Tah1 helix-swap dimerization prevents mixed Hsp90 co-chaperone complexes

Specific co-chaperone adaptors facilitate the recruitment of client proteins to the Hsp90 system. Tah1 binds the C-terminal conserved MEEVD motif of Hsp90, thus linking an eclectic set of client proteins to the R2TP complex for their assembly and regulation by Hsp90. Rather than the normal complement of seven α-helices seen in other tetratricopeptide repeat (TPR) domains, Tah1 unusually consists of the first five only. Consequently, the methionine of the MEEVD peptide remains exposed to solvent when bound by Tah1. In solution Tah1 appears to be predominantly monomeric, and recent structures have failed to explain how Tah1 appears to prevent the formation of mixed TPR domain-containing complexes such as Cpr6-(Hsp90)2-Tah1. To understand this further, the crystal structure of Tah1 in complex with the MEEVD peptide of Hsp90 was determined, which shows a helix swap involving the fifth α-helix between two adjacently bound Tah1 molecules. Dimerization of Tah1 restores the normal binding environment of the bound Hsp90 methionine residue by reconstituting a TPR binding site similar to that in seven-helix-containing TPR domain proteins. Dimerization also explains how other monomeric TPR-domain proteins are excluded from forming inappropriate mixed co-chaperone complexes

Multi-chaperone function modulation and association with cytoskeletal proteins are key features of the function of AIP in the pituitary gland

Despite the well-recognized role of loss-of-function mutations of the aryl hydrocarbon receptor interacting protein gene (AIP) predisposing to pituitary adenomas, the pituitary-speci c function of this tumor suppressor remains an enigma. To determine the repertoire of interacting partners for the AIP protein in somatotroph cells, wild-type and variant AIP proteins were used for pull-down/quantitative mass spectrometry experiments against lysates of rat somatotropinoma-derived cells; relevant ndings were validated by co-immunoprecipitation and co-localization. Global gene expression was studied in AIP mutation positive and negative pituitary adenomas via RNA microarrays. Direct interaction with AIP was con rmed for three known and six novel partner proteins. Novel interactions with HSPA5 and HSPA9, together with known interactions with HSP90AA1, HSP90AB1 and HSPA8, indicate that the function/ stability of multiple chaperone client proteins could be perturbed by a de cient AIP co-chaperone function. Interactions with TUBB, TUBB2A, NME1 and SOD1 were also identi ed. The AIP variants p.R304* and p.R304Q showed impaired interactions with HSPA8, HSP90AB1, NME1 and SOD1; p.R304* also displayed reduced binding to TUBB and TUBB2A, and AIP-mutated tumors showed reduced TUBB2A expression. Our ndings suggest that cytoskeletal organization, cell motility/adhesion, as well as oxidative stress responses, are functions that are likely to be involved in the tumor suppressor activity of AIP

Structure of the TPR Domain of AIP: Lack of Client Protein Interaction with the C-Terminal alpha-7 Helix of the TPR Domain of AIP Is Sufficient for Pituitary Adenoma Predisposition

PMCID: PMC3534021This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Impact of Auto-evaluation Tests as Part of the Continuous Evaluation in Programming Courses

The continuous evaluation allows for the assessment of the progressive assimilation of concepts and the competences that must be achieved in a course. There are several ways to implement such contin uous evaluation system. We propose auto-evaluation tests as a valuable tool for the student to judge his level of knowledge. Furthermore, these tests are also used as a small part of the continuous evaluation process, encouraging students to learn the concepts seen in the course, as they have the feeling that the time dedicated to this study will have an assured reward, binge able to answer correctly the questions in the continuous evaluation exams. New technologies are a great aid to improve the auto evaluation experience both for the students and the teachers. In this research work we have compared the results obtained in courses where auto-evaluation tests were provided against courses where they were not provided, showing how the tests improve a set of quality metrics in the results of the courseMinisterio de Economía y Competitividad TIN2017-88209-C2-

Cooperation of local motions in the Hsp90 molecular chaperone ATPase mechanism

The Hsp90 chaperone is a central node of protein homeostasis activating a large number of diverse client proteins. Hsp90 functions as a molecular clamp that closes and opens in response to the binding and hydrolysis of ATP. Crystallographic studies define distinct conformational states of the mechanistic core implying structural changes that have not yet been observed in solution. Here, we engineered one-nanometer fluorescence probes based on photo-induced electron transfer into yeast Hsp90 to observe these motions. We found that the ATPase activity of the chaperone was reflected in the kinetics of specific structural rearrangements at remote positions that acted cooperatively. Nanosecond single-molecule fluorescence fluctuation analysis uncovered that critical structural elements that undergo rearrangement are mobile on a sub-millisecond time scale. We identified a two-step mechanism for lid closure over the nucleotide-binding pocket. The activating co-chaperone Aha1 mobilizes the lid of apo Hsp90, suggesting an early role in the catalytic cycle

Rapid proteasomal degradation of mutant proteins is the primary mechanism leading to tumorigenesis in patients with missense AIP mutations

