Lost in translation: Threonine dictates aggregation of CAT tails.

In this issue of Molecular Cell, Chang et al. elaborate on the mechanisms by which CAT tail aggregation is mediated through threonine residues and how it contributes to the perturbation of proteostasis.

Genome-wide screen identifies new set of genes for improved heterologous laccase expression in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is widely used as a host cell for recombinant protein production due to its fast growth, cost-effective culturing, and ability to secrete large and complex proteins. However, one major drawback is the relatively low yield of produced proteins compared to other host systems. To address this issue, we developed an overlay assay to screen the yeast knockout collection and identify mutants that enhance recombinant protein production, specifically focusing on the secretion of the Trametes trogii fungal laccase enzyme.

Shift of the insoluble content of the proteome in the aging mouse brain.

During aging, the cellular response to unfolded proteins is believed to decline, resulting in diminished proteostasis. In model organisms, such as proteostatic decline with age has been linked to proteome solubility shifts and the onset of protein aggregation. However, this correlation has not been extensively characterized in aging mammals.

Comparison of Biomolecular Condensate Localization and Protein Phase Separation Predictors.

Research in the field of biochemistry and cellular biology has entered a new phase due to the discovery of phase separation driving the formation of biomolecular condensates, or membraneless organelles, in cells. The implications of this novel principle of cellular organization are vast and can be applied at multiple scales, spawning exciting research questions in numerous directions. Of fundamental importance are the molecular mechanisms that underly biomolecular condensate formation within cells and whether insights gained into these mechanisms provide a gateway for accurate predictions of protein phase behavior.

Identification of newly translated thermo-sensitive proteins using pulse SILAC mass spectrometry and the GAL promoter system.

Some newly translated proteins are more susceptible to misfolding and aggregation upon heat shock in comparison to other proteins. To study these newly translated thermo-sensitive proteins on a proteomic scale, we present here a protocol that combines pulse-SILAC with biochemical fractionation for mass spectrometry analysis, followed by an orthogonal validation protocol for selected candidates using the GAL promoter system in Saccharomyces cerevisiae. This approach can be further developed to study other stresses and specific post-translational modifications or adapted to mammalian cells.

Pulse labeling reveals the tail end of protein folding by proteome profiling.

Accurate and efficient folding of nascent protein sequences into their native states requires support from the protein homeostasis network. Herein we probe which newly translated proteins are thermo-sensitive, making them susceptible to misfolding and aggregation under heat stress using pulse-SILAC mass spectrometry. We find a distinct group of proteins that is highly sensitive to this perturbation when newly synthesized but not once matured.

Protein interaction networks in neurodegenerative diseases: From physiological function to aggregation.

The accumulation of protein inclusions is linked to many neurodegenerative diseases that typically develop in older individuals, due to a combination of genetic and environmental factors. In rare familial neurodegenerative disorders, genes encoding for aggregation-prone proteins are often mutated. While the underlying mechanism leading to these diseases still remains to be fully elucidated, efforts in the past 20 years revealed a vast network of protein-protein interactions that play a major role in regulating the aggregation of key proteins associated with neurodegeneration.

Isolation of Detergent Insoluble Proteins from Mouse Brain Tissue for Quantitative Analysis Using Data Independent Acquisition (DIA).

Enrichment of detergent insoluble proteins is a commonly used technique for analyzing proteins that may be aggregating in disease or with age. However, various methods for enriching for these proteins are used. Here we present a method using a mild detergent (Triton X-100) and high centrifugation speed (20,000 × g) allowing for sufficient protein extraction and enrichment for large protein assemblies.

GraPES: The Granule Protein Enrichment Server for prediction of biological condensate constituents.

Phase separation-based condensate formation is a novel working paradigm in biology, helping to rationalize many important cellular phenomena including the assembly of membraneless organelles. Uncovering the functional impact of cellular condensates requires a better knowledge of these condensates' constituents. Herein, we introduce the webserver GraPES (Granule Protein Enrichment Server), a user-friendly online interface containing the MaGS and MaGSeq predictors, which provide propensity scores for proteins' localization into cellular condensates.

Protein feature analysis of heat shock induced ubiquitination sites reveals preferential modification site localization.

Protein aggregation is indicative of failing protein quality control systems. These systems are responsible for the refolding or degradation of aberrant and misfolded proteins. Heat stress can cause proteins to misfold, triggering cellular responses including a marked increase in the ubiquitination of proteins.

Proteomic analysis reveals the direct recruitment of intrinsically disordered regions to stress granules in .

Stress granules (SGs) are stress-induced membraneless condensates that store non-translating mRNA and stalled translation initiation complexes. Although metazoan SGs are dynamic compartments where proteins can rapidly exchange with their surroundings, yeast SGs seem largely static. To gain a better understanding of yeast SGs, we identified proteins that sediment after heat shock using mass spectrometry.

Protein Aggregation Triggered by Rewired Protein Homeostasis during Neuronal Differentiation.

In this issue of Molecular Cell, Vonk et al. (2020) and Thiruvalluvan et al. (2020) identify key chaperones that confer resistance to protein aggregation in neural stem cells and become reduced upon differentiation.

Distinct Features of Stress Granule Proteins Predict Localization in Membraneless Organelles.

