Elastase inhibition by the C-terminal domains of α-crystallin and small heat-shock protein

α-Crystallin, an abundant eye-lens protein and a stress protein in other tissues, shows structural and functional similarities with the small heat-shock proteins. One of the properties in common is the inhibition of elastase. We now report that the separated subunits of α-crystallin, αA and αB,also exhibit elastase inhibition, whereas phosphorylation of these subunits apparently has no influence on the inhibitory capacity. Furthermore, for both αA-crystallin and mouse HSP25 the putative C-terminal structural domain, comprising the major region of homology between these proteins, is sufficient to give elastase inhibition. With database search no homology could be found between the three proteins under investigation and any of the known consensus sequences of proteinase inhibitor families.     

Comparison of the homologous carboxy-terminal domain and tail of α-crystallin and small heat shock protein

The C-terminal domain and tail, which is the most conserved region of the -crystallin/small heat shock protein (HSP) family, was obtained from rat A-crystallin, bovine B-crystallin and mouse HSP25. All three domains have primarily -sheet conformation and less than 10% of -helix, like the proteins from which they are derived. Whereas the C-terminal part of A-crystallin forms dimers or tetramers, the corresponding regions of B-crystallin and HSP25 form larger aggregates. The heat-protective activity, recently described for the -crystallin/small HSP family, is not retained in the C-terminal domain and tail. In the course of this study some differences with the previously published sequence of HSP25 were observed, and a revision is proposed.Abbreviations A2Dt residues 64–173 of rat -crystallin - B2Dt residues 70–175 of bovine B-crystallin - bp base pair - HSP2Dt residues 92–209 of HSP25 - HSP(s) heat shock protein(s) - HSP25 mouse small HSP - PCR polymerase chain reaction - PMSF phenylmethylsulfonyl chloride - SDS sodium dodecyl sulfate; polyacrylamide - WSF water-soluble fraction     

Evolution and functional diversification of the small heat shock protein/α-crystallin family in higher plants

Small heat shock proteins (sHSPs) are chaperones that play an important role in stress tolerance. They consist of an alpha-crystallin domain (ACD) flanked by N- and C-terminal regions. However, not all proteins that contain an ACD, hereafter referred to as ACD proteins, are sHSPs because certain ACD proteins are known to have different functions. Furthermore, since not all ACD proteins have been identified yet, current classifications are incomplete. A total of 17 complete plant proteomes were screened for the presence of ACD proteins by HMMER profiling and the identified ACD protein sequences were classified by maximum likelihood phylogeny. Differences among and within groups were analysed, and levels of functional constraint were determined. There are 29 different classes of ACD proteins, eight of which contain classical sHSPs and five likely chaperones. The other classes contain proteins with uncharacterised or poorly characterised functions. N- and C-terminal sequences are conserved within the phylogenetic classes. Phylogenetics suggests a single duplication of the CI sHSP ancestor that occurred prior to the speciation of mono- and dicotyledons. This was followed by a number of more recent duplications that resulted in the presence of many paralogues. The results suggest that N- and C-terminal sequences of sHSPs play a role in class-specific functionality and that non-sHSP ACD proteins have conserved but unexplored functions, which are mainly determined by subsequences other than that of the ACD.     

Structural and mechanistic implications of metal binding in the small heat-shock protein αB-crystallin

