Biomedical subjects

William E Balch

Publications and source records attributed to William E Balch.

At least 19 recordsLinked to original sources

PubMed2006

Hsp90 cochaperone Aha1 downregulation rescues misfolding of CFTR in cystic fibrosis.

The pathways that distinguish transport of folded and misfolded cargo through the exocytic (secretory) pathway of eukaryotic cells remain unknown. Using proteomics to assess global cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein interactions (the CFTR interactome), we show that Hsp90 cochaperones modulate Hsp90-dependent stability of CFTR protein folding in the endoplasmic reticulum (ER). Cell-surface rescue of the most common disease variant that is restricted to the ER, DeltaF508, can be initiated by partial siRNA silencing of the Hsp90 cochaperone ATPase regulator Aha1. We propose that failure of DeltaF508 to achieve an energetically favorable fold in response to the steady-state dynamics of the chaperone folding environment (the "chaperome") is responsible for the pathophysiology of CF. The activity of cargo-associated chaperome components may be a common mechanism regulating folding for ER exit, providing a general framework for correction of misfolding disease.

Animals↗

PubMed2006

Domain interdependence in the biosynthetic assembly of CFTR.

The dimerization of their two nucleotide binding domains (NBDs) in a so-called "nucleotide-sandwich" is the hallmark of ATP cassette binding (ABC) proteins and the basis of their catalytic activities. The major disease-causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR or ABCC7), deletion of Phe508 in NBD1, does not grossly alter the structure of that domain but prevents conformational maturation of the whole CFTR protein, possibly by disrupting the native interaction between NBD1 and NBD2. However, the role of inter-domain interactions in CFTR folding has been brought into question by a recent report that all CFTR domains fold independently. Here we show that in addition to domain folding, correct inter-domain assembly is essential to form a stable unit that satisfies endoplasmic reticulum (ER) quality control. N-terminal domains depend on their more C-terminal neighbors, most essentially the second membrane-spanning domain (MSD2) but significantly, not NBD2. Wild-type C-terminal truncation constructs, completely devoid of NBD2 are transported out of the ER and to the cell surface where they form characteristic CFTR chloride channels with low open probability. The DeltaNBD2 wild-type protein matures and has similar stability as its full-length counterpart. Therefore, the catalytically crucial inter-NBD associations are not required to satisfy ER quality control mechanisms. The DeltaF508 mutation arrests the maturation of DeltaNBD2 just as it does full-length CFTR, indicating that DeltaF508 perturbs other portions of the molecule in addition to NBD2. We find that the mutation prevents formation of a compact MSD1, reflected in its susceptibility to protease digestion. This perturbation of MSD1 may in turn prevent its normal integration with MSD2. The dispensability of NBD2 in the folding of more N-terminal domains stands in contrast to the known hypersensitivity to proteolysis of NBD2 in the DeltaF508 protein.

Adenosine Triphosphate↗

PubMed2006

Chemical chaperones and permissive temperatures alter localization of Gaucher disease associated glucocerebrosidase variants.

Point mutations in the lysosomal hydrolase, glucocerebrosidase (GC), can cause Gaucher disease, a common lysosomal storage disease. Several clinically important GC mutations impede folding in the endoplasmic reticulum (ER) and target these enzymes for ER-associated degradation (ERAD). The removal of these misfolded proteins decreases the lysosomal concentration of GC, which results in glucosylceramide accumulation. The most common GC variant, N370S, and other clinically relevant variants, G202R and L444P, exhibit different cellular localization patterns in patient-derived fibroblasts. We show that these distributions can be altered by manipulation of the ER folding environment, either by chemical chaperones or by temperature shifts. N370S, L444P, and G202R GC are destabilized in the neutral pH environment of the ER, rendering them prone to ERAD. Fibroblasts harboring the G202R and L444P GC mutations grown at 30 degrees C localize the mutant proteins to the lysosome, and this increases total GC activity. Both of these temperature-sensitive mutants appear to be stable at 37 degrees C once they are trafficked to the low pH environment of the lysosome. Chemical chaperones correct the ER instability and significant ER retention of G202R GC. N370S is also destabilized under ER simulating conditions, a deficiency that is corrected by chemical chaperone binding. These data clearly show manipulating the ER environment with chemical chaperones increases the lysosomal concentration of partially active GC variants and suggest that small molecules could be used to treat Gaucher disease.

