Characterization of p97 mutations causing multisystem proteinopathy support a gain-of-function model for pathology

Valosin‐containing protein (VCP, or p97) is an ATPase essential in numerous protein quality control (PQC) pathways, such as ER‐associated degradation. p97 functions as a segregase, extracting ubiquitylated proteins from membranes or complexes so they can be degraded by the proteasome. However, the complexity of native p97 PQC substrates has stymied the detailed biochemical study of this function. Previously, to address this problem, we developed an in vitro p97 substrate based on an ubiquitin fusion degradation (UFD) pathway substrate, Ub‐GFP, and showed that the unfolding of this substrate by p97 is dependent upon extensive substrate ubiquitylation, the p97 adaptors NPLOC4‐UFD1L, and ATP hydrolysis. Here, we make use of this system, employing an updated version of this substrate, to explore how mutations in p97 that cause multisystem proteinopathy (MSP) affect substrate processing. Previous studies have shown that MSP mutants have higher basal ATP rates than wild type yet cause deficiencies in many p97‐dependent pathways, creating controversy as to whether these dominantly inherited mutations cause disease through a gain‐of‐function or a loss‐of‐function. We have now analyzed seven distinct MSP mutants, all of which showed modestly improved unfolding of our model substrate over wild type p97, providing evidence that the increased ATPase activity leads to a gain‐of‐function. Furthermore, we showed evidence that p97 inhibitors may restore proper p97 function to MSP mutants, suggesting a potential treatment strategy for p97 diseases

From a microscopic solution to a continuum description of active particles with a recoil interaction in one dimension

We consider a model system of persistent random walkers that can jam, pass through each other or jump apart (recoil) on contact. In a continuum limit, where particle motion between stochastic changes in direction becomes deterministic, we find that the stationary inter-particle distribution functions are governed by an inhomogeneous fourth-order differential equation. Our main focus is on determining the boundary conditions that these distribution functions should satisfy. We find that these do not arise naturally from physical considerations, but need to be carefully matched to functional forms that arise from the analysis of an underlying discrete process. The inter-particle distribution functions, or their first derivatives, are generically found to be discontinuous at the boundaries.Comment: 16 pages; 5 figures; published in PR

Combinatorial mappings of exclusion processes

We review various combinatorial interpretations and mappings of stationary-state probabilities of the totally asymmetric, partially asymmetric and symmetric simple exclusion processes (TASEP, PASEP, SSEP respectively). In these steady states, the statistical weight of a configuration is determined from a matrix product, which can be written explicitly in terms of generalised ladder operators. This lends a natural association to the enumeration of random walks with certain properties. Specifically, there is a one-to-many mapping of steady-state configurations to a larger state space of discrete paths, which themselves map to an even larger state space of number permutations. It is often the case that the configuration weights in the extended space are of a relatively simple form (e.g., a Boltzmann-like distribution). Meanwhile, various physical properties of the nonequilibrium steady state - such as the entropy - can be interpreted in terms of how this larger state space has been partitioned. These mappings sometimes allow physical results to be derived very simply, and conversely the physical approach allows some new combinatorial problems to be solved. This work brings together results and observations scattered in the combinatorics and statistical physics literature, and also presents new results. The review is pitched at statistical physicists who, though not professional combinatorialists, are competent and enthusiastic amateurs.Comment: 56 pages, 21 figure

Exact joint density-current probability function for the asymmetric exclusion process

We study the asymmetric exclusion process with open boundaries and derive the exact form of the joint probability function for the occupation number and the current through the system. We further consider the thermodynamic limit, showing that the resulting distribution is non-Gaussian and that the density fluctuations have a discontinuity at the continuous phase transition, while the current fluctuations are continuous. The derivations are performed by using the standard operator algebraic approach, and by the introduction of new operators satisfying a modified version of the original algebra.Comment: 4 pages, 3 figure

Exact probability function for bulk density and current in the asymmetric exclusion process

We examine the asymmetric simple exclusion process with open boundaries, a paradigm of driven diffusive systems, having a nonequilibrium steady state transition. We provide a full derivation and expanded discussion and digression on results previously reported briefly in M. Depken and R. Stinchcombe, Phys. Rev. Lett. {\bf 93}, 040602, (2004). In particular we derive an exact form for the joint probability function for the bulk density and current, both for finite systems, and also in the thermodynamic limit. The resulting distribution is non-Gaussian, and while the fluctuations in the current are continuous at the continuous phase transitions, the density fluctuations are discontinuous. The derivations are done by using the standard operator algebraic techniques, and by introducing a modified version of the original operator algebra. As a byproduct of these considerations we also arrive at a novel and very simple way of calculating the normalization constant appearing in the standard treatment with the operator algebra. Like the partition function in equilibrium systems, this normalization constant is shown to completely characterize the fluctuations, albeit in a very different manner.Comment: 10 pages, 4 figure

Characterization of p97 mutations causing multisystem proteinopathy support a gain-of-function model for pathology