The human small heat-shock protein αB-crystallin (αB) rescues misfolded proteins from irreversible aggregation during cellular stress. Binding of Cu(II) was shown to modulate the oligomeric architecture and the chaperone activity of αB. However, the mechanistic basis of this stimulation is so far not understood. We provide here first structural insights into this Cu(II)-mediated modulation of chaperone function using NMR spectroscopy and other biophysical approaches. We show that the α-crystallin domain is the elementary Cu(II)-binding unit specifically coordinating one Cu(II) ion with picomolar binding affinity. Putative Cu(II) ligands are His(83), His(104), His(111), and Asp(109) at the dimer interface. These loop residues are conserved among different metazoans, but also for human αA-crystallin, HSP20, and HSP27. The involvement of Asp(109) has direct implications for dimer stability, because this residue forms a salt bridge with the disease-related Arg(120) of the neighboring monomer. Furthermore, we observe structural reorganization of strands β2-β3 triggered by Cu(II) binding. This N-terminal region is known to mediate both the intermolecular arrangement in αB oligomers and the binding of client proteins. In the presence of Cu(II), the size and the heterogeneity of αB multimers are increased. At the same time, Cu(II) increases the chaperone activity of αB toward the lens-specific protein β(L)-crystallin. We therefore suggest that Cu(II) binding unblocks potential client binding sites and alters quaternary dynamics of both the dimeric building block as well as the higher order assemblies of αB.     

Three-dimensional structure of α-crystallin domain dimers of human small heat shock proteins HSPB1 and HSPB6

Small heat shock proteins (sHSPs) are a family of evolutionary conserved ATP-independent chaperones. These proteins share a common architecture defined by a signature α-crystallin domain (ACD) flanked by highly variable N- and C-terminal extensions. The ACD, which has an immunoglobulin-like fold, plays an important role in sHSP assembly. This domain mediates dimer formation of individual protomers, which then may assemble into larger oligomers. In vertebrate sHSPs, the dimer interface is formed by the symmetrical antiparallel pairing of two β-strands (β7), generating an extended β-sheet on one face of the ACD dimer. Recent structural studies of isolated ACDs from a number of vertebrate sHSPs suggest a variability in the register of the β7/β7 strand interface, which may, in part, give rise to the polydispersity often associated with the full-length proteins. To further analyze the structure of ACD dimers, we have employed a combination of X-ray crystallography and solution small-angle X-ray scattering (SAXS) to study the ACD-containing fragments of human HSPB1 (HSP27) and HSPB6 (HSP20). Unexpectedly, the obtained crystal structure of the HSPB1 fragment does not reveal the typical β7/β7 dimers but, rather, hexamers formed by an asymmetric contact between the β4 and the β7 strands from adjacent ACDs. Nevertheless, in solution, both ACDs form stable dimers via the symmetric antiparallel interaction of β7 strands. Using SAXS, we show that it is possible to discriminate between different putative registers of the β7/β7 interface, with the results indicating that, under physiological conditions, there is only a single register of the strands for both proteins.     

The RAF family：an expanding network of post—traslational controls and protein—protein interactions

Protein kinase RAF is strategically located in the “Ras-MAP-kinase signal transduction pathway“,a principle system which transmits signals from growth factor receptors to the nucleus,resulting in cell proliferation.Growth factor responses are mediated in part by activation of Ras,which in turn activates RAF to phosphorylate MEK,its downstream substrate.MEK activates MAPkinase to in fluence nuclear events.it is clear.however,that a network of signals other than those carred by Ras plays a role in RAF regulation.These orthogonal influences are mediated bu:serine/threonine kinases,tyrosine kinases,and protein-protein interactions.As a further complication to the RAF network,three isoforms of RAF have been established which have divergent N-terminal regulatory domains,Whereas these divergent regulatory domains implicate isoform-specific functions,no clear evidence or hypothesis for distinct functions for individual isoforms has been presented.Recently,“isoform-specific protein interactions“have been identified among numerous proteins interacting with RAF,These studies may serve to delineate independent functions for RAF isoforms.     

The PUF family of RNA-binding proteins: does evolutionarily conserved structure equal conserved function?

