Laurence H Pearl

..The central role of the Hsp90-Cdc37 chaperone complex makes it an important target for future anti-cancer drug development...

..We also identify the gaps in our current understanding and highlight important topics for future research...

..Additionally, we describe the roles of the plethora of cochaperones with which Hsp90 cooperates and growing insights into their biochemical mechanisms, which come from crystal structures of Hsp90 cochaperone complexes...

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..These data reveal a cyclic regulatory mechanism for Cdc37, in which its constitutive phosphorylation is reversed by targeted dephosphorylation in Hsp90 complexes...

..Quantitative analysis suggests that the interaction of GSK3beta with the axin scaffold enhances phosphorylation of beta-catenin by >20 000-fold...

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..This interaction fixes the lid segment in an open conformation, inserts an arginine side chain into the ATP binding pocket to disable catalysis, and prevents trans-activating interaction of the N domains...

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..Contrary to expectations, the closed Hsp90 would not enclose its client proteins but provides a bipartite binding surface whose formation and disruption are coupled to the chaperone ATPase cycle...

..Biochemical data suggest that this dimer is the biologically active state promoted by ATM-phosphorylation, and also suggests a mechanism for dimerisation-driven activation of Chk2 by trans-phosphorylation...

..However, disrupting phosphopeptide binding slows formation of ssDNA-binding protein (Rpa1/Rad11) foci and reduces levels of Rad22(Rad52) recombination foci, indicating a DNA repair defect...

..Comparison with the crystal structure of Hsp90 allows us to identify the locations of Cdc37 and Cdk4 in the complex and suggests a mechanism by which conformational changes in the kinase are coupled to the Hsp90 ATPase cycle...

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..Biochemical analysis reveals a single repair enzyme-binding site on 9-1-1 that can be blocked competitively by the PCNA-binding cell-cycle regulator p21(cip1/waf1)...

..Consequently, our findings imply that CHK2 inhibitors may exert therapeutic activity in combination with PARP inhibitors...

..The structure shows the molecular determinants of this important protein-protein interaction and explains the effects of both cancer-associated truncating mutants in PALB2 and missense mutations in the amino-terminal region of BRCA2...

..The high-resolution crystal structure of a PNK-FHA-XRCC1 phosphopeptide complex reveals the basis for this unusual bis-phosphopeptide recognition, which is probably a common feature of the known XRCC1-associating end-processing enzymes...

..Some initial structure-activity relationships are discussed, as well as the crystal structure of CCT018159 bound to Hsp90...

..Additionally, this analysis suggests a coherent conformation for the Rb holoprotein in which RbN and pocket domains directly interact, and which can be modulated through ligand binding and possibly Rb phosphorylation...

..Together with the absence of specificity subsites in the structure, this suggests an electrostatic sensing mechanism for communicating the modification status of vicinal residues as part of the syntax of the 'histone code.'..

..This structure indicates a more invasive interaction with dsDNA than observed with other UDGs and reveals an elegant water displacement/replacement mechanism that allows SMUG1 to exclude thymine from its active site while accepting HmU...

..However, attempting the discovery of stable core complexes using informed trial-and-error approaches can prove time and resource intensive...

..Together, our data indicate a substantial remodelling of DNA-PK holo-enzyme upon autophosphorylation, which is crucial to the release of protein factors from a repaired DNA double-strand break...

..In addition to showing mechanistic inhibition of CHK2 in HT29 human colon cancer cells, a concentration dependent radioprotective effect in mouse thymocytes was demonstrated for the potent inhibitor 46 (CCT241533)...

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..6 A (1 A=0.1 nm). Structure-based sequence analysis reveals several features that may be involved in mediating protein-protein interactions...

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..Expressing this protein in bacteria could facilitate studies investigating how this molecule interacts with aPL...

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..8 A resolution, and compare this to the catalytic fragment of PARP-1, with an emphasis on providing a possible framework for rational drug design in order to develop future isoform-specific inhibitors...

..We show that the site in the second but not third BRCT domain in the N-terminus of TopBP1, provides specific interaction with a phosphorylated motif at pSer387 in Rad9, which can be generated by CK2...

..Studies of PLCepsilon regulation suggest that, in a cellular context, the RA2 domain, in a mode specific to PLCepsilon, has a role in membrane targeting with further regulatory impact on PLC activity...

..To the best of our knowledge, this is the first demonstration that it is possible for Hsp90 inhibitor resistance to arise by subtle alteration to the structure of Hsp90 itself...

..Compounds from this series showed activity in cell-based mechanistic assays for inhibition of CHK2...

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..These studies identify human Ire1α as a target for development of ATP-competitive inhibitors that will modulate the UPR in human cells, which has particular relevance for myeloma and other secretory malignancies...

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..We also propose a NHEJ model where protein-protein interactions alter substantially the architecture of DNA-PK dimers at DSBs, to trigger subsequent interactions or enzymatic reactions...

..The location of the evolutionarily conserved N-terminus of Cct5 on the outside of the barrel, confirmed by mutational studies, is unique to eukaryotic cytosolic chaperonins...

