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Structural studies of human small heat shock proteins and their complexes

Final Report Summary - SHSPCOMPLEX (Structural studies of human small heat shock proteins and their complexes)

The collective goal of the project is to obtain detailed molecular insight into the structure and activity of human small heat shock proteins (sHSPs). These proteins are an important, conserved component of the cellular chaperone machinery. Members of the sHSP family function, in an ATP-independent manner, as “holdases” binding to partially unfolded proteins and preventing these species from aggregating. Importantly they act as a primary buffer against protein aggregation under stress conditions where the ATP-dependent refoldases, such as HSP70 and HSP90, may be overloaded or even inhibited. Our interest in sHSPs stem from the diverse roles they play in human health. Up-regulation of activity has been associated with protection of some tumors against chemotherapeutic reagents, and high levels of these proteins are commonly found in amyloid plaques. Additionally dominant inherited mutations have been identified for a number of the human
family members that have been linked to inherited peripheral neuropathies, myobrillar myopathy and cataract.

The project involved the detailed molecular study of a specific member of the human small heat shock protein repertoire, namely HSPB6 (also known as HSP20). Earlier studies suggested that this particular sHSP differed dramatically from prototypical orthologues, such as HSPB1 (HSP27) and the two α-crystallins, in that it forms small homo-oligomers in solution rather than the large polydisperse assemblies that are typically ascribed to this family of proteins. In addition to this structurally defining property, HSPB6 has also been described to form a number of stable hetero-complexes. These complexes include hetero-assemblies with other sHSPs, possibly resulting in the formation sHSP species with mixed or new chaperoning properties, as well as complexes with the 14-3-3 adapter protein and the co-chaperone protein Bag3. The overall goal of the work was to develop a better understanding of the structure and function of these various complexes.

Essential to the success of the project was a detailed description of the structure of HSPB6 alone. Like other sHSP members this proved to be challenging. Exhaustive attempts to crystallise the full-length protein failed therefore a hybrid structural approach was employed. Initially a proteolytically stable fragment corresponding to the conserved α-crystallin domain (ACD) flanked by the full C-terminus and a portion of the N-terminus was identified. A construct corresponding to this fragment was cloned and recombinantly produced in E. coli, purified and ultimately yielded suitably diffracting crystals the structure of which was solved. To extend this atomic resolution structure to the full-length protein the isolated HSPB6, and a number of deletion mutants, were characterized by small-angle X-ray scattering (SAXS). Using a new algorithm developed specifically for this project an ensemble of biologically relevant all-atom models were generated that described the scattering properties of the protein. Importantly the models showed that in solution HSPB6 is a dimer mediated by the core ACD where the N- and C-terminal regions are largely disordered. This work, plus a separate combined crystallographic and SAXS study on the isolated ACDs of both HSPB6 and the orthologue HSPB1 have been published in peer reviewed journals. During the course of the project dimeric members of the sHSP superfamily were also reported to be present in bacteria and plants. The cross-species conservation of these smaller sHSP assemblies adds extra emphasis to the likely importance of HSPB6 in humans, the results therefore should have a broad application to understanding this important class of chaperones.

Building upon the structural characterization of HSPB6 a comprehensive study was performed to identify the sequence determinants of chaperone activity. Initial analyses showed that the disordered N-terminal region of this protein (corresponding to 70 residues) was necessary for preventing the aggregation of a number of standard substrates, therefore many deletion and single site mutants were constructed and characterized to discern which residues within this region of the protein were necessary for activity. A number of sites were identified that were important but, most intriguingly, a highly conserved consecutive stretch of residues was shown to have a role in negatively regulating HSPB6 activity. These results, which have been published in a peer reviewed journal, highlight a complex mechanism of regulation in a region of the protein that has until now proven a challenge to study by standard biophysical techniques. This mode of regulation may extend to other family members, a possibility that is currently being investigated. Importantly the results allude to the prospect of externally modulating the activity of these proteins. This may be of particular interest in cancer treatment where elevated levels of these proteins have been linked to increased resistance to chemotherapeutic treatment, suppression of activity could thus be used in combination therapy to improve drug response. On the flipside a chemically induced increase in the activity of these proteins may prove beneficial under stress conditions, such as that resulting to tissue from ischemia/reperfusion injury.

In addition to the results described above as part of the grant mandate the recipient participated in a number of external collaborations. These partnerships, formed with other laboratories within the host institute, and with other institutes at the national, European and international level have provided the researcher the opportunity to share expertise and skills that had been previously been acquired with a broad group of peers. These efforts have culminated in additional publications that fall outside the core objectives of the project and include a diverse range of topics. These include but are not limited to: (1) solution structure studies of multivalent antagonists that target lectins on the surface of pathogenic bacteria, which are considered as putative compounds for the treatment of urinary tract infection. (2) The high resolution X-ray crystallographic study of the major secreted protein ASP-1 from the nematode Ostertagia ostertagi, one of the most prevalent gastrointestinal parasites that is responsible for major losses in cattle productivity worldwide. The results of which are being employed to identify the target receptor of this protein as well as engineer sequence-optimized vaccines for yielding an elevated immune response of the host towards the parasite. (3) Structural and biochemical characterization of the metacaspase domain of MALT1, an important drug target for treatment of cancer and inflammatory bowel disease. The results for these various projects have direct and far-reaching prospects in the development of treatments for a variety of different diseases, both in humans and in economically important livestock, the incidence of which have considerable economic impact on national budgets in terms of lost output, and the expense of current medication regimes.