Drosophila Pumilio (Pum) protein is a founder member of a novel family of RNA-binding proteins, known as the PUF family. The PUF proteins constitute an evolutionarily highly conserved family of proteins present from yeast to humans and plants, and are characterized by a highly conserved C-terminal RNA-binding domain, composed of eight tandem repeats. The conserved biochemical features and genetic function of PUF family members have emerged from studies of model organisms. PUF proteins bind to related sequence motifs in the 3' untranslated region (3'UTR) of specific target mRNAs and repress their translation. Frequently, PUF proteins function asymmetrically to create protein gradients, thus causing asymmetric cell division and regulating cell fate specification. Thus, it was recently proposed that the primordial role of PUF proteins is to sustain mitotic proliferation of stem cells. Here we review the evolution, conserved genetic and biochemical properties of PUF family of proteins, and discuss protein interactions, upstream regulators and downstream targets of PUF proteins. We also suggest that a conserved mechanism of PUF function extends to the newly described mammalian members of the PUF family (human PUM1 and PUM2, and mouse Pum1 and Pum2), that show extensive homology to Drosophila Pum, and could have an important role in cell development, fate specification and differentiation.     

The small members of the JMJD protein family: Enzymatic jewels or jinxes?

Jumonji C domain-containing (JMJD) proteins are mostly epigenetic regulators that demethylate histones. However, a hitherto neglected subfamily of JMJD proteins, evolutionarily distant and characterized by their relatively small molecular weight, exerts different functions by hydroxylating proteins and RNA. Recently, unsuspected proteolytic and tyrosine kinase activities were also ascribed to some of these small JMJD proteins, further increasing their enzymatic versatility. Here, we discuss the ten human small JMJD proteins (HIF1AN, HSPBAP1, JMJD4, JMJD5, JMJD6, JMJD7, JMJD8, RIOX1, RIOX2, TYW5) and their diverse physiological functions. In particular, we focus on the roles of these small JMJD proteins in cancer and other maladies and how they are modulated in diseased cells by an altered metabolic milieu, including hypoxia, reactive oxygen species and oncometabolites. Because small JMJD proteins are enzymes, they are amenable to inhibition by small molecules and may represent novel targets in the therapy of cancer and other diseases.     

The roles of the conserved tyrosine in the β2-α2 loop of the prion protein

ABSTRACT

Prions cause neurodegenerative diseases for which no cure exists. Despite decades of research activities the function of the prion protein (PrP) in mammalians is not known. Moreover, little is known on the molecular mechanisms of the self-assembly of the PrP from its monomeric state (cellular PrP, PrP^C) to the multimeric state. The latter state includes the toxic species (scrapie PrP, PrP^Sc) knowledge of which would facilitate the development of drugs against prion diseases. Here we analyze the role of a tyrosine residue (Y169) which is strictly conserved in mammalian PrPs. Nuclear magnetic resonance (NMR) spectroscopy studies of many mammalian PrP^C proteins have provided evidence of a conformational equilibrium between a 3₁₀-helical turn and a type I β turn conformation in the β2-α2 loop (residues 165–175). In vitro cell-free experiments of the seeded conversion of PrP^C indicate that non-aromatic residues at position 169 reduce the formation of proteinase K-resistant PrP. Recent molecular dynamics (MD) simulations of monomeric PrP and several single-point mutants show that Y169 stabilizes the 3₁₀-helical turn conformation more than single-point mutants at position 169 or residues in contact with it. In the 3₁₀-helical turn conformation the hydrophobic and aggregation-prone segment 169-YSNQNNF-175 is buried and thus not-available for self-assembly. From the combined analysis of simulation and experimental results it emerges that Y169 is an aggregation gatekeeper with a twofold role. Mutations related to 3 human prion diseases are interpreted on the basis of the gatekeeper role in the monomeric state. Another potential role of the Y169 side chain is the stabilization of the ordered aggregates, i.e., reduction of frangibility of filamentous protofibrils and fibrils, which is likely to reduce the generation of toxic species.     

