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The dorsolateral prefrontal cortex (DLPFC) has a crucial role in cognitive functioning and negative symptoms in schizophrenia. However, limited information of altered protein networks is available in this region in schizophrenia. We performed a proteomic analysis using single-shot liquid chromatography-tandem mass spectrometry of grey matter of postmortem DLPFC in chronic schizophrenia subjects (n = 20) and unaffected subjects (n = 20) followed by bioinformatic analysis to identify altered protein networks in schizophrenia (PXD024939 identifier in ProteomeXchange repository). Our results displayed a proteome profile in the DLPFC of 1989 proteins. 43 proteins were found significantly altered in schizophrenia. Analysis of this panel showed an enrichment of biological processes implicated in vesicle-mediated transport, processing and antigen presentation via MHC class II, intracellular transport and selenium metabolism. The enriched identified pathways were MHC class II antigen presentation, vesicle-mediated transport, Golgi ER retrograde transport, Nef mediated CD8 downregulation and the immune system. All these enriched categories were found to be downregulated. Furthermore, our network analyses showed crosstalk between proteins involved in MHC class II antigen presentation, membrane trafficking, Golgi-to-ER retrograde transport, Nef-mediated CD8 downregulation and the immune system with only one module built by 13 proteins. RAB7A showed eight interactions with proteins of all these pathways. Our results provide an altered molecular network involved in immune response in the DLPFC in schizophrenia with a central role of RAB7A. These results suggest that RAB7A or other proteins of this network could be potential targets for novel pharmacological strategies in schizophrenia for improving cognitive and negative symptoms.

This article enquires into states’ obligations to provide remedies for domestic violence when the right-claimants are in a context of precarious, insecure or irregular migration status (“migration status precarity”). Through an exploration of the laws and policies in place in two case studies, Ireland and Spain, this article questions the role of law in ensuring that barriers posed by migration statuses do not hamper women’s access to remedies for intimate partner violence. This article posits that, in the case studies examined, the criminalisation of domestic violence and coercive control does not automatically serve as an avenue to remedies when it intersects with migration status. Rather, it can be a tool of (negative) regulation of women’s right to live a life free from gender-based violence. It argues that the states’ lack of remedying an unequal access to remedies due to hurdles posed by migration statuses creates distinct categories of more or less deserving victims/survivors depending on their citizenship and migration statuses. Received: 24 June 2021 Accepted: 22 February 2022

AbstractTransfer RNAs (tRNAs) are the adaptor molecules required for reading the genetic code and producing proteins. Transfer RNA variants can lead to genome-wide mistranslation, the misincorporation of amino acids not specified by the standard genetic code into nascent proteins. While genome sequencing has identified putative mistranslating transfer RNA variants in human populations, little is known regarding how mistranslation affects multicellular organisms. Here, we create a multicellular model of mistranslation by integrating a serine transfer RNA variant that mistranslates serine for proline ( tRNA UGG,G26A SerDrosophila melanogaster tRNA UGG,G26A Ser tRNA UGG,G26A Ser tRNA UGG,G26A Ser

AbstractMistranslation, the misincorporation of an amino acid not specified by the “standard” genetic code, occurs in all organisms. tRNA variants that increase mistranslation arise spontaneously and engineered tRNAs can achieve mistranslation frequencies approaching 10% in yeast and bacteria. Interestingly, human genomes contain tRNA variants with the potential to mistranslate. Cells cope with increased mistranslation through multiple mechanisms, though high levels cause proteotoxic stress. The goal of this study was to compare the genetic interactions and the impact on transcriptome and cellular growth of two tRNA variants that mistranslate at a similar frequency but create different amino acid substitutions in Saccharomyces cerevisiae.

Data-independent acquisition (DIA)-based proteomics often relies on mass spectrum libraries from data-dependent acquisition experiments. Here, the authors present a method to generate DIA-based chromatogram libraries, enabling DIA-only workflows and detecting more peptides than with spectrum libraries alone.