CONTEXT The pathogenic effect of AIP mutations (AIPmuts) in pituitary adenomas is incompletely understood. We have identified the primary mechanism of loss of function for missense AIPmuts. OBJECTIVE To analyze the mechanism/speed of protein turnover of wild-type (WT) and missense AIP variants, correlating protein half-life with clinical parameters. DESIGN Half-life and protein-protein interaction experiments and cross-sectional analysis of AIPmut positive patients' data were performed. SETTING Clinical academic research institution. PATIENTS Data was obtained from our cohort of pituitary adenoma patients and literature-reported cases. INTERVENTIONS Protein turnover of endogenous AIP in two cell lines and fifteen AIP variants overexpressed in HEK293 cells was analyzed via cycloheximide chase and proteasome inhibition. GST pull-down and quantitative mass spectrometry identified proteins involved in AIP degradation; results were confirmed by co-immunoprecipitation and gene knockdown. Relevant clinical data was collected. MAIN OUTCOME MEASURES Half-life of WT and mutant AIP proteins and its correlation with clinical parameters. RESULTS Endogenous AIP half-life was similar in HEK293 and lymphoblastoid cells (43.5 and 32.7h). AIP variants were divided in stable proteins (median 77.7h [IQR 60.7-92.9]), and those with short (27h [21.6-28.7]) or very short (7.7h [5.6-10.5]) half-life; proteasomal inhibition rescued the rapid degradation of mutant proteins. The experimental half-life significantly correlated with age at diagnosis of acromegaly/gigantism (r=0.411, P=0.002). The FBXO3-containing SCF complex was identified as the E3 ubiquitin-ligase recognizing AIP. CONCLUSIONS AIP is a stable protein, driven to ubiquitination by the SCF complex. Enhanced proteasomal degradation is a novel pathogenic mechanism for AIPmuts, with direct implications for the phenotype

Primary cilia respond to fluid shear stress and mediate flow-induced calcium deposition in osteoblasts

Bone turnover in vivo is regulated by mechanical forces such as shear stress originating from interstitial oscillatory fluid flow (OFF), and bone cells in vitro respond to mechanical loading. However, the mechanisms by which bone cells sense mechanical forces, resulting in increased mineral deposition, are not well understood. The aim of this study was to investigate the role of the primary cilium in mechanosensing by osteoblasts. MLO-A5 murine osteoblasts were cultured in monolayer and subjected to two different OFF regimens: 5 short (2 h daily) bouts of OFF followed by morphological analysis of primary cilia; or exposure to chloral hydrate to damage or remove primary cilia and 2 short bouts (2 h on consecutive days) of OFF. Primary cilia were shorter and there were fewer cilia per cell after exposure to periods of OFF compared with static controls. Damage or removal of primary cilia inhibited OFF-induced PGE2 release into the medium and mineral deposition, assayed by Alizarin red staining. We conclude that primary cilia are important mediators of OFF-induced mineral deposition, which has relevance for the design of bone tissue engineering strategies and may inform clinical treatments of bone disorders causes by load-deficiency.—Delaine-Smith, R. M., Sittichokechaiwut, A., Reilly, G. C. Primary cilia respond to fluid shear stress and mediate flow-induced calcium deposition in osteoblasts

Dihydropyridine derivatives modulate heat shock responses and have a neuroprotective effect in a transgenic mouse model of Alzheimer’s disease

Heat shock proteins (Hsps) have chaperone activity and play a pivotal role in the homeostasis of proteins by preventing misfolding, by clearing aggregated and damaged proteins from cells and by maintaining proteins in an active state. Alzheimer’s disease (AD) is thought to be caused by β- amyloid peptide that triggers tau hyperphosphorylation, which is neurotoxic. Although proteostasis capacity declines with age and facilitates the manifestation of neurodegenerative diseases such as AD, the upregulation of chaperones improves prognosis. Our research goal is to identify potent Hsp co-inducers that enhance protein homeostasis for the treatment of AD, especially 1,4-dihydropyridine derivatives optimized for their ability to modulate cellular stress responses. Based on favorable toxicological data and Hsp co-inducing activity, LA1011 was selected for the in vivo analysis of its neuroprotective effect in the APPxPS1 mouse model of AD. Here, we report that 6 months of LA1011 administration effectively improved the spatial learning and memory functions in wild type mice and eliminated neurodegeneration in double mutant mice. Furthermore, Hsp co-inducer therapy preserves the number of neurons, increases dendritic spine density, and reduces tau pathology and amyloid plaque formation in transgenic AD mice. In conclusion, the Hsp co-inducer LA1011 is neuroprotective and therefore is a potential pharmaceutical candidate for the therapy of neurodegenerative diseases, particularly AD

Recognition of BRAF by CDC37 and re‐evaluation of the activation mechanism for the Class 2 BRAF‐L597R mutant

The kinome specific co-chaperone, CDC37 (cell division cycle 37), is responsible for delivering BRAF (B-Rapidly Accelerated Fibrosarcoma) to the Hsp90 (heat shock protein 90) complex, where it is then translocated to the RAS (protooncogene product p21) complex at the plasma membrane for RAS mediated dimerization and subsequent activation. We identify a bipartite interaction between CDC37 and BRAF and delimitate the essential structural elements of CDC37 involved in BRAF recognition. We find an extended and conserved CDC37 motif, 20HPNID---SL--W31, responsible for recognizing the C-lobe of BRAF kinase domain, while the c-terminal domain of CDC37 is responsible for the second of the bipartite interaction with BRAF. We show that dimerization of BRAF, independent of nucleotide binding, can act as a potent signal that prevents CDC37 recognition and discuss the implications of mutations in BRAF and the consequences on signaling in a clinical setting, particularly for class 2 BRAF mutations