Mutant glucocerebrosidase in Gaucher disease recruits Hsp27 to the Hsp90 chaperone complex for proteasomal degradation

Gaucher disease is caused by mutations of the GBA1 gene, which encodes the lysosomal anchored gluococerebrosidase (GCase). GBA1 mutations commonly result in protein misfolding, abnormal chaperone recognition, and premature degradation, but are less likely to affect catalytic activity. In the present study, we demonstrate that the Hsp90/HOP/Cdc37 complex recruits Hsp27 after recognition of GCase mutants with subsequent targeting of GCase mutant peptides to degradation mechanisms such as VCP and the 26S proteasome. Inhibition of Hsp27 not only increased the quantity of enzyme but also enhanced GCase activity in fibroblasts derived from patients with Gaucher disease. These findings provide insight into a possible therapeutic strategy for protein misfolding diseases by correcting chaperone binding and altering subsequent downstream patterns of protein degradation.Gaucher disease (GD) is an autosomal recessive disorder caused by mutations in the GBA1 gene encoding the lysosomal enzyme, glucocerebrosidase (GCase). Genetic alterations in GBA1 lead to quantitative losses of GCase and accumulation of toxic amounts of glucocerebroside. As a consequence, patients suffer widespread organ and metabolic dysfunction. Among the 360 mutations that have been identified in GD, the majority of nucleotide changes are missense mutations, in which a single amino acid is substituted by another (1, 2). However, instead of catastrophic loss of intrinsic enzymatic catalytic function, most GBA1 mutations lead to changes in the natural conformation of the peptide during protein folding, followed by retention within the endoplasmic reticulum (ER) (3–6). Subsequently, the unnatural peptide conformation affects chaperone recognition, which then stimulates premature protein degradation via mechanisms, involving c-cbl associated proteasomal complexes and the E3 ligases Parkin and Itch (4, 7, 8). Identifying key chaperone proteins that determine GCase proteostasis is a plausible approach for developing targeted therapies in GD especially in cases of disease manifestations in the central nervous system where standard enzyme replacement treatment is ineffective.Heat shock protein 27 (Hsp27, also known as heat shock protein beta-1, HSPB1) is a chaperone protein of the small heat shock protein group that also includes ubiquitin, α-crystallin, and Hsp20 among others. In addition to its chaperone activities, Hsp27 is involved in thermotolerance, inhibition of apoptosis, and regulation of cell development and differentiation (9). Hsp27 acts as an ATP-independent chaperone by inhibiting protein aggregation and stabilizing partially denatured proteins ensuring proper refolding by the Hsp70 complex. In addition, Hsp27 may be a key player in proteasomal activation. For example, Hsp27 is integral to degradation of phosphorylated I-κBα in the proteasome (10). In addition, Hsp27 has been shown to interact with p27^Kip1 and to guide its degradation (11). These findings imply that Hsp27 plays a significant role in the proteasomal degradation of misfolded proteins in mammalian cells.We explored potential binding partners to mutant GCases using an unbiased mass spectrometric approach (20535800). We found that Hsp27 was a previously unidentified binding partner that specifically recognized two common mutations for type I (N370S/N370S) and type II/III (L444P/L444P) GD, but not its wild-type counterpart. When presented with the mutant GCase, the Hsp90 complex recruited Hsp27 before induction of the VCP/proteasomal degradation pathway. Targeting Hsp27 not only rescued quantitative enzymatic levels but also increased the catalytic activity of GCase in patient derived fibroblasts. These findings suggest that Hsp27 may be a useful target for the treatment of GD.     

Mitochondrial Hsp90 is a ligand-activated molecular chaperone coupling ATP binding to dimer closure through a coiled-coil intermediate

Heat-shock protein of 90 kDa (Hsp90) is an essential molecular chaperone that adopts different 3D structures associated with distinct nucleotide states: a wide-open, V-shaped dimer in the apo state and a twisted, N-terminally closed dimer with ATP. Although the N domain is known to mediate ATP binding, how Hsp90 senses the bound nucleotide and facilitates dimer closure remains unclear. Here we present atomic structures of human mitochondrial Hsp90_N (TRAP1_N) and a composite model of intact TRAP1 revealing a previously unobserved coiled-coil dimer conformation that may precede dimer closure and is conserved in intact TRAP1 in solution. Our structure suggests that TRAP1 normally exists in an autoinhibited state with the ATP lid bound to the nucleotide-binding pocket. ATP binding displaces the ATP lid that signals the cis-bound ATP status to the neighboring subunit in a highly cooperative manner compatible with the coiled-coil intermediate state. We propose that TRAP1 is a ligand-activated molecular chaperone, which couples ATP binding to dramatic changes in local structure required for protein folding.Heat-shock protein of 90 kDa (Hsp90) is a conserved ATP-dependent molecular chaperone (1–4), which together with heat-shock protein of 70 kDa (Hsp70) (5–7) and a cohort of cochaperones (8–10), promotes the late-stage folding of Hsp90 client proteins (11). It is presumed that almost 400 different proteins, including a majority of signaling and tumor promoting proteins, depend on cytosolic Hsp90 for folding (12). Consequently, the ability to inactivate multiple oncogenic pathways simultaneously has made Hsp90 a major target for drug development (13), with several Hsp90 inhibitors currently undergoing clinical trials (14).Hsp90 chaperones display conformational plasticity in solution (2, 15, 16), with different adenine nucleotides either facilitating or stabilizing distinct Hsp90 dimer conformations (17–19). Interestingly, apo Hsp90 forms a wide-open, V-shaped dimer with the N domains separated by as much as 101 Å (18). This open conformation is markedly distinct from the intertwined, N-terminally closed dimer with ATP bound (20, 21). Because the open-state dimer cannot signal the nucleotide status between neighboring subunits, an intermediate conformation preceding dimer closure must exist, which so far has remained elusive.Apart from cytosolic Hsp90s, Hsp90 homologs are found in the endoplasmic reticulum, chloroplasts, and mitochondria (Fig. S1) (22). The tumor necrosis factor receptor-associated protein 1 (TRAP1) is the mitochondrial Hsp90 paralog, which prevents apoptosis and protects mitochondria against oxidative damage (23–25). TRAP1 is widely expressed in many tumors (24, 26, 27), but not in mitochondria of most normal tissues (24), benign prostatic hyperplasia (26), or highly proliferating, nontransformed cells (27). Notably, it was found that TRAP1 not only promotes neoplastic growth, but also confers tumorigenic potential on nontransformed cells (27), indicating a major role of TRAP1 in tumorigenesis, although TRAP1’s specific function remains poorly understood (28).Open in a separate windowFig. S1.Multiple sequence alignment of the N-terminal nucleotide-binding domain of TRAP1 (mitochondria), HtpG (eubacteria), Hsp90 (cytosolic), and Grp94 (endoplasmic reticulum) from Homo sapiens (Hs), Danio rerio (Dr), E. coli (Ec), and S. cerevisiae (Sc). Residues conserved across sequences of representative members are highlighted in green, and those that are similar are in yellow. Residues not found in the mature protein are shown in lowercase letters. The N strap, ATP lid, and G1 and G2 boxes are marked. Residues that define the G1 and G2 box motifs are in red. Asp158, Phe183, Gln200, and Phe201 are surrounded by a gray box.TRAP1 is a multidomain protein consisting of an N-terminal or N domain (TRAP1_N), a middle domain (TRAP1_M), and a C-terminal or C domain (TRAP1_C), but it lacks the charged linker found in eukaryotic Hsp90 paralogs. Human TRAP1 is preceded by a mitochondrial localization sequence (MLS) of 59 residues that are cleaved off during import (29). The mature form of TRAP1 is a homodimer held together by TRAP1_C, with a second, ATP binding-dependent dimer interface in TRAP1_N. The crystal structures of zebrafish TRAP1 (zTRAP1) and zebrafish and human TRAP1_NM bound to ADPNP were recently reported (21, 30), and are largely consistent with our current understanding of Hsp90 chaperones. In the ATP-bound state, the N-terminal extension (known as the N strap) straddles the N domain of the neighboring subunit, thereby stabilizing the structure of the closed-state dimer (21, 30). The ordered segment of the N strap significantly lengthens the previously observed β-strand swap and may function as a regulatory element that controls TRAP1 function (16, 21). Interestingly, intact zTRAP1-ADPNP crystallized as an asymmetric dimer that could support a sequential ATP hydrolysis mechanism (31, 32); however, no asymmetric nucleotide binding was observed, and no molecular contacts between cis-bound ADPNP and the N domain of the neighboring subunit were seen in TRAP1 (21, 30) and other known Hsp90 structures (18, 20, 33), leaving open the question of how TRAP1 senses and signals the nucleotide-bound status between subunits.Here we present atomic structures of human TRAP1_N (hTRAP1_N) alone and in complex with ADPNP. Unexpectedly, we found that unliganded hTRAP1_N forms a previously unobserved coiled-coil dimer that is distinct from the proposed open-state and closed-state conformations (16, 21, 30). Importantly, intact hTRAP1 forms a similar coiled-coil dimer in solution, but only in the absence of ATP. Our findings show that ATP binding triggers a dramatic change in local structure and displaces the ATP lid, which is bound to the ATP-binding pocket, indicating that TRAP1 normally exists in an autoinhibited state. Strikingly, mutations of conserved residues that impair lid binding stimulate the hTRAP1 ATPase activity in a highly cooperative manner, supporting a previously unknown role of the ATP lid in signaling the cis-bound nucleotide status to the trans subunit, which is compatible with the coiled-coil dimer. Finally, we demonstrate that TRAP1 folding requires ATP and the functional cooperation of the mitochondrial Hsp70 chaperone system, supporting the existence of a mitochondrial Hsp90-Hsp70 supercomplex that may present a new target for drug development.     

