Single-tissue proteomics in reveals proteins resident in intestinal lysosome-related organelles.

The nematode intestine is the primary site for nutrient uptake and storage as well as the synthesis of biomolecules; lysosome-related organelles known as gut granules are important for many of these functions. Aspects of intestine biology are not well understood, including the export of the nutrients it imports and the molecules it synthesizes, as well as the complete functions and protein content of the gut granules. Here, we report a mass spectrometry (MS)-based proteomic analysis of the intestine of the and of its gut granules.

The fatty liver disease-causing protein PNPLA3-I148M alters lipid droplet-Golgi dynamics.

Nonalcoholic fatty liver disease, recently renamed metabolic dysfunction-associated steatotic liver disease (MASLD), is a progressive metabolic disorder that begins with aberrant triglyceride accumulation in the liver and can lead to cirrhosis and cancer. A common variant in the gene , encoding the protein PNPLA3-I148M, is the strongest known genetic risk factor for MASLD. Despite its discovery 20 y ago, the function of PNPLA3, and now the role of PNPLA3-I148M, remain unclear.

The fatty liver disease-causing protein PNPLA3-I148M alters lipid droplet-Golgi dynamics.

Non-alcoholic fatty liver disease (NAFLD), recently renamed metabolic dysfunction-associated steatotic liver disease (MASLD), is a progressive metabolic disorder that begins with aberrant triglyceride accumulation in the liver and can lead to cirrhosis and cancer. A common variant in the gene , encoding the protein PNPLA3-I148M, is the strongest known genetic risk factor for MASLD to date. Despite its discovery twenty years ago, the function of PNPLA3, and now the role of PNPLA3-I148M, remain unclear.

Acquired resistance to KRAS G12C small-molecule inhibitors via genetic/nongenetic mechanisms in lung cancer.

Inherent or acquired resistance to sotorasib poses a substantialt challenge for NSCLC treatment. Here, we demonstrate that acquired resistance to sotorasib in isogenic cells correlated with increased expression of integrin β4 (ITGB4), a component of the focal adhesion complex. Silencing ITGB4 in tolerant cells improved sotorasib sensitivity, while overexpressing ITGB4 enhanced tolerance to sotorasib by supporting AKT-mTOR bypass signaling.

Pervasive SUMOylation of heterochromatin and piRNA pathway proteins.

Genome regulation involves complex protein interactions that are often mediated through post-translational modifications (PTMs). SUMOylation-modification by the small ubiquitin-like modifier (SUMO)-has been implicated in numerous essential processes in eukaryotes. In , SUMO is required for viability and fertility, with its depletion from ovaries leading to heterochromatin loss and ectopic transposon and gene activation.

Functional glycoproteomics by integrated network assembly and partitioning.

The post-translational modification (PTM) of proteins by O-linked β--acetyl-D-glucosamine (O-GlcNAcylation) is widespread across the proteome during the lifespan of all multicellular organisms. However, nearly all functional studies have focused on individual protein modifications, overlooking the multitude of simultaneous O-GlcNAcylation events that work together to coordinate cellular activities. Here, we describe etworking of nteractors and ubstrats (NISE), a novel, systems-level approach to rapidly and comprehensively monitor O-GlcNAcylation across the proteome.

Quantitative measurement of the requirement of diverse protein degradation pathways in MHC class I peptide presentation.

Peptides from degradation of intracellular proteins are continuously displayed by major histocompatibility complex (MHC) class I. To better understand origins of these peptides, we performed a comprehensive census of the class I peptide repertoire in the presence and absence of ubiquitin-proteasome system (UPS) activity upon developing optimized methodology to enrich for and quantify these peptides. Whereas most class I peptides are dependent on the UPS for their generation, a surprising 30%, enriched in peptides of mitochondrial origin, appears independent of the UPS.

Tidyproteomics: an open-source R package and data object for quantitative proteomics post analysis and visualization.