The protein–protein interaction network of the human Sirtuin family

Protein–protein interaction networks are useful for studying human diseases and to look for possible health care through a holistic approach. Networks are playing an increasing and important role in the understanding of physiological processes such as homeostasis, signaling, spatial and temporal organizations, and pathological conditions. In this article we show the complex system of interactions determined by human Sirtuins (Sirt) largely involved in many metabolic processes as well as in different diseases. The Sirtuin family consists of seven homologous Sirt-s having structurally similar cores but different terminal segments, being rather variable in length and/or intrinsically disordered. Many studies have determined their cellular location as well as biological functions although molecular mechanisms through which they act are actually little known therefore, the aim of this work was to define, explore and understand the Sirtuin-related human interactome. As a first step, we have integrated the experimentally determined protein–protein interactions of the Sirtuin-family as well as their first and second neighbors to a Sirtuin-related sub-interactome. Our data showed that the second-neighbor network of Sirtuins encompasses 25% of the entire human interactome, and exhibits a scale-free degree distribution and interconnectedness among top degree nodes. Moreover, the Sirtuin sub interactome showed a modular structure around the core comprising mixed functions. Finally, we extracted from the Sirtuin sub-interactome subnets related to cancer, aging and post-translational modifications for information on key nodes and topological space of the subnets in the Sirt family network.     

The products of the “heat-shock” loci of Drosophila hydei

Antibody was raised against total Drosophila hydei embryonic cellular protein with a molecular weight between 65,000 and 70,000 dalton. This antiserum reacted with the 70,000 MW heat-shock peptide found, in ³⁵S labelled cell extracts of heat-shocked D. hydei tissue culture cells or salivary glands. — The antibody was coupled to Sepharose 4B and this material was used to absorb polysomes obtained from tissue culture cells incubated at 37° C in the presence of tritiated RNA precursors. The relative concentrations of various RNA species complementary to the heat-shock loci 2-32A, 2-36A, and 2-48C in either bound, non-bound, or total polysomal material was then determined by in situ hybridization. The RNA species complementary to locus 2-36A was found to be enriched in the bound polysomal material.     

The β1 domain of protein G can replace the chorismate mutase domain of the T-protein

T-protein is composed of chorismate mutase (AroQ(T)) fused to the N-terminus of prephenate dehydrogenase (TyrA). Here, we report the replacement of AroQ(T) with the β1-domain of protein G (Gβ1). The TyrA domain shows a strong dehydrogenase activity within the context of this fusion, and our data indicate that Gβ1-TyrA folds into a dimeric conformation. Amino acid substitutions in the Gβ1 domain of Gβ1-TyrA identified residues involved in stabilizing the TyrA dimeric conformation. Gβ1 substitutions in the N-terminal β-hairpin eliminated Gβ1-TyrA expression, whereas Gβ1-TyrA tolerated Gβ1 substitutions in the C-terminal β-hairpin and in the α-helix. All of the characterized variants folded into a dimeric conformation. The importance of the β2-strand in forming a Gβ1 homo-dimerization interface explains the relevance of the first-β-hairpin in stabilizing the dimeric TyrA protein.     

How reliably can we predict the reliability of protein structure predictions?

Background  

Comparative methods have been the standard techniques for in silico protein structure prediction. The prediction is based on a multiple alignment that contains both reference sequences with known structures and the sequence whose unknown structure is predicted. Intensive research has been made to improve the quality of multiple alignments, since misaligned parts of the multiple alignment yield misleading predictions. However, sometimes all methods fail to predict the correct alignment, because the evolutionary signal is too weak to find the homologous parts due to the large number of mutations that separate the sequences.     

A computational tool to predict the evolutionarily conserved protein–protein interaction hot-spot residues from the structure of the unbound protein

Identifying hot-spot residues – residues that are critical to protein–protein binding – can help to elucidate a protein’s function and assist in designing therapeutic molecules to target those residues. We present a novel computational tool, termed spatial-interaction-map (SIM), to predict the hot-spot residues of an evolutionarily conserved protein–protein interaction from the structure of an unbound protein alone. SIM can predict the protein hot-spot residues with an accuracy of 36–57%. Thus, the SIM tool can be used to predict the yet unknown hot-spot residues for many proteins for which the structure of the protein–protein complexes are not available, thereby providing a clue to their functions and an opportunity to design therapeutic molecules to target these proteins.     

The fight of the Tudor domain “Royal family” for a broken DNA throne

Comment on: Mallette FA, et al. EMBO J 2012; In press; PMID:22373579; 10.1038/emboj.2012.47.