CRINEPT-TROSY NMR reveals p53 core domain bound in an unfolded form to the chaperone Hsp90

The molecular chaperone Hsp90 sequesters oncogenic mutants of the tumor suppressor p53 that have unstable core domains. It is not known whether p53 is bound in an unfolded, partly folded, or distorted structure, as is unknown for the structure of any bound substrate of Hsp90. It is a particularly difficult problem to analyze in detail the structures of large complexes in which one component is (partly) unfolded. We have shown by transverse relaxation-optimized NMR spectroscopy combined with cross-correlated relaxation-enhanced polarization transfer (CRINEPT-TROSY) that p53 core domain bound in an approximately 200-kDa complex with Hsp90 was predominantly unfolded lacking helical or sheet secondary structure. This mode of binding might be a general feature of substrates of Hsp90.     

A novel role for DYX1C1, a chaperone protein for both Hsp70 and Hsp90, in breast cancer

Aims  With three consecutive tetratricopeptide repeat (TPR) motifs at its C-terminus essential for neuronal migration, and a p23 domain at its N-terminus, DYX1C1 was the first gene proposed to have a role in developmental dyslexia. In this study, we attempted to identify the potential interaction of DYX1C1 and heat shock protein, and the role of DYX1C1 in breast cancer. Main methods  GST pull-down, a yeast two-hybrid system, RT-PCR, site-directed mutagenesis approach. Key findings  Our study initially confirmed DYX1C1, a dyslexia related protein, could interact with Hsp70 and Hsp90 via GST pull-down and a yeast two-hybrid system. And we verified that EEVD, the C-terminal residues of DYX1C1, is responsible for the identified association. Further, DYX1C1 mRNA was significantly overexpressed in malignant breast tumor, linking with the up-regulated expression of Hsp70 and Hsp90. Significance  These results suggest that DYX1C1 is a novel Hsp70 and Hsp90-interacting co-chaperone protein and its expression is associated with malignancy. Yuxin Chen, Muzi Zhao, Saiqun Wang contributed equally to this work.     