Recently generated proteomic data provides unprecedented insight into stress granule composition and stands as fruitful ground for further analysis. Stress granules are stress-induced biological assemblies that are of keen interest due to being linked to both long-term cell viability and a variety of protein aggregation-based diseases. Herein, we compile recently published stress granule composition data, formed specifically through heat and oxidative stress, for both mammalian (Homo sapiens) and yeast (Saccharomyces cerevisiae) cells.

Unfolding or aggregation, that is the question.

Cellular processes accompanying protein aggregation are diverse and entangled, making it difficult to investigate the underlying molecular processes in a time-resolved way. Gottlieb, Thompson, and colleagues address this shortcoming using a chemical biology approach to monitor ubiquitination within the first 10 min after the initiation of protein aggregation. Intriguingly, unfolding rather than aggregation seems to trigger the observed events.

Phase separation and clustering of an ABC transporter in .

Phase separation drives numerous cellular processes, ranging from the formation of membrane-less organelles to the cooperative assembly of signaling proteins. Features such as multivalency and intrinsic disorder that enable condensate formation are found not only in cytosolic and nuclear proteins, but also in membrane-associated proteins. The ABC transporter Rv1747, which is important for () growth in infected hosts, has a cytoplasmic regulatory module consisting of 2 phosphothreonine-binding Forkhead-associated domains joined by an intrinsically disordered linker with multiple phospho-acceptor threonines.

Prion-dependent proteome remodeling in response to environmental stress is modulated by prion variant and genetic background.

A number of fungal proteins are capable of adopting multiple alternative, self-perpetuating prion conformations. These prion variants are associated with functional alterations of the prion-forming protein and thus the generation of new, heritable traits that can be detrimental or beneficial. Here we sought to determine the extent to which the previously-reported ZnCl-sensitivity trait of yeast harboring the [PSI] prion is modulated by genetic background and prion variant, and whether this trait is accompanied by prion-dependent proteomic changes that could illuminate its physiological basis.

Exploring the Rampant Expansion of Ubiquitin Proteomics.

The ubiquitin proteasome system can arguably affect all cellular proteins with few exceptions. In addition to regulating many pathways such as cell cycle progression, inflammation, gene expression, DNA repair, and vesicle trafficking-to just name a few-ubiquitination can occur to any nascent or newly translated protein that misfolds. In the past years, substantial progress has been achieved in advancing our global understanding of the ubiquitinome-the ensemble of ubiquitinated proteins within a cell-using mass spectrometry-based proteomics.

A Method to Monitor Protein Turnover by Flow Cytometry and to Screen for Factors that Control Degradation by Fluorescence-Activated Cell Sorting.

The protein quality control network consists of multiple proteins or protein complexes that monitor proteome integrity by mediating protein folding and the removal of proteins that cannot be folded. An integral component of this network is the ubiquitin-proteasome system, which controls the degradation of thousands of cellular proteins. A number of questions remain unanswered regarding the degradation of misfolded proteins.

Insulin-like growth factor 1 receptor stabilizes the ETV6-NTRK3 chimeric oncoprotein by blocking its KPC1/Rnf123-mediated proteasomal degradation.

Many oncogenes, including chimeric oncoproteins, require insulin-like growth factor 1 receptor (IGF1R) for promoting cell transformation. The ETS variant 6 (ETV6)-neurotrophic receptor tyrosine kinase 3 (NTRK3) (EN) chimeric tyrosine kinase is expressed in mesenchymal, epithelial, and hematopoietic cancers and requires the IGF1R axis for transformation. However, current models of IGF1R-mediated EN activation are lacking mechanistic detail.

Elucidation of the 14-3-3ζ interactome reveals critical roles of RNA-splicing factors during adipogenesis.

Adipogenesis involves a complex signaling network requiring strict temporal and spatial organization of effector molecules. Molecular scaffolds, such as 14-3-3 proteins, facilitate such organization, and we have previously identified 14-3-3ζ as an essential scaffold in adipocyte differentiation. The interactome of 14-3-3ζ is large and diverse, and it is possible that novel adipogenic factors may be present within it, but this possibility has not yet been tested.

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.

Recurrent background mutations in WHI2 impair proteostasis and degradation of misfolded cytosolic proteins in Saccharomyces cerevisiae.

Proteostasis promotes viability at both the cellular and organism levels by maintaining a functional proteome. This requires an intricate protein quality control (PQC) network that mediates protein folding by molecular chaperones and removes terminally misfolded proteins via the ubiquitin proteasome system and autophagy. How changes within the PQC network can perturb proteostasis and shift the balance between protein folding and proteolysis remain poorly understood.

Deubiquitinase activity is required for the proteasomal degradation of misfolded cytosolic proteins upon heat-stress.

Elimination of misfolded proteins is crucial for proteostasis and to prevent proteinopathies. Nedd4/Rsp5 emerged as a major E3-ligase involved in multiple quality control pathways that target misfolded plasma membrane proteins, aggregated polypeptides and cytosolic heat-induced misfolded proteins for degradation. It remained unclear how in one case cytosolic heat-induced Rsp5 substrates are destined for proteasomal degradation, whereas other Rsp5 quality control substrates are otherwise directed to lysosomal degradation.