectively, connected by a short inter-domain loop. Alpha crystallin domains can dimerize through the formation of an inter-subunit composite β-sheet (5). This feature of dimerization is conserved throughout sHsps. The C-terminal region after α-crystallin domain is involved in stabilizing the oligomer. The orientation of the C-terminal region is flexible (8). Recently, it has been shown that contacts between a conserved Ile-Xxx-Ile/Val23 motif in the C-terminal region and a hydrophobic patch in α-crystallin domains of a neighboring subunit are critical for oligomer formation (1, 5, 8). The N-terminal region is highly variable in both sequence and length. It varies from 24 residues in C. Elegans Ηsp12.2 to 247 residues in S. Cerevisiae, Hsp42. The N-terminal regions due to their increased flexibility are partially resolved in the available crystal structure (7). In wheat Hsp16.9, the N-terminal regions are also important for stabilizing the oligomer. The N-terminal arms of the subunits from the two disks intertwine to form pairs of knot-like structure, and the hydrophobic contacts in these knots are buried inside the oligomer. Hence, from structural analyses, it is evident that residues of all three regions of sHsps are required for oligomerization (1). Although α-crystallin domain is necessary for dimer formation and thus assembles the basic building block, both flanking regions promote the formation of higher-order structures (8).

Mode of Action

sHsps bind denatured proteins and prevent their irreversible aggregation (6). Bovine α-crystallin and murine Hsp25 was the first sHsps reported to have chaperone activity (3). In contrast to other classes of molecular chaperones, several non-native polypeptide chains are bound by each oligomeric sHsp complex. A ratio of up to one non-native model-substrate protein per dimeric subunit has been reported. In yeast, it has been shown that approximately one-third of the cytosolic proteins are maintained in soluble state by sHsps under heat-shock conditions and is protected from precipitation. The complexes between substrates and sHsps are very stable at physiological temperatures (1). Upon presence of excess non-native proteins, larger assemblies of substrate proteins and sHsps have been observed. These are reminiscent of the ‘heat-shock granules’ which are large, ordered structures found upon heat shock in plants (6). Several hydrophobic sites have been postulated to be involved in the chaperone function and complex formation of sHsps, including the N-terminal and C-terminal regions. It is observed that the hydrophobic N-terminal regions are integrated into the oligomeric assembly and contribute to substrate binding and chaperone activity (9). However, until a date, it has not been possible to distinguish clearly the parts of sHsps that are necessary for chaperone function, and those involved in oligomer interactions or to determine whether there is substantial crosstalk between these two functions (5, 9). The unfolded proteins and sHsps stably interact with each other forming substrate–chaperone complexes. The non-native proteins are neither transferred between different sHsp complexes nor spontaneously released (6). Here, Hsp70 is required for the refolding of protein from insoluble aggregates consisting of sHsps and model substrate proteins. Hsp70 allows reactivation of the sHsp-bound proteins in the presence of ATP and sometimes also involve members of the Hsp100 family, including ClpB and Hsp104 in E. Coli. In E. Coli,it is shown that bound non-native proteins were specifically transferred to the DnaK-DnaJ-GrpE chaperone system and reactivated (2,3).

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sHsp oligomers complexes with denatured protein to prevent aggregation

A conserved trait of molecular chaperones is the existence of states of low and high affinity for non-native proteins (2). In the case of ATP-dependent chaperones, the shift between the two functional states is governed by ATP binding and hydrolysis. Although there have been reports of ATP binding by α-crystallin, it is perceived that ATP does not have a direct role in the regulation of sHsp chaperone activity (7). For sHsps, shifting between active and inactive states involves a different mechanism. Notably, many sHsps are not constitutively functioning; they are specifically activated upon the introduction of stress conditions such as elevated temperatures. sHsp complexes are dynamic structures and exchange subunits constantly to form hetero-oligomeric assemblies with other sHsp species present in the same compartment (5). It is possible that these hetero-oligomeric complexes possess binding-specificities different from the homo-oligomeric complexes. This dynamic behavior of sHsps could allow the substrate-binding sites that are buried in the oligomeric complex, to become exposed (9). The spontaneous dissociation-reassociation process might be a ‘sensing’ mechanism to monitor the presence of non-native proteins in the cellular environment. However, recent data challenge the concept and suggest that dissociation and subunit exchange do not correlate with chaperone activity, and the activation process involves rearrangements within the sHsp oligomeric structure (1,9).