Maturation of the tyrosine kinase c-src as a kinase and as a substrate depends on the molecular chaperone Hsp90

Although Hsp90 displays general chaperone activity in vitro, few substrates of the chaperone have been identified in vivo, and the characteristics that render these substrates dependent on Hsp90 remain elusive. To investigate this issue, we exploited a paradoxical observation: several unrelated oncogenic viral tyrosine kinases, including v-src, attain their native conformation after association with Hsp90, yet their nearly identical cellular homologs interact only weakly with the chaperone. It has been controversial whether Hsp90 is vital for normal maturation of the cellular kinases or is simply binding a misfolded subfraction of the proteins. By modulating Hsp90 levels in Saccharomyces cerevisiae, we determined that Hsp90 is indeed necessary for the maturation of c-src (the normal homolog of v-src). c-src maturation is, however, less sensitive to Hsp90 perturbations than is v-src maturation. Dependence of the two proteins on Hsp90 does not correspond to their relative efficiency in reaching their final destination (the plasma membrane); we observed that in yeast, unlike in vertebrate cells, neither c-src nor v-src concentrate in the membrane. Expression of different v/c-src chimeras in cells carrying wild-type or temperature-sensitive Hsp90 alleles revealed that the difference between the proteins instead arises from multiple, naturally occurring mutations in the C-terminal region of v-src.     

The BRAF(V600E) mutation is associated with malignant ultrasonographic features in thyroid nodules

Context Several ultrasonographic (US) features of thyroid nodules have been reported to predict malignancy. The BRAF^V600E mutation is a useful diagnostic marker for differentiating papillary thyroid carcinoma from benign thyroid nodules, especially in BRAF^V600E‐prevalent populations such as in Korea. Objective To evaluate the association of BRAF^V600E mutation with US features of thyroid nodules in predicting the malignancy of thyroid nodules in Korean patients. Design A total of 991 thyroid nodules from 823 patients in fine‐needle aspiration biopsy (FNAB) specimens were investigated. The relationship between US features and the presence of BRAF^V600E mutation by pyrosequencing method was prospectively analysed. Results The BRAF^V600E mutation was associated with the following US features: solid composition [odds ratio (OR) 20·338; 95% confidence interval (CI): 4·952–83·532; P < 0·001], marked hypoechogenicity (OR 30·744; 95% CI: 15·951–59·255; P < 0·001), irregular margin (OR 9·889; 95% CI: 7·005–13·859; P < 0·001), taller‐than‐wide shape (OR 6·031; 95% CI: 4·343–8·376; P < 0·001) and the presence of microcalcifications (OR 6·664; 95% CI: 4·604–9·648; P < 0·001). The BRAF^V600E mutation with malignant US features in FNAB enhanced the diagnostic accuracy compared with cytologic diagnosis alone (94·3%vs 69·7%). Conclusion The BRAF^V600E mutation is significantly associated with malignant US features, such as solid composition, marked hypoechogenicity, irregular margin, taller‐than‐wide shape and the presence of microcalcifications. The application of BRAF^V600E mutation analysis in US‐guided FNAB can improve the diagnostic accuracy of thyroid nodules.     

Hsp40 proteins phase separate to chaperone the assembly and maintenance of membraneless organelles

Membraneless organelles contain a wide spectrum of molecular chaperones, indicating their important roles in modulating the metastable conformation and biological function of membraneless organelles. Here we report that class I and II Hsp40 (DNAJ) proteins possess a high ability of phase separation rendered by the flexible G/F-rich region. Different Hsp40 proteins localize in different membraneless organelles. Specifically, human Hdj1 (DNAJB1), a class II Hsp40 protein, condenses in ubiquitin (Ub)-rich nuclear bodies, while Hdj2 (DNAJA1), a class I Hsp40 protein, condenses in nucleoli. Upon stress, both Hsp40 proteins incorporate into stress granules (SGs). Mutations of the G/F-rich region not only markedly impaired Hdj1 phase separation and SG involvement and disrupted the synergistic phase separation and colocalization of Hdj1 and fused in sarcoma (FUS) in cells. Being cophase separated with FUS, Hdj1 stabilized the liquid phase of FUS against proceeding into amyloid aggregation in vitro and alleviated abnormal FUS aggregation in cells. Moreover, Hdj1 uses different domains to chaperone FUS phase separation and amyloid aggregation. This paper suggests that phase separation is an intrinsic property of Hsp40 proteins, which enables efficient incorporation and function of Hsp40 in membraneless organelles and may further mediate the buildup of chaperone network in membraneless organelles.