Valosin‐containing protein (VCP, or p97) is an ATPase essential in numerous protein quality control (PQC) pathways, such as ER‐associated degradation. p97 functions as a segregase, extracting ubiquitylated proteins from membranes or complexes so they can be degraded by the proteasome. However, the complexity of native p97 PQC substrates has stymied the detailed biochemical study of this function. Previously, to address this problem, we developed an in vitro p97 substrate based on an ubiquitin fusion degradation (UFD) pathway substrate, Ub‐GFP, and showed that the unfolding of this substrate by p97 is dependent upon extensive substrate ubiquitylation, the p97 adaptors NPLOC4‐UFD1L, and ATP hydrolysis. Here, we make use of this system, employing an updated version of this substrate, to explore how mutations in p97 that cause multisystem proteinopathy (MSP) affect substrate processing. Previous studies have shown that MSP mutants have higher basal ATP rates than wild type yet cause deficiencies in many p97‐dependent pathways, creating controversy as to whether these dominantly inherited mutations cause disease through a gain‐of‐function or a loss‐of‐function. We have now analyzed seven distinct MSP mutants, all of which showed modestly improved unfolding of our model substrate over wild type p97, providing evidence that the increased ATPase activity leads to a gain‐of‐function. Furthermore, we showed evidence that p97 inhibitors may restore proper p97 function to MSP mutants, suggesting a potential treatment strategy for p97 diseases

Transcriptional landscape of Aspergillus niger at breaking of conidial dormancy revealed by RNA-sequencing

Background Genome-wide analysis was performed to assess the transcriptional landscape of germinating A. niger conidia using both next generation RNA-sequencing and GeneChips. The metabolism of storage compounds during conidial germination was also examined and compared to the transcript levels from associated genes. Results The transcriptome of dormant conidia was shown to be highly differentiated from that of germinating conidia and major changes in response to environmental shift occurred within the first hour of germination. The breaking of dormancy was associated with increased transcript levels of genes involved in the biosynthesis of proteins, RNA turnover and respiratory metabolism. Increased transcript levels of genes involved in metabolism of nitrate at the onset of germination implies its use as a source of nitrogen. The transcriptome of dormant conidia contained a significant component of antisense transcripts that changed during germination. Conclusion Dormant conidia contained transcripts of genes involved in fermentation, gluconeogenesis and the glyoxylate cycle. The presence of such transcripts in dormant conidia may indicate the generation of energy from non-carbohydrate substrates during starvation-induced conidiation or for maintenance purposes during dormancy. The immediate onset of metabolism of internal storage compounds after the onset of germination, and the presence of transcripts of relevant genes, suggest that conidia are primed for the onset of germination. For some genes, antisense transcription is regulated in the transition from resting conidia to fully active germinants

Multisystem Proteinopathy Mutations in VCP/p97 Increase NPLOC4·UFD1L Binding and Substrate Processing

Valosin-containing protein (VCP)/p97 is an essential ATP-dependent protein unfoldase. Dominant mutations in p97 cause multisystem proteinopathy (MSP), a disease affecting the brain, muscle, and bone. Despite the identification of numerous pathways that are perturbed in MSP, the molecular-level defects of these p97 mutants are not completely understood. Here, we use biochemistry and cryoelectron microscopy to explore the effects of MSP mutations on the unfoldase activity of p97 in complex with its substrate adaptor NPLOC4⋅UFD1L (UN). We show that all seven analyzed MSP mutants unfold substrates faster. Mutant homo- and heterohexamers exhibit tighter UN binding and faster substrate processing. Our structural studies suggest that the increased UN affinity originates from a decoupling of p97's nucleotide state and the positioning of its N-terminal domains. Together, our data support a gain-of-function model for p97-UN-dependent processes in MSP and underscore the importance of N-terminal domain movements for adaptor recruitment and substrate processing by p97

Multisystem Proteinopathy Mutations in VCP/p97 Increase NPLOC4·UFD1L Binding and Substrate Processing

Valosin-containing protein (VCP)/p97 is an essential ATP-dependent protein unfoldase. Dominant mutations in p97 cause multisystem proteinopathy (MSP), a disease affecting the brain, muscle, and bone. Despite the identification of numerous pathways that are perturbed in MSP, the molecular-level defects of these p97 mutants are not completely understood. Here, we use biochemistry and cryoelectron microscopy to explore the effects of MSP mutations on the unfoldase activity of p97 in complex with its substrate adaptor NPLOC4⋅UFD1L (UN). We show that all seven analyzed MSP mutants unfold substrates faster. Mutant homo- and heterohexamers exhibit tighter UN binding and faster substrate processing. Our structural studies suggest that the increased UN affinity originates from a decoupling of p97's nucleotide state and the positioning of its N-terminal domains. Together, our data support a gain-of-function model for p97-UN-dependent processes in MSP and underscore the importance of N-terminal domain movements for adaptor recruitment and substrate processing by p97

A framework for deriving semantic web services

Web service-based development represents an emerging approach for the development of distributed information systems. Web services have been mainly applied by software practitioners as a means to modularize system functionality that can be offered across a network (e.g., intranet and/or the Internet). Although web services have been predominantly developed as a technical solution for integrating software systems, there is a more business-oriented aspect that developers and enterprises need to deal with in order to benefit from the full potential of web services in an electronic market. This ‘ignored’ aspect is the representation of the semantics underlying the services themselves as well as the ‘things’ that the services manage. Currently languages like the Web Services Description Language (WSDL) provide the syntactic means to describe web services, but lack in providing a semantic underpinning. In order to harvest all the benefits of web services technology, a framework has been developed for deriving business semantics from syntactic descriptions of web services. The benefits of such a framework are two-fold. Firstly, the framework provides a way to gradually construct domain ontologies from previously defined technical services. Secondly, the framework enables the migration of syntactically defined web services toward semantic web services. The study follows a design research approach which (1) identifies the problem area and its relevance from an industrial case study and previous research, (2) develops the framework as a design artifact and (3) evaluates the application of the framework through a relevant scenario