Animals↗

PubMed2006

The Hsp90 chaperone complex regulates GDI-dependent Rab recycling.

Rab GTPase regulated hubs provide a framework for an integrated coding system, the membrome network, that controls the dynamics of the specialized exocytic and endocytic membrane architectures found in eukaryotic cells. Herein, we report that Rab recycling in the early exocytic pathways involves the heat-shock protein (Hsp)90 chaperone system. We find that Hsp90 forms a complex with guanine nucleotide dissociation inhibitor (GDI) to direct recycling of the client substrate Rab1 required for endoplasmic reticulum (ER)-to-Golgi transport. ER-to-Golgi traffic is inhibited by the Hsp90-specific inhibitors geldanamycin (GA), 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), and radicicol. Hsp90 activity is required to form a functional GDI complex to retrieve Rab1 from the membrane. Moreover, we find that Hsp90 is essential for Rab1-dependent Golgi assembly. The observation that the highly divergent Rab GTPases Rab1 involved in ER-to-Golgi transport and Rab3A involved in synaptic vesicle fusion require Hsp90 for retrieval from membranes lead us to now propose that the Hsp90 chaperone system may function as a general regulator for Rab GTPase recycling in exocytic and endocytic trafficking pathways involved in cell signaling and proliferation.

Animals↗

PubMed2006

Heparin accelerates gelsolin amyloidogenesis.

The chemical environment of the extracellular matrix may influence the tissue-selective deposition observed there in gelsolin amyloid disease. Previously, we have identified the proteases that generate the amyloidogenic fragments from the full-length gelsolin variants and demonstrated that heparin is capable of accelerating gelsolin amyloidogenesis. Herein, we identify the structural features of heparin that promote the 8 kDa disease-associated gelsolin fragments (residues 173-243) generated at the cell surface to form amyloid. In conjunction with electron microscopy analyses, our kinetic studies demonstrate that heparin efficiently accelerates the formation of gelsolin amyloid by enabling intermolecular beta-sheet formation. The use of heparin analogues reveals that sulfation is important in accelerating amyloidogenesis and that the extent of acceleration is proportional to the molecular weight of heparin. In addition, heparin accelerated aggregation at both early and late stages of amyloidogenesis. Dynamic light scattering coupled to size exclusion chromatography showed that heparin promotes the formation of soluble aggregates. Collectively, these data reveal that heparin templates fibril formation and affords solubility to the aggregating peptides through its sulfated structure. By extension, the biochemical results herein suggest that tissue-selective deposition characteristic of the gelsolin amyloidoses is likely influenced by the extracellular localization of distinct glycosaminoglycans.

Amyloid↗

PubMed2006

Structure of the Sec13/31 COPII coat cage.

Endomembranes of eukaryotic cells are dynamic structures that are in continuous communication through the activity of specialized cellular machineries, such as the coat protein complex II (COPII), which mediates cargo export from the endoplasmic reticulum (ER). COPII consists of the Sar1 GTPase, Sec23 and Sec24 (Sec23/24), where Sec23 is a Sar1-specific GTPase-activating protein and Sec24 functions in cargo selection, and Sec13 and Sec31 (Sec13/31), which has a structural role. Whereas recent results have shown that Sec23/24 and Sec13/31 can self-assemble to form COPII cage-like particles, we now show that Sec13/31 can self-assemble to form minimal cages in the absence of Sec23/24. We present a three-dimensional reconstruction of these Sec13/31 cages at 30 A resolution using cryo-electron microscopy and single particle analysis. These results reveal a novel cuboctahedron geometry with the potential to form a flexible lattice and to generate a diverse range of containers. Our data are consistent with a model for COPII coat complex assembly in which Sec23/24 has a non-structural role as a multivalent ligand localizing the self-assembly of Sec13/31 to form a cage lattice driving ER cargo export.