Membraneless organelles in cells are highly diverse and fulfill important biological functions under both normal and stress conditions (1, 2). Protein phase separation can drive the assembly of membraneless organelles, which incorporates hundreds of different proteins and nucleic acids (3–6). Membraneless organelles feature a physical property in between liquid and solid and present as a metastable state of complex supermolecular assembly (7). The maintenance of membraneless organelles in the liquidlike state rather than spontaneous solidification or dissociation is essential for membraneless organelles to fulfill their biological functions (8, 9). Failure of the conformational maintenance of membraneless organelles results in pathological protein aggregation, which is closely associated with various neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (10–14).Molecular chaperones are key components of the quality control system in cells to maintain protein homeostasis (proteostasis). They assist client proteins in folding and translocation and maintain clients in the native conformation avoiding misfolding and aggregation (15). Proteomic analysis of SGs has identified a wide spectrum of molecular chaperones, including Hsp40 (DNAJ), Hsp70, Hsp90, and small Hsps (5, 16), which indicate the importance of chaperones in the assembly and maintenance of membraneless organelles. Studies on yeast Hsp40 proteins (e.g., Sis1 and Ydj1) reveal that they accumulate in SGs and directly influence the assembly, dynamics and clearance of SGs (17). Human Hsp40 proteins, e.g., Hdj1 (DNAJB1) and Hdj2 (DNAJA1), cannot only inhibit the aggregation of a variety of amyloid proteins including α-synuclein, Τau, and polyQ in vitro (18–20) and rescue the cytotoxicity of polyQ and SG-associated RNA-binding proteins including fused in sarcoma (FUS) and TAR DNA-binding protein 43 (TDP-43) in cellular and animal disease models (21–23). However, the mechanism of how Hsp40s rescue the cytotoxicity of these proteins remains unknown. Moreover, genetic defects of Hsp40 proteins have been identified in neurodegenerative diseases including ALS (24), Parkinson’s disease (25), cerebellar ataxia (26), and distal hereditary motor neuron neuropathy (27). These evidences suggest that Hsp40 proteins play an important role in maintaining the neuronal proteostasis by regulating protein assembly and preventing pathological amyloid aggregation.In this paper, we find that human class I and II Hsp40 proteins—Hdj2 and Hdj1 localize differently in nucleoli and Ub-rich nuclear bodies, respectively, under normal conditions. Under stress, both chaperones condense in SGs in the cytoplasm. We show that the close association of Hdj1 and Hdj2 with various membraneless organelles attributes to their phase separation property via the flexible G/F-rich region, which we show is a common property shared by class I and II Hsp40 proteins in different organisms. Furthermore, we find that Hdj1 synergistically phase separates with FUS and maintains the phase-separated state of FUS from proceeding to amyloid aggregation. Interestingly, Hdj1 employs distinctive mechanisms to chaperone the different states (i.e., soluble and phase-separated states) of FUS. This paper suggests that phase separation renders the localization and function of Hsp40 in various membraneless organelles, which may further recruit cochaperones (e.g., Hsp70) for the function and regulation of membraneless organelles.     

Clinical significance of immunohistochemistry to detect BRAF V600E mutant protein in thyroid tissues

This study investigated the feasibility of using immunohistochemistry (IHC) instead of PCR to detect BRAF V600E mutant protein in papillary thyroid carcinoma (PTC), and to determine the value of using preoperative BRAF V600E mutant protein by IHC to assist in the diagnosis of thyroid nodule patients with Hashimoto''s thyroiditis (HT).The expression of BRAFV600E mutant protein was measured in 23 cases of HT+PTC, 31 cases of PTC, and 28 cases of HT by IHC, followed by PCR in the same samples for validation. SPSS 19.0 software was used for statistical analysis.The sensitivity and specificity of IHC to detect BRAF V600E mutation were 100% and 42.86%, respectively. In addition, the mutation rate of BRAF V600E protein in the HT+PTC group (34.78%, 8/23) was lower than that in the PTC group (80.65%, 25/31).The application of IHC to detect BRAF V600E mutant protein has good sensitivity but not specificity to diagnose PTC. IHC can be used as a preliminary screening method to detect BRAF V600E mutation. The strongly positive (+++) staining of IHC potently indicated BRAF V600E gene mutation. For suspicious thyroid nodules combined with HT, the detection of BRAF V600E mutant protein with IHC alone is not of great significance for differentiating benign and malignant nodules.     

ZAP-70 is a novel conditional heat shock protein 90 (Hsp90) client: inhibition of Hsp90 leads to ZAP-70 degradation, apoptosis, and impaired signaling in chronic lymphocytic leukemia

The zeta-associated protein of 70 kDa (ZAP-70) is expressed in patients with aggressive chronic lymphocytic leukemia (CLL). We found that ZAP-70+ CLL cells expressed activated heat-shock protein 90 (Hsp90) with high binding affinity for Hsp90 inhibitors, such as 17-allyl-amino-demethoxy-geldanamycin (17-AAG), whereas normal lymphocytes or ZAP-70- CLL cells expressed nonactivated Hsp90. Activated Hsp90 bound and stabilized ZAP-70, which behaved like an Hsp90 client protein only in CLL cells. Treatment with Hsp90 inhibitors such as 17-AAG and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) induced ZAP-70 degradation and apoptosis in CLL cells but not in T cells, and also impaired B-cell receptor signaling in leukemia cells. Transduction of ZAP-70- CLL cells with an adenovirus encoding ZAP-70 activated Hsp90 and specifically rendered the leukemia cells sensitive to 17-AAG. These data indicate that Hsp90 is necessary for ZAP-70 expression and activity; that ZAP-70 is unique among Hsp90 clients, in that its chaperone-dependency is conditional on the cell type in which it is expressed; and also that ZAP-70 is required for cell survival and signaling in CLL. Additionally, ZAP-70 expression in CLL cells confers markedly heightened sensitivity to 17-AAG or 17-DMAG, suggesting that these or other Hsp90 inhibitors could be valuable therapeutically in patients with aggressive CLL.     

BRAF(V600E) mutation and the biology of papillary thyroid cancer

BRAF((V600E)) mutation is the most frequent genetic alteration in papillary thyroid carcinomas (PTCs) that are 80-90% of all thyroid cancers. We evaluated the relationship between BRAF((V600E)) and tumor, host, and environmental factors in PTCs from all geographical areas of Sicily. By PCR, BRAF((V600E)) was investigated in a series of 323 PTCs diagnosed in 2002-2005. The correlation between clinicopathological tumor, host, and environmental characteristics and the presence of BRAF((V600E)) were evaluated by both univariate and multivariate analyses. BRAF((V600E)) was found in 38.6% PTCs, with a 52% frequency in the classical PTCs and 26.4% in the tall cell variant. Univariate analysis indicated that BRAF((V600E)) was associated with greater tumor size (P=0.0048), extra-thyroid invasion (P<0.0001), and cervical lymph nodal metastases (P=0.0001). Multivariate logistic regression analysis confirmed that BRAF((V600E)) was an independent predictor of extra-thyroid invasion (P=0.0001) and cervical lymph nodal metastasis (P=0.0005). The association between BRAF((V600E)) and extra-thyroid invasion was also found in micro-PTCs (P=0.006). In 60 classical PTCs, BRAF((V600E)) was positively correlated with matrix metalloproteinase-9 expression (P=0.0047), suggesting a possible mechanism for BRAF((V600E)) effect on PTC invasiveness. No association was found between BRAF((V600E)) and patient age, gender, or iodine intake. In contrast, a strong association was found with residency in Eastern Sicily (P<0.0001 compared with Western Sicily). These results indicate that BRAF((V600E)) mutation is a marker of aggressive disease in both micro- and macro-PTCs. Moreover, for the first time, a possible link between BRAF((V600E)) mutation and environmental carcinogens is suggested.