Background: The analysis of mass spectrometry-based quantitative proteomics data can be challenging given the variety of established analysis platforms, the differences in reporting formats, and a general lack of approachable standardized post-processing analyses such as sample group statistics, quantitative variation and even data filtering. We developed tidyproteomics to facilitate basic analysis, improve data interoperability and potentially ease the integration of new processing algorithms, mainly through the use of a simplified data-object.

Results: The R package tidyproteomics was developed as both a framework for standardizing quantitative proteomics data and a platform for analysis workflows, containing discrete functions that can be connected end-to-end, thus making it easier to define complex analyses by breaking them into small stepwise units.

secreted cytotoxins are competition sensing signals for .

Coinfection with two notorious opportunistic pathogens, the Gram-negative and Gram-positive , dominates chronic pulmonary infections. While coinfection is associated with poor patient outcomes, the interspecies interactions responsible for such decline remain unknown. Here, we dissected molecular mechanisms of interspecies sensing between and .

Mechanisms of chlorate toxicity and resistance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that often encounters hypoxic/anoxic environments within the host, which increases its tolerance to many conventional antibiotics. Toward identifying novel treatments, we explored the therapeutic potential of chlorate, a pro-drug that kills hypoxic/anoxic, antibiotic-tolerant P. aeruginosa populations.

MTCH2 is a mitochondrial outer membrane protein insertase.

In the mitochondrial outer membrane, α-helical transmembrane proteins play critical roles in cytoplasmic-mitochondrial communication. Using genome-wide CRISPR screens, we identified mitochondrial carrier homolog 2 (MTCH2), and its paralog MTCH1, and showed that it is required for insertion of biophysically diverse tail-anchored (TA), signal-anchored, and multipass proteins, but not outer membrane β-barrel proteins. Purified MTCH2 was sufficient to mediate insertion into reconstituted proteoliposomes.

Adaptive exchange sustains cullin-RING ubiquitin ligase networks and proper licensing of DNA replication.

Cop9 signalosome (CSN) regulates the function of cullin-RING E3 ubiquitin ligases (CRLs) by deconjugating the ubiquitin-like protein NEDD8 from the cullin subunit. To understand the physiological impact of CSN function on the CRL network and cell proliferation, we combined quantitative mass spectrometry and genome-wide CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) screens to identify factors that modulate cell viability upon inhibition of CSN by the small molecule CSN5i-3. CRL components and regulators strongly modulated the antiproliferative effects of CSN5i-3, and in addition we found two pathways involved in genome integrity, SCF-APC/C-GMNN and CUL4-SETD8, that contribute substantially to the toxicity of CSN inhibition.

System-wide analyses reveal essential roles of N-terminal protein modification in bacterial membrane integrity.

The removal of the N-terminal formyl group on nascent proteins by peptide deformylase (PDF) is the most prevalent protein modification in bacteria. PDF is a critical target of antibiotic development; however, its role in bacterial physiology remains a long-standing question. This work used the time-resolved analyses of the translatome and proteome to investigate the consequences of PDF inhibition.

Surface cysteines could protect the SARS-CoV-2 main protease from oxidative damage.

The SARS-CoV-2 main protease (M) is responsible for cleaving twelve nonstructural proteins from the viral polyprotein. M, a cysteine protease, is characterized by a large number of noncatalytic cysteine (Cys) residues, none involved in disulfide bonds. In the absence of a tertiary-structure stabilizing role for these residues, a possible alternative is that they are involved in redox processes.

A proteome-wide map of 20(S)-hydroxycholesterol interactors in cell membranes.

Oxysterols (OHCs) are hydroxylated cholesterol metabolites that play ubiquitous roles in health and disease. Due to the non-covalent nature of their interactions and their unique partitioning in membranes, the analysis of live-cell, proteome-wide interactions of OHCs remains an unmet challenge. Here, we present a structurally precise chemoproteomics probe for the biologically active molecule 20(S)-hydroxycholesterol (20(S)-OHC) and provide a map of its proteome-wide targets in the membranes of living cells.

Amino Acid Analog Induces Stress Response in Marine .