Animals↗

PubMed2006

The integration of cell and chemical biology in protein folding.

Eukaryotic cells are specialized, interdependent functional units of complex tissues that are composed of metabolically integrated systems defined by chemically distinct organelles that operate as reaction vessels. It is now clear that the small-molecule and polymer-based composition of these organelles plays a crucial role in generating and maintaining protein folds and functions through the systems chemistry of the local environments.

Biochemical Phenomena↗

PubMed2006

The COPII cage: unifying principles of vesicle coat assembly.

Communication between compartments of the exocytic and endocytic pathways in eukaryotic cells involves transport carriers - vesicles and tubules - that mediate the vectorial movement of cargo. Recent studies of transport-carrier formation in the early secretory pathway have provided new insights into the mechanisms of cargo selection by coat protein complex-II (COPII) adaptor proteins, the construction of cage-protein scaffolds and fission. These studies are beginning to produce a unifying molecular and structural model of coat function in the formation and fission of vesicles and tubules in endomembrane traffic.

Animals↗

PubMed2006

Functional amyloid formation within mammalian tissue.

Amyloid is a generally insoluble, fibrous cross-beta sheet protein aggregate. The process of amyloidogenesis is associated with a variety of neurodegenerative diseases including Alzheimer, Parkinson, and Huntington disease. We report the discovery of an unprecedented functional mammalian amyloid structure generated by the protein Pmel17. This discovery demonstrates that amyloid is a fundamental nonpathological protein fold utilized by organisms from bacteria to humans. We have found that Pmel17 amyloid templates and accelerates the covalent polymerization of reactive small molecules into melanin-a critically important biopolymer that protects against a broad range of cytotoxic insults including UV and oxidative damage. Pmel17 amyloid also appears to play a role in mitigating the toxicity associated with melanin formation by sequestering and minimizing diffusion of highly reactive, toxic melanin precursors out of the melanosome. Intracellular Pmel17 amyloidogenesis is carefully orchestrated by the secretory pathway, utilizing membrane sequestration and proteolytic steps to protect the cell from amyloid and amyloidogenic intermediates that can be toxic. While functional and pathological amyloid share similar structural features, critical differences in packaging and kinetics of assembly enable the usage of Pmel17 amyloid for normal function. The discovery of native Pmel17 amyloid in mammals provides key insight into the molecular basis of both melanin formation and amyloid pathology, and demonstrates that native amyloid (amyloidin) may be an ancient, evolutionarily conserved protein quaternary structure underpinning diverse pathways contributing to normal cell and tissue physiology.

Amyloid↗

PubMed2005

Metalloendoprotease cleavage triggers gelsolin amyloidogenesis.

Amyloid diseases like Alzheimer's disease and familial amyloidosis of Finnish type (FAF) stem from endoproteolytic cleavage of a precursor protein to generate amyloidogenic peptides that accumulate as amyloid deposits in a tissue-specific manner. FAF patients deposit both 8 and 5 kDa peptides derived from mutant (D187Y/N) plasma gelsolin in the extracellular matrix (ECM). The first of two aberrant sequential proteolytic events is executed by furin to yield a 68 kDa (C68) secreted fragment. We now identify the metalloprotease MT1-matrix metalloprotease (MMP), an integral membrane protein active in the ECM, as a protease that processes C68 to the amyloidogenic peptides. We further demonstrate that ECM components are capable of accelerating gelsolin amyloidogenesis. Proteolysis by MT1-MMP-like proteases proximal to the unique chemical environment of the ECM offers an explanation for the tissue-specific deposition observed in FAF and provides critical insight into new therapeutic strategies.

Amyloid↗

PubMed2005

Large-scale profiling of Rab GTPase trafficking networks: the membrome.

Rab GTPases and SNARE fusion proteins direct cargo trafficking through the exocytic and endocytic pathways of eukaryotic cells. We have used steady state mRNA expression profiling and computational hierarchical clustering methods to generate a global overview of the distribution of Rabs, SNAREs, and coat machinery components, as well as their respective adaptors, effectors, and regulators in 79 human and 61 mouse nonredundant tissues. We now show that this systems biology approach can be used to define building blocks for membrane trafficking based on Rab-centric protein activity hubs. These Rab-regulated hubs provide a framework for an integrated coding system, the membrome network, which regulates the dynamics of the specialized membrane architecture of differentiated cells. The distribution of Rab-regulated hubs illustrates a number of facets that guides the overall organization of subcellular compartments of cells and tissues through the activity of dynamic protein interaction networks. An interactive website for exploring datasets comprising components of the Rab-regulated hubs that define the membrome of different cell and organ systems in both human and mouse is available at http://www.membrome.org/.

Animals↗

PubMed2005

The biological and chemical basis for tissue-selective amyloid disease.

Factors controlling the onset and progression of extracellular amyloid diseases remain largely unknown. Central to disease etiology is the efficiency of the endoplasmic reticulum (ER) machinery that targets destabilized mutant proteins for degradation and the enhanced tendency of these variants to aggregate if secreted. We demonstrate that mammalian cells secrete numerous transthyretin (TTR) disease-associated variants with wild-type efficiency in spite of compromised folding energetics. Only the most highly destabilized TTR variants are subjected to ER-associated degradation (ERAD) and then only in certain tissues, providing insight into tissue selective amyloidosis. Rather than a "quality control" standard based on wild-type stability, we find that ER-assisted folding (ERAF), based on global protein energetics, determines the extent of export. We propose that ERAF (influenced by the energetics of the protein fold, chaperone enzyme distributions, and metabolite chaperones) in competition with ERAD defines the unique secretory aptitude of each tissue.

Amyloidosis↗

PubMed2005

A new pharmacology--drugging stressed folding pathways.

Folding in the endoplasmic reticulum (ER) must couple protein-synthesis pathways operating outside of the compartment with ER-assisted folding (ERAF) pathways in the lumen. Chaperone-mediated folding imbalances that are associated with numerous misfolding diseases, including diabetes, trigger the unfolded-protein response (UPR), using both transcriptional and translational pathways to correct the problem. Recent work suggests that small-molecule inhibitors could be useful to help rebalance protein synthesis with ERAF pathways through the ribosomal initiating factor eIF2alpha. Reprogramming stress pathways with drugs provides a potential new approach for balancing ER-protein load with cellular-folding capacity, thus correcting disease.

Animals↗

PubMed2005

Exploring trafficking GTPase function by mRNA expression profiling: use of the SymAtlas web-application and the Membrome datasets.

Despite complete sequencing of the human and mouse genomes, functional annotation of novel gene function still remains a major challenge in mammalian biology. Emerging strategies to help elucidate unknown gene function include the analysis of tissue-specific patterns of mRNA expression. A recent study investigated the steady-state mRNA expression profiling of the vast majority of protein-encoding human and mouse genes across a panel of 79 human and 61 mouse nonredundant tissues. The microarray data from this study constitutes the Genomics Institute of Novartis Foundation (GNF) Human and Mouse Gene Atlases and is publicly available for exploration through the SymAtlas web-application (http://symatlas.gnf.org/). We have recently reported the use of these data and hierarchical clustering algorithms to generate a global overview of the distribution of Rabs, SNAREs, and coat machinery components, as well as their respective adaptors, effectors, and regulators. This systems biology approach led us to propose Rab-centric protein activity hubs as a framework for an integrated coding system, the membrome network, which orchestrates the dynamics of specialized membrane architecture of differentiated cells. Here, we describe the use of the SymAtlas web-application and the Membrome datasets to help explore trafficking GTPase function. The human and mouse membrome datasets are available through the Membrome homepage (http://www.membrome.org/) and correspond to subsets of the SymAtlas content restricted to known membrane trafficking components. Considering the fragmentary nature of the current reductionist approaches in elucidating trafficking component functions, the membrome datasets provide a more focused systems biology perspective that not only complements our current understanding of transport in complex tissues but also provides an integrated perspective of Rab activity in controlling membrane architecture.

Animals↗

PubMed2005

Use of Hsp90 inhibitors to disrupt GDI-dependent Rab recycling.

Guanine nucleotide dissociation inhibitor (GDI) is a central regulator of Rab GTPase family members. GDI recycles Rab proteins from the membrane and sequesters the inactive GDP-bound form of Rab in the cytosol for use in multiple rounds of transport. The balance between the membrane-bound form of Rab and the cytosolic reserve pool of the Rab-GDI complex is critical for vesicular trafficking between membrane compartments. Recycling of Rab GTPases is likely to require a membrane-bound complex of GDI, Hsp90, and Rab given that alphaGDI-dependent recycling of Rab3A at the synapse and neurotransmitter transmitter release is inhibited by Hsp90-specific inhibitors. Here we describe methods required for establishing the dependence of Rab recycling pathways on Hsp90 in vitro.

Animals↗

PubMed2005

Recombinant production in baculovirus-infected insect cells and purification of the mammalian Sec13/Sec31 complex.

Membrane traffic along the eukaryotic secretory pathway starts with the selective packing of biosynthetic cargo into nascent vesicles that are forming on the endoplasmic reticulum (ER). This process is mediated by the coat protein complex II (COPII) machinery, which at the minimum, comprises the Sar1 GTPase and the cytosolic protein complexes Sec23/Sec24 (Sec23/24) and Sec13/Sec31 (Sec13/31). While the components of the basic COPII machinery are highly conserved from yeast to human, it is now clearly evident that the overall process is under tighter spatial and temporal regulation in higher eukaryotes. Here we describe recombinant production in baculovirus-infected insect cells and subsequent purification to homogeneity of the mammalian Sec13/31 complex for biochemical and biophysical characterization.

Animals↗

PubMed2005

Purification and properties of mammalian Sec23/24 from insect cells.

The Sec23/24 complex is a large heterodimeric protein involved in COPII vesicle biogenesis. The individual mammalian protein subunits are too large for expression in bacterial systems. This article details the use of the Bac-to-Bac baculovirus coexpression system in insect cells for both the human Sec23A and Sec24C. This strategy results in high yields of pure, functional protein and can be adapted for the purification of other Sec23/24 isoforms for their biochemical and biological characterization.

Animals↗

PubMed2005

The role of ARF1 and rab GTPases in polarization of the Golgi stack.

The organization and sorting of proteins within the Golgi stack to establish and maintain its cis to trans polarization remains an enigma. The function of Golgi compartments involves coat assemblages that facilitate vesicle traffic, Rab-tether-SNAP receptor (SNARE) machineries that dictate membrane identity, as well as matrix components that maintain structure. We have investigated how the Golgi complex achieves compartmentalization in response to a key component of the coat complex I (COPI) coat assembly pathway, the ARF1 GTPase, in relationship to GTPases-regulating endoplasmic reticulum (ER) exit (Sar1) and targeting fusion (Rab1). Following collapse of the Golgi into the ER in response to inhibition of activation of ARF1 by Brefeldin A, we found that Sar1- and Rab1-dependent Golgi reformation took place at multiple peripheral and perinuclear ER exit sites. These rapidly converged into immature Golgi that appeared as onion-like structures composed of multiple concentrically arrayed cisternae of mixed enzyme composition. During clustering to the perinuclear region, Golgi enzymes were sorted to achieve the degree of polarization within the stack found in mature Golgi. Surprisingly, we found that sorting of Golgi enzymes into their subcompartments was insensitive to the dominant negative GTP-restricted ARF1 mutant, a potent inhibitor of COPI coat disassembly and vesicular traffic. We suggest that a COPI-independent, Rab-dependent mechanism is involved in the rapid reorganization of resident enzymes within the Golgi stack following synchronized release from the ER, suggesting an important role for Rab hubs in directing Golgi polarization.

ADP-Ribosylation Factor 1↗