Hsp104 and Potentiated Variants Can Operate as Distinct Nonprocessive Translocases.
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ABSTRACT: Heat shock protein (Hsp) 104 is a hexameric ATPases associated with diverse cellular activities motor protein that enables cells to survive extreme stress. Hsp104 couples the energy of ATP binding and hydrolysis to solubilize proteins trapped in aggregated structures. The mechanism by which Hsp104 disaggregates proteins is not completely understood but may require Hsp104 to partially or completely translocate polypeptides across its central channel. Here, we apply transient state, single turnover kinetics to investigate the ATP-dependent translocation of soluble polypeptides by Hsp104 and Hsp104A503S, a potentiated variant developed to resolve misfolded conformers implicated in neurodegenerative disease. We establish that Hsp104 and Hsp104A503S can operate as nonprocessive translocases for soluble substrates, indicating a "partial threading" model of translocation. Remarkably, Hsp104A503S exhibits altered coupling of ATP binding to translocation and decelerated dissociation from polypeptide substrate compared to Hsp104. This altered coupling and prolonged substrate interaction likely increases entropic pulling forces, thereby enabling more effective aggregate dissolution by Hsp104A503S.
Project description:There are no therapies that reverse the proteotoxic misfolding events that underpin fatal neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). Hsp104, a conserved hexameric AAA+ protein from yeast, solubilizes disordered aggregates and amyloid but has no metazoan homolog and only limited activity against human neurodegenerative disease proteins. Here, we reprogram Hsp104 to rescue TDP-43, FUS, and ?-synuclein proteotoxicity by mutating single residues in helix 1, 2, or 3 of the middle domain or the small domain of nucleotide-binding domain 1. Potentiated Hsp104 variants enhance aggregate dissolution, restore proper protein localization, suppress proteotoxicity, and in a C. elegans PD model attenuate dopaminergic neurodegeneration. Potentiating mutations reconfigure how Hsp104 subunits collaborate, desensitize Hsp104 to inhibition, obviate any requirement for Hsp70, and enhance ATPase, translocation, and unfoldase activity. Our work establishes that disease-associated aggregates and amyloid are tractable targets and that enhanced disaggregases can restore proteostasis and mitigate neurodegeneration.
Project description:Cardiolipin (CL) is a universal component of energy generating membranes. In most bacteria, it is synthesized via the condensation of two molecules phosphatidylglycerol (PG) by phospholipase D-type cardiolipin synthases (PLD-type Cls). In the plant pathogen and natural genetic engineer Agrobacterium tumefaciens CL comprises up to 15% of all phospholipids in late stationary growth phase. A. tumefaciens harbors two genes, atu1630 (cls1) and atu2486 (cls2), coding for PLD-type Cls. Heterologous expression of either cls1 or cls2 in Escherichia coli resulted in accumulation of CL supporting involvement of their products in CL synthesis. Expression of cls1 and cls2 in A. tumefaciens is constitutive and irrespective of the growth phase. Membrane lipid profiling of A. tumefaciens mutants suggested that Cls2 is required for CL synthesis at early exponential growth whereas both Cls equally contribute to CL production at later growth stages. Contrary to many bacteria, which suffer from CL depletion, A. tumefaciens tolerates large changes in CL content since the CL-deficient cls1/cls2 double mutant showed no apparent defects in growth, stress tolerance, motility, biofilm formation, UV-stress and tumor formation on plants.
Project description:In vivo extracellular recording studies have traditionally shown that dopamine (DA) transiently inhibits prefrontal cortex (PFC) neurons, yet recent biophysical measurements in vitro indicate that DA enhances the evoked excitability of PFC neurons for prolonged periods. Moreover, although DA neurons apparently encode stimulus salience by transient alterations in firing, the temporal properties of the PFC DA signal associated with various behaviors is often extraordinarily prolonged. The present study used in vivo electrophysiological and electrochemical measures to show that the mesocortical system produces a fast non-DA-mediated postsynaptic response in the PFC that appears to be initiated by glutamate. In contrast, short burst stimulation of mesocortical DA neurons that produced transient (<4 s) DA release in the PFC caused a simultaneous reduction in spontaneous firing (consistent with extracellular in vivo recordings) and a form of DA-induced potentiation in which evoked firing was increased for tens of minutes (consistent with in vitro measurements). We suggest that the mesocortical system might transmit fast signals about reward or salience via corelease of glutamate, whereas the simultaneous prolonged DA-mediated modulation of firing biases the long-term processing dynamics of PFC networks.
Project description:Heat shock protein 104 (HSP104) is a conserved AAA+ protein disaggregase, can disassemble the toxic aggregates formed by different amyloid proteins, and is protective in various animal models associated with amyloid-related diseases. Extensive studies have attempted to elucidate how HSP104 disassembles the aggregated form of clients. Here, we found that HSP104 exhibits a potent holdase activity that does not require energy, prevents the soluble form of amyloid clients from aggregating, and differs from HSP104's disaggregase activity. Using cryo-EM, NMR, and additional biophysical approaches, we found that HSP104 utilizes its small subdomain of nucleotide-binding domain 2 (ssNBD2) to capture the soluble amyloid client (K19 of Tau) independent of its ATP hydrolysis activity. Our results indicate that HSP104 utilizes two fundamental distinct mechanisms to chaperone different forms of amyloid client and highlight the important yet previously unappreciated function of ssNBD2 in chaperoning amyloid client and thereby preventing pathological aggregation.
Project description:In cells, membrane tubes are extracted by molecular motors. Although individual motors cannot provide enough force to pull a tube, clusters of such motors can. Here, we investigate, using a minimal in vitro model system, how the tube pulling process depends on fundamental properties of the motor species involved. Previously, it has been shown that processive motors can pull tubes by dynamic association at the tube tip. We demonstrate that, remarkably, nonprocessive motors can also cooperatively extract tubes. Moreover, the tubes pulled by nonprocessive motors exhibit rich dynamics as compared to those pulled by their processive counterparts. We report distinct phases of persistent growth, retraction, and an intermediate regime characterized by highly dynamic switching between the two. We interpret the different phases in the context of a single-species model. The model assumes only a simple motor clustering mechanism along the length of the entire tube and the presence of a length-dependent tube tension. The resulting dynamic distribution of motor clusters acts as both a velocity and distance regulator for the tube. We show the switching phase to be an attractor of the dynamics of this model, suggesting that the switching observed experimentally is a robust characteristic of nonprocessive motors. A similar system could regulate in vivo biological membrane networks.
Project description:Key cellular processes such as cell division, membrane compartmentalization, and intracellular transport rely on motor proteins. Motors have been studied in detail on the single motor level such that information on their step size, stall force, average run length, and processivity are well known. However, in vivo, motors often work together, so that the question of their collective coordination has raised great interest. Here, we specifically attach motors to giant vesicles and examine collective motor dynamics during membrane tube formation. Image correlation spectroscopy reveals directed motion as processive motors walk at typical speeds (< or = 500 nm/s) along an underlying microtubule and accumulate at the tip of the growing membrane tube. In contrast, nonprocessive motors exhibit purely diffusive behavior, decorating the entire length of a microtubule lattice with diffusion constants at least 1000 times smaller than a freely-diffusing lipid-motor complex in a lipid bilayer (1 microm(2)/s); fluorescence recovery after photobleaching experiments confirm the presence of the slower-moving motor population at the microtubule-membrane tube interface. We suggest that nonprocessive motors dynamically bind and unbind to maintain a continuous interaction with the microtubule. This dynamic and continuous interaction is likely necessary for nonprocessive motors to mediate bidirectional membrane tube dynamics reported previously.
Project description:The transcription factor PU.1 occupies a central role in controlling myeloid and early B-cell development, and its correct lineage-specific expression is critical for the differentiation choice of hematopoietic progenitors. However, little is known of how this tissue-specific pattern is established. We previously identified an upstream regulatory cis element whose targeted deletion in mice decreases PU.1 expression and causes leukemia. We show here that the upstream regulatory cis element alone is insufficient to confer physiologic PU.1 expression in mice but requires the cooperation with other, previously unidentified elements. Using a combination of transgenic studies, global chromatin assays, and detailed molecular analyses we present evidence that PU.1 is regulated by a novel mechanism involving cross talk between different cis elements together with lineage-restricted autoregulation. In this model, PU.1 regulates its expression in B cells and macrophages by differentially associating with cell type-specific transcription factors at one of its cis-regulatory elements to establish differential activity patterns at other elements.
Project description:The threat of viral pandemics demands a comprehensive understanding of evolution at the host-pathogen interface. Here, we show that the accessibility of adaptive mutations in influenza nucleoprotein at fever-like temperatures is mediated by host chaperones. Particularly noteworthy, we observe that the Pro283 nucleoprotein variant, which (1) is conserved across human influenza strains, (2) confers resistance to the Myxovirus resistance protein A (MxA) restriction factor, and (3) critically contributed to adaptation to humans in the 1918 pandemic influenza strain, is rendered unfit by heat shock factor 1 inhibition-mediated host chaperone depletion at febrile temperatures. This fitness loss is due to biophysical defects that chaperones are unavailable to address when heat shock factor 1 is inhibited. Thus, influenza subverts host chaperones to uncouple the biophysically deleterious consequences of viral protein variants from the benefits of immune escape. In summary, host proteostasis plays a central role in shaping influenza adaptation, with implications for the evolution of other viruses, for viral host switching, and for antiviral drug development.
Project description:Cellulases hydrolyze ?-1,4 glycosidic linkages in cellulose, which are among the most prevalent and stable bonds in Nature. Cellulases comprise many glycoside hydrolase families and exist as processive or nonprocessive enzymes. Product inhibition negatively impacts cellulase action, but experimental measurements of product-binding constants vary significantly, and there is little consensus on the importance of this phenomenon. To provide molecular level insights into cellulase product inhibition, we examine the impact of product binding on processive and nonprocessive cellulases by calculating the binding free energy of cellobiose to the product sites of catalytic domains of processive and nonprocessive enzymes from glycoside hydrolase families 6 and 7. The results suggest that cellobiose binds to processive cellulases much more strongly than nonprocessive cellulases. We also predict that the presence of a cellodextrin bound in the reactant site of the catalytic domain, which is present during enzymatic catalysis, has no effect on product binding in nonprocessive cellulases, whereas it significantly increases product binding to processive cellulases. This difference in product binding correlates with hydrogen bonding between the substrate-side ligand and the cellobiose product in processive cellulase tunnels and the additional stabilization from the longer tunnel-forming loops. The hydrogen bonds between the substrate- and product-side ligands are disrupted by water in nonprocessive cellulase clefts, and the lack of long tunnel-forming loops results in lower affinity of the product ligand. These findings provide new insights into the large discrepancies reported for binding constants for cellulases and suggest that product inhibition will vary significantly based on the amount of productive binding for processive cellulases on cellulose.