Alterations in the cortico-cerebellar-thalamic-cortical circuit might underlie the diversity of symptoms in schizophrenia. However, molecular changes in cerebellar neuronal circuits, part of this network, have not yet been fully determined. Using LC-MS/MS, we screened altered candidates in pooled grey matter of cerebellum from schizophrenia subjects who committed suicide (n = 4) and healthy individuals (n = 4). Further validation by immunoblotting of three selected candidates was performed in two cohorts comprising schizophrenia (n = 20), non-schizophrenia suicide (n = 6) and healthy controls (n = 21). We found 99 significantly altered proteins, 31 of them previously reported in other brain areas by proteomic studies. Transport function was the most enriched category, while cell communication was the most prevalent function. For validation, we selected the vacuolar proton pump subunit 1 (VPP1), from transport, and two EF-hand calcium-binding proteins, calmodulin and parvalbumin, from cell communication. All candidates showed significant changes in schizophrenia (n = 7) compared to controls (n = 7). VPP1 was altered in the non-schizophrenia suicide group and increased levels of parvalbumin were linked to antipsychotics. Further validation in an independent cohort of non-suicidal chronic schizophrenia subjects (n = 13) and non-psychiatric controls (n = 14) showed that parvalbumin was increased, while calmodulin was decreased in schizophrenia. Our findings provide evidence of calcium-binding protein dysregulation in the cerebellum in schizophrenia, suggesting an impact on normal calcium-dependent synaptic functioning of cerebellar circuits. Our study also links VPP1 to suicide behaviours, suggesting a possible impairment in vesicle neurotransmitter refilling and release in these phenotypes.

Abstract Recent developments in proteomics have enabled signaling studies where > 10,000 phosphosites can be routinely identified and quantified. Yet, current analyses are limited in throughput, reproducibility, and robustness, hampering experiments that involve multiple perturbations, such as those needed to map kinase–substrate relationships, capture pathway crosstalks, and network inference analysis. To address these challenges, we introduce rapid‐robotic phosphoproteomics (R2‐P2), an end‐to‐end automated method that uses magnetic particles to process protein extracts to deliver mass spectrometry‐ready phosphopeptides. R2‐P2 is rapid, robust, versatile, and high‐throughput. To showcase the method, we applied it, in combination with data‐independent acquisition mass spectrometry, to study signaling dynamics in the mitogen‐activated protein kinase (MAPK) pathway in yeast. Our results reveal broad and specific signaling events along the mating, the high‐osmolarity glycerol, and the invasive growth branches of the MAPK pathway, with robust phosphorylation of downstream regulatory proteins and transcription factors. Our method facilitates large‐scale signaling studies involving hundreds of perturbations opening the door to systems‐level studies aiming to capture signaling complexity.

Despite the growing importance of the cerebellum as a region highly vulnerable to accumulating molecular errors in schizophrenia, limited information is available regarding altered molecular networks with potential therapeutic targets. To identify altered networks, we conducted one-shot liquid chromatography–tandem mass spectrometry in postmortem cerebellar cortex in schizophrenia and healthy individuals followed by bioinformatic analysis (PXD024937 identifier in ProteomeXchange repository). A total of 108 up-regulated proteins were enriched in stress-related proteins, half of which were also enriched in axonal cytoskeletal organization and vesicle-mediated transport. A total of 142 down-regulated proteins showed an enrichment in proteins involved in mitochondrial disease, most of which were also enriched in energy-related biological functions. Network analysis identified a mixed module of mainly axonal-related pathways for up-regulated proteins with a high number of interactions for stress-related proteins. Energy metabolism and neutrophil degranulation modules were found for down-regulated proteins. Further, two double-hit postnatal stress murine models based on maternal deprivation combined with social isolation or chronic restraint stress were used to investigate the most robust candidates of generated networks. CLASP1 from the axonal module in the model of maternal deprivation was combined with social isolation, while YWHAZ was not altered in either model. METTL7A from the degranulation pathway was reduced in both models and was identified as altered also in previous gene expression studies, while NDUFB9 from the energy network was reduced only in the model of maternal deprivation combined with social isolation. This work provides altered stress- and mitochondrial disease-related proteins involved in energy, immune and axonal networks in the cerebellum in schizophrenia as possible novel targets for therapeutic interventions and suggests that METTL7A is a possible relevant altered stress-related protein in this context.

Triple-negative breast cancer is a heterogeneous disease characterized