Characterizing the cell-level metabolic trade-offs that phytoplankton exhibit in response to changing environmental conditions is important for predicting the impact of these changes on marine food web dynamics and biogeochemical cycling. The time-selective proteome-labeling approach, bioorthogonal noncanonical amino acid tagging (BONCAT), has potential to provide insight into differential allocation of resources at the cellular level, especially when coupled with proteomics. However, the application of this technique in marine phytoplankton remains limited.

LONP1 and mtHSP70 cooperate to promote mitochondrial protein folding.

Most mitochondrial precursor polypeptides are imported from the cytosol into the mitochondrion, where they must efficiently undergo folding. Mitochondrial precursors are imported as unfolded polypeptides. For proteins of the mitochondrial matrix and inner membrane, two separate chaperone systems, HSP60 and mitochondrial HSP70 (mtHSP70), facilitate protein folding.

Enzyme Replacement Therapy for Mucopolysaccharidosis IIID using Recombinant Human α--Acetylglucosamine-6-Sulfatase in Neonatal Mice.

There is currently no cure or effective treatment available for mucopolysaccharidosis type IIID (MPS IIID, Sanfilippo syndrome type D), a lysosomal storage disorder (LSD) caused by the deficiency of α--acetylglucosamine-6-sulfatase (GNS). The clinical symptoms of MPS IIID, like other subtypes of Sanfilippo syndrome, are largely localized to the central nervous system (CNS), and any treatments aiming to ameliorate or reverse the catastrophic and fatal neurologic decline caused by this disease need to be delivered across the blood-brain barrier. Here, we report a proof-of-concept enzyme replacement therapy (ERT) for MPS IIID using recombinant human α--acetylglucosamine-6-sulfatase (rhGNS) intracerebroventricular (ICV) delivery in a neonatal MPS IIID mouse model.

Activin-A limits Th17 pathogenicity and autoimmune neuroinflammation via CD39 and CD73 ectonucleotidases and Hif1-α-dependent pathways.

In multiple sclerosis (MS), Th17 cells are critical drivers of autoimmune central nervous system (CNS) inflammation and demyelination. Th17 cells exhibit functional heterogeneity fostering both pathogenic and nonpathogenic, tissue-protective functions. Still, the factors that control Th17 pathogenicity remain incompletely defined.

Nano volume fractionation strategy for dilute-and-shoot injections in off-line loss-less proteomic workflows for extensive protein identifications of ultra-low sample amounts.

A proteomic workflow for a simple loss-less manual nano-fractionation (300 nL/fraction) for low µg sample amounts which avoids the need to dry down or transfer fractions to autosampler vials is shown to be feasible. It is demonstrated that the conventional procedure of drying samples down followed by reconstitution negatively affects the number of protein and peptide identifications. Furthermore, these losses seem to disproportionately affect hydrophobic peptides from the drying down and reconstitution step.

Sperm can act as vectors for HIV-1 transmission into vaginal and cervical epithelial cells.

Problem: Sperm are the major cells in semen. Human sperm possess a number of HIV-1 gp120 binding ligands including sulfogalactosylglycerolipid (SGG). However, the mechanisms of how sperm capture HIV-1 onto their surface are unclear.

Identification and characterization of β-sitosterol target proteins.

β-Sitosterol is the most abundant plant sterol in the human diet. It is also the major component of several traditional medicines, including saw palmetto and devil's claw. Although β-sitosterol is effective against enlarged prostate in human clinical trials and has anti-cancer and anti-inflammatory activities, the mechanisms of action are poorly understood.

Drug affinity responsive target stability (DARTS) for small-molecule target identification.

Drug affinity responsive target stability (DARTS) is a relatively quick and straightforward approach to identify potential protein targets for small molecules. It relies on the protection against proteolysis conferred on the target protein by interaction with a small molecule. The greatest advantage of this method is being able to use the native small molecule without having to immobilize or modify it (e.

The metabolite α-ketoglutarate extends lifespan by inhibiting ATP synthase and TOR.

Metabolism and ageing are intimately linked. Compared with ad libitum feeding, dietary restriction consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits.