Joerg Gsponer

Classification of Intrinsically Disordered Regions and Proteins.

1.1. Uncharacterized Protein Segments Are a Source of Functional Novelty Over the past decade, we have observed a massive increase in the amount of information describing protein sequences from a variety of organisms.1,2 While this may reflect the diversity in sequence space, and possibly also in function space,3 a large proportion of the sequences lacks any useful function annotation.4,5 Often these sequences are annotated as putative or hypothetical proteins, and for the majority their functions still remain unknown.6,7 Suggestions about potential protein function, primarily molecular function, often come from computational analysis of their sequences. For instance, homology detection allows for the transfer of information from well-characterized protein segments to those with similar sequences that lack annotation of molecular function.8−10 Other aspects of function, such as the biological processes proteins participate in, may come from genetic- and disease-association studies, expression and interaction network data, and comparative genomics approaches that investigate genomic context.11−17 Characterization of unannotated and uncharacterized protein segments is expected to lead to the discovery of novel functions as well as provide important insights into existing biological processes. In addition, it is likely to shed new light on molecular mechanisms of diseases that are not yet fully understood. Thus, uncharacterized protein segments are likely to be a large source of functional novelty relevant for discovering new biology.

A high-throughput approach for measuring temporal changes in the interactome.

Interactomes are often measured using affinity purification–mass spectrometry (AP-MS) or yeast two-hybrid approaches, but these methods do not provide stoichiometric or temporal information. We combine quantitative proteomics and size-exclusion chromatography to map 291 coeluting complexes. This method allows mapping of an interactome to the same depth and accuracy as AP-MS with less work and without overexpression or tagging. The use of triplex labeling enables monitoring of interactome rearrangements.

Protein synthesis rate is the predominant regulator of protein expression during differentiation

External perturbations, by forcing cells to adapt to a new environment, often elicit large‐scale changes in gene expression resulting in an altered proteome that improves the cells fitness in the new conditions. Steady‐state levels of a proteome depend on transcription, the levels of transcripts, translation and protein degradation but system‐level contribution that each of these processes make to the final protein expression change has yet to be explored. We therefore applied a systems biology approach to characterize the regulation of protein expression during cellular differentiation using quantitative proteomics. As a general rule, it seems that protein expression during cellular differentiation is largely controlled by changes in the relative synthesis rate, whereas the relative degradation rate of the majority of proteins stays constant. In these data, we also observe that the proteins in defined sub‐structures of larger protein complexes tend to have highly correlated synthesis and degradation rates but that this does not necessarily extend to the holo‐complex. Finally, we provide strong evidence that the generally poor correlation observed between transcript and protein levels can fully be explained once the protein synthesis and degradation rates are taken into account.

Rsp5/Nedd4 is the main ubiquitin ligase that targets cytosolic misfolded proteins following heat stress

The heat-shock response is a complex cellular program that induces major changes in protein translation, folding and degradation to alleviate toxicity caused by protein misfolding. Although heat shock has been widely used to study proteostasis, it remained unclear how misfolded proteins are targeted for proteolysis in these conditions. We found that Rsp5 and its mammalian homologue Nedd4 are important E3 ligases responsible for the increased ubiquitylation induced by heat stress. We determined that Rsp5 ubiquitylates mainly cytosolic misfolded proteins upon heat shock for proteasome degradation. We found that ubiquitylation of heat-induced substrates requires the Hsp40 co-chaperone Ydj1 that is further associated with Rsp5 upon heat shock. In addition, ubiquitylation is also promoted by PY Rsp5-binding motifs found primarily in the structured regions of stress-induced substrates, which can act as heat-induced degrons. Our results support a bipartite recognition mechanism combining direct and chaperone-dependent ubiquitylation of misfolded cytosolic proteins by Rsp5.