..The structure reveals the presence of a Hoogsteen base pair within the deaminated recognition sequence and the substantial distortions of the DNA that accompany Vsr binding to product sites...

..The structure of the middle region of Hsp90 will also accelerate our understanding of client protein interactions since this region is implicated in their recognition and in particular their active-site openings...

..Our studies reveal a surprisingly specific developmental role for a single Hsp90 gene in a regulatory pathway controlling late steps in sarcomere assembly...

..Structural studies of GSK3beta complexes are contributing to our understanding of the phosphorylation-independent mechanism that insulates the Wnt and insulin/growth factor pathways...

..The assay was robust and reproducible (2-8% CV) and used to screen a compound collection of approximately 56,000 compounds in 384-well format with Z' factors between 0.6 and 0.8...

..Analysis of missense mutations in the C. elegans Groucho homolog UNC-37 identifies sites of interaction with repression effectors, and suggests an induced fit binding site for eh1 domains of Engrailed-type transcription factors...

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..This difference in binding ability may help to distinguish those dsDNA antibodies that are more pathogenic...

..The site and nature of substrate interaction are thus profoundly different between CCT and its eubacterial homologue GroEL, consistent with their different functions in general versus specific protein folding assistance...

..Similar to the Nth/MutY enzymes, the Family 4 UDG centers probably play a structural rather than a catalytic role...

..Together our work provides novel insight into the molecular mechanisms governing cyclin-CDK function and regulation and defines the biological forces that may have driven evolution of viral cyclins...

..Surprisingly, we find no genetic interaction between a non-Axin-binding GSK-3 mutant and T-cell factor activity, arguing that Axin interactions alone cannot explain the regulation of T-cell factor-mediated gene expression...

..The identification of these Hsp90 cochaperone activators adds to the complex roles of cochaperones in regulating the ATPase-coupled conformational changes of the Hsp90 chaperone cycle...

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..Several studies have made use of the electron microscope to reveal the three-dimensional architecture of DNA-PK and the structural basis for the recognition of damaged DNA and the activation of DNA-PK's kinase activity...

..Two prominent hydrophobic pockets in the gap between helices alpha2 and alpha4 suggest a potential ligand-binding characteristic for this globular domain...

..We suggest that the observed domain movements might help the binding and maintenance of DNA-PKcs' interaction with DNA at the sites of damage, and that these conformational changes activate the kinase...

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..However, unlike Sti1/Hop/p60, which can displace geldanamycin upon binding to Hsp90, Cdc37p/p50(cdc37) forms a stable complex with geldanamycin-bound Hsp90 and may be sequestered in geldanamycin-inhibited Hsp90 complexes in vivo...

..These data indicated that Sba1 and Aha1 regulate Hsp90 by influencing the conformational state of the "ATP lid" and consequent N-terminal dimerization, whereas Sti1 does not...

..Comparison of DNA-PKcs with another PIKK DNA repair factor, ATM, defines a common architecture for this important protein family...

..It has been possible to identify a mechanism whereby the catalytic residue Lys 249 is "primed" for nucleophilic attack of the N-glycosidic bond...

..This could imply a role for Nse4 in maintenance of higher order chromosome structure...

..Our structural and functional analysis explains the rigid conservation of the WRPW motif, the sequence flexibility of eh1 motifs, and other aspects of repressor recognition by Gro in vivo...

..Based on the presented structural and functional data, a model for specific activation segment phosphorylation at non-consensus substrate sites is proposed that is likely to be common to other kinases from diverse subfamilies...

..Compound 40f is retained in tumors in vivo when administered i.p., as evaluated by cassette dosing in tumor-bearing mice. In a human colon cancer xenograft model, 40f inhibits tumor growth by approximately 50%...

..e., depletion of client proteins with upregulation of Hsp70...

..As a proof of principle, we applied CDH to p85alpha, successfully identifying soluble and highly expressed constructs encapsulating all the known globular domains, and immediately suitable for downstream applications...

..In the present study we also show that Tah1 can affect the ATPase activity of Hsp90, in common with some other TPR-domain proteins...

..Computer-generated models of the three-dimensional structures of the auto-antibodies and their hybrids, suggest predominant interaction of their light chains with domain IV of beta2-GPI...

..Complementation of the slt2Delta yeast defect by ERK5 expression establishes a new tool with which to screen for novel agonists and antagonists of ERK5 signaling as well as for isolating mutant forms of ERK5...

..It is shown that these mutations render at least one Hsp90-dependent process (deactivation of heat-induced heat shock factor activity) more sensitive to drug inhibition in vivo...

..A number of potentially novel cochaperone interactions were also identified, providing a framework for these to be analyzed further using other techniques...

..The interaction between Hsp90 and (Y-P,T-P)Slt2p, characterized in this study, is probably essential in this Hsp90 facilitation of the Rlm1p activation by Slt2p...

..The possible mechanisms that such antibodies may employ to recognise their antigens, are discussed...