Role in Cell Physiology

One of the most classical examples of sHsp is the α-crystallin which forms the major protein component of a mammalian eye lens. It exerts a protective role in maintenance of lens structure and prevents protein crystallins to aggregate (1, 2). It is present therein as a polydisperse hetero-oligomer of αA- and αB-crystallins, each of a molecular weight of about 20 kDa. Besides the lens, the αB subunit is expressed in many other tissues, including the brain, lungs, spleen, cardiac and skeletal muscles, where it functions as a chaperone by interacting with several partially folded target proteins (9). Alpha crystallin was found to prevent the formation of amyloid fibrils by various proteins (e.g. amyloid β-peptide, apolipoprotein C-II, α-synuclein, κ-casein). It has been shown that α-crystallin inhibits a formation of amyloid fibrils by Aβ-(1–40) and apolipoprotein C-II due to interaction of the chaperone with the fibril nucleus, and does not inhibit the relatively rapid fibril elongation upon nucleation. αB-Crystallin also prevents the fibril growth of β2-microglobulin under acidic conditions (4).


Interaction with Cytoskeleton

sHSPs also associate with microfilaments, intermediate filaments and microtubules, the cytoskeletal elements of eukaryotic cells and increase their stability (5). Up-regulation of αB-crystallin stabilizes glial fibrillary acidic protein (GFAP) filaments, while Hsp27 and αB-crystallin interact with intermediate filaments, preventing network formation and regulating spatial organization. αB-crystallin and desmin, a type III intermediate filament, couples, specifically within cardiac muscle cells (1). Hence, sHSPs are involved in maintenance and remodeling of the cytoskeleton.

Interaction with Membranes

Nonameric Hsp16.3, a major membrane protein of Mycobacterium tuberculosis, generates dimers upon combining with positively charged lipid layers, and is thought to protect the bacterium from reactive oxygen species of the macrophage defence system (7). A portion of the lens α-crystallin resides in the plasma membrane suggesting that maintenance of membranes is an important sHSP action under physiological conditions (5). Hsp17 modulates the physical state of thylakoid membranes, a notion supported by increased membrane fluidity in mutants lacking the chaperone. Further, Hsp17 and α-crystallin in concert with molecular chaperones such as GroEL, might regulate membrane fluidity by stabilizing the liquid crystalline state and lowering heat induced hyper fluidity (3).

Role of sHsps in nucleus

Tomato Hsp16.1-CIII contains a nuclear localization signal between β-strands 5 and 6 of α-crystallin domain and resides mainly in nuclei, though it localizes to the cytoplasm by joining with other sHSPs. Hsp27 migrates into transfected A549 cell nuclei during stress (9). Rat myocardial cell Hsp20 occupies nuclei upon heat shock. In normal non-stressed tissues, human testis specific HspB9 occurs predominately in nuclei. However, the consequences of nuclear localization of these sHsps are unknown. Hsp27, Hsp25 and αB-crystallin bind to nucleoli and speckles (intranuclear structures populated by splicing factors), and possibly regulate the molecular processes or protecting proteins (6). Interesting functional relationships between sHSPs and nucleic acids are observed in studies where a mouse α-crystallin binds αD/E/F-crystallin genes and bovine lens α-crystallin recognizes single and double-stranded DNA. sHSPs may also protect messenger RNAs (mRNAs) during stress-induced translational arrest, through an unclear mechanism (5). Therefore, sHSPs interact with many essential molecules comprising different cell compartments and processes. They are fundamentally important to all organisms either as a first line of defense during stress or in pursuit of normal affairs.