Recognition Pliability Is Coupled to Structural Heterogeneity: A Calmodulin Intrinsically Disordered Binding Region Complex

Protein interactions within regulatory networks should adapt in a spatiotemporal-dependent dynamic environment, in order to process and respond to diverse and versatile cellular signals. However, the principles governing recognition pliability in protein complexes are not well understood. We have investigated a region of the intrinsically disordered protein myelin basic protein (MBP(145-165)) that interacts with calmodulin, but that also promiscuously binds other biomolecules (membranes, modifying enzymes). To characterize this interaction, we implemented an NMR spectroscopic approach that calculates, for each conformation of the complex, the maximum occurrence based on recorded pseudocontact shifts and residual dipolar couplings. We found that the MBP(145-165)-calmodulin interaction is characterized by structural heterogeneity. Quantitative comparative analysis indicated that distinct conformational landscapes of structural heterogeneity are sampled for different calmodulin-target complexes. Such structural heterogeneity in protein complexes could potentially explain the way that transient and promiscuous protein interactions are optimized and tuned in complex regulatory networks.

A defect in ATP‐citrate lyase links acetyl‐CoA production, virulence factor elaboration and virulence in Cryptococcus neoformans

The interaction of Cryptococcus neoformans with phagocytic cells of the innate immune system is a key step in disseminated disease leading to meningoencephalitis in immunocompromised individuals. Transcriptional profiling of cryptococcal cells harvested from cell culture medium or from macrophages found differential expression of metabolic and other functions during fungal adaptation to the intracellular environment. We focused on the ACL1 gene for ATP‐citrate lyase, which converts citrate to acetyl‐CoA, because this gene showed elevated transcript levels in macrophages and because of the importance of acetyl‐CoA as a central metabolite. Mutants lacking ACL1 showed delayed growth on medium containing glucose, reduced cellular levels of acetyl‐CoA, defective production of virulence factors, increased susceptibility to the antifungal drug fluconazole and decreased survival within macrophages. Importantly, acl1 mutants were unable to cause disease in a murine inhalation model, a phenotype that was more extreme than other mutants with defects in acetyl‐CoA production (e.g. an acetyl‐CoA synthetase mutant). Loss of virulence is likely due to perturbation of critical physiological interconnections between virulence factor expression and metabolism in C. neoformans. Phylogenetic analysis and structural modelling of cryptococcal Acl1 identified three indels unique to fungal protein sequences; these differences may provide opportunities for the development of pathogen‐specific inhibitors.

System-wide Analysis Reveals Intrinsically Disordered Proteins Are Prone to Ubiquitylation after Misfolding Stress

Damaged and misfolded proteins that are no longer functional in the cell need to be eliminated. Failure to do so might lead to their accumulation and aggregation, a hallmark of many neurodegenerative diseases. Protein quality control pathways play a major role in the degradation of these proteins, which is mediated mainly by the ubiquitin proteasome system. Despite significant focus on identifying ubiquitin ligases involved in these pathways, along with their substrates, a systems-level understanding of these pathways has been lacking. For instance, as misfolded proteins are rapidly ubiquitylated, unconjugated ubiquitin is rapidly depleted from the cell upon misfolding stress; yet it is unknown whether certain targets compete more efficiently to be ubiquitylated. Using a system-wide approach, we applied statistical and computational methods to identify characteristics enriched among proteins that are further ubiquitylated after heat shock. We discovered that distinct populations of structured and, surprisingly, intrinsically disordered proteins are prone to ubiquitylation. Proteomic analysis revealed that abundant and highly structured proteins constitute the bulk of proteins in the low-solubility fraction after heat shock, but only a portion is ubiquitylated. In contrast, ubiquitylated, intrinsically disordered proteins are enriched in the low-solubility fraction after heat shock. These proteins have a very low abundance in the cell, are rarely encoded by essential genes, and are enriched in binding motifs. In additional experiments, we confirmed that several of the identified intrinsically disordered proteins were ubiquitylated after heat shock and demonstrated for two of them that their disordered regions are important for ubiquitylation after heat shock. We propose that intrinsically disordered regions may be recognized by the protein quality control machinery and thereby facilitate the ubiquitylation of proteins after heat shock.