sHsps in Protein Conformation Disorders and Cancer

Upregulation and accumulation αB-crystallin and Hsp27 into inclusion bodies are often observed upregulated in many protein conformation diseases. Alpha beta-crystallin and Hsp27 accumulate in Rosenthal fibers of Alexander disease, cortical lewy bodies, Alzheimer disease plaques, neurofibrillary tangles as well as in synuclein, deposit associated to Parkinson disease or myopathy-associated inclusion body (9). Other studies have reported that aB-crystallin is present in reactive glia in Creutzfeldt-Jakob disease, and a high prevalence of anti-alpha-crystallin antibodies has been described in multiple sclerosis, which correlates with severity and activity of the disease (4). Mutations in the small heat shock protein HSP22 (HSPB8) is associated with the inherited peripheral motor neuron disorders distal hereditary motor neuropathy type II and axonal Charcot-Marie-Tooth disease type 2L. These αB-crystallinopathies are a special case of protein conformation disease, which resulted from the misfolding and progressive aggregation of mutated αB-crystallin to which subsequently associate desmin filaments to form αB-crystallin/desmin/amyloid positive aggresomes (1). These aggregates can by themselves be toxic, inhibiting the ubiquitin–proteasomal system of protein degradation and causing deficits in mitochondrial function (4). Recently, two mutations in αB-crystallin gene have been identified that are responsive for dominant cataract and for cardiomyopathy. At the biochemical level, mutations in αB-crystallin have been found to modify the properties of αB-crystallin such as its oligomerization and in vitro chaperone-like activity and to increase its affinity to desmin (5,9).

Hsp27 is found to be overexpressed in a variety of cancers and was suggested to play a major role in tumor development by blocking apoptosis (1). In addition, Hsp27 can also suppress the senescence program (10). Depletion of Hsp27 in highly transformed cells caused activation of the p53 pathway, induction of p21 and expression of typical signs of cell senescence (1). On the other hand, overexpression of Hsp27 in immortalized mammary epithelium cells caused suppression of the senescence program activated by genotoxic drugs and oxidants (5). Further, appears to play a major role in cell migration and metastases through its interaction with the actin cytoskeleton and potentiating expression of metalloproteases MMP2 and MMP9 that are essential for metastases (10). Additionally, overexpression of a close homolog of Hsp27, αB-crystallin has been observed to transform immortalized human mammary epithelial cells (9). It induces EGF- and anchorage-independent growth, increases cell migration and invasion, and constitutively activates the MAPK kinase/ERK (MEK/ERK) pathway (1,5). Hence; further analysis on the modification and mode of action of sHsps will provide considerable insights on the maintenance of oncogenetic pathways during tumour progression.

References

  1. Haslbeck, M., Franzmann, T., Weinfurtner, D. and Buchner, J. (2005) Some like it hot: the structure and function of small heat-shock proteins. Nat Struct Mol Biol, 12, 842-846.
  2. Lund, P.A. (2001) Molecular chaperones in the cell. Oxford University Press, Oxford.
  3. Bukau, B., Weissman, J. and Horwich, A. (2006) Molecular chaperones and protein quality control. Cell, 125, 443-451.
  4. Clark, J.I. and Muchowski, P.J. (2000) Small heat-shock proteins and their potential role in human disease. Curr Opin Struct Biol, 10, 52-59.
  5. Sun, Y. and MacRae, T.H. (2005) Small heat shock proteins: molecular structure and chaperone function. Cell Mol Life Sci, 62, 2460-2476.
  6. U Jakob, M Gaestel, K Engel and J Buchner (1993) Small heat shock proteins are molecular chaperones. J Biol Chem, 268, 1517-1520.
  7. Pierre Poulain, Jean-Christophe Gelly, Delphine Flatters (2010) Detection and Architecture of Small Heat Shock Protein Monomers PLoS ONE, 5(4), e9990.
  8. Kyeong Kyu Kim, Rosalind Kim and Sung-Hou Kim (1998) Crystal structure of a small heat-shock protein Nature, 394, 595-599.
  9. Basha E, O'Neill H, Vierling E. (2012) Small heat shock proteins and α-crystallins: dynamic proteins with flexible functions. Trends Biochem Sci., 37(3), 106-117.
  10. Steffi Oesterreich, Chye-Ning Weng, Ming Qiu, Susan G. Hilsenbeck, C. Kent Osborne, and Suzanne A. W. Fuqua (1993) The Small Heat Shock Protein hsp27 Is Correlated with Growth and Drug Resistance in Human Breast Cancer Cell Lines Cancer Res, 53, 4443-4448.
  11. Lindquist S., Craig E. A., (1988) THE HEAT-SHOCK PROTEINS. Annu. Rev. Genet., 22, 631-677

Database Statistics

Total Number of Genomes
277
Numbers in Bacteria
257
Numbers in Archaea
80
Numbers in Protists
35
Numbers in Fungi
64
Numbers in Algae
18
Numbers in Plants
492
Numbers in Animals
382
Total Number of sHsp Proteins
1328