Topological dissection of the membrane transport protein Mhp1 derived from cysteine accessibility and mass spectrometry

Cys accessibility and quantitative intact mass spectrometry (MS) analyses have been devised to study the topological transitions of Mhp1, the membrane protein for sodium-linked transport of hydantoins from Microbacterium liquefaciens. Mhp1 has been crystallized in three forms (outward-facing open, outward-facing occluded with substrate bound, and inward-facing open). We show that one natural cysteine residue, Cys327, out of three, has an enhanced solvent accessibility in the inward-facing (relative to the outward-facing) form. Reaction of the purified protein, in detergent, with the thiol-reactive N-ethylmalemide (NEM), results in modification of Cys327, suggesting that Mhp1 adopts predominantly inward-facing conformations. Addition of either sodium ions or the substrate 5-benzyl-l-hydantoin (L-BH) does not shift this conformational equilibrium, but systematic co-addition of the two results in an attenuation of labeling, indicating a shift toward outward-facing conformations that can be interpreted using conventional enzyme kinetic analyses. Such measurements can afford the Km for each ligand as well as the stoichiometry of ion–substrate-coupled conformational changes. Mutations that perturb the substrate binding site either result in the protein being unable to adopt outward-facing conformations or in a global destabilization of structure. The methodology combines covalent labeling, mass spectrometry, and kinetic analyses in a straightforward workflow applicable to a range of systems, enabling the interrogation of changes in a protein’s conformation required for function at varied concentrations of substrates, and the consequences of mutations on these conformational transitions.

An atlas of protein-protein interactions across mammalian tissues

Cellular processes arise from the dynamic organization of proteins in networks of physical interactions. Mapping the complete network of biologically relevant protein-protein interactions, the interactome, has therefore been a central objective of high-throughput biology. Yet, because widely used methods for high-throughput interaction discovery rely on heterologous expression or genetically manipulated cell lines, the dynamics of protein interactions across physiological contexts are poorly understood. Here, we use a quantitative proteomic approach combining protein correlation profiling with stable isotope labelling of mammals (PCP SILAM) to map the interactomes of seven mouse tissues. The resulting maps provide the first proteome-scale survey of interactome dynamics across mammalian tissues, revealing over 27,000 unique interactions with an accuracy comparable to the highest-quality human screens. We identify systematic suppression of cross-talk between the evolutionarily ancient housekeeping interactome and younger, tissue-specific modules. Rewiring of protein interactions across tissues is widespread, and is poorly predicted by gene expression or coexpression. Rewired proteins are tightly regulated by multiple cellular mechanisms and implicated in disease. Our study opens up new avenues to uncover regulatory mechanisms that shape in vivo interactome responses to physiological and pathophysiological stimuli in mammalian systems.

The [ PSI + ] yeast prion does not wildly affect proteome composition whereas selective pressure exerted on [ PSI + ] cells can promote aneuploidy

The yeast Sup35 protein is a subunit of the translation termination factor, and its conversion to the [PSI+] prion state leads to more translational read-through. Although extensive studies have been done on [PSI+], changes at the proteomic level have not been performed exhaustively. We therefore used a SILAC-based quantitative mass spectrometry approach and identified 4187 proteins from both [psi−] and [PSI+] strains. Surprisingly, there was very little difference between the two proteomes under standard growth conditions. We found however that several [PSI+] strains harbored an additional chromosome, such as chromosome I. Albeit, we found no evidence to support that [PSI+] induces chromosomal instability (CIN). Instead we hypothesized that the selective pressure applied during the establishment of [PSI+]-containing strains could lead to a supernumerary chromosome due to the presence of the ade1-14 selective marker for translational read-through. We therefore verified that there was no prevalence of disomy among newly generated [PSI+] strains in absence of strong selection pressure. We also noticed that low amounts of adenine in media could lead to higher levels of mitochondrial DNA in [PSI+] in ade1-14 cells. Our study has important significance for the establishment and manipulation of yeast strains with the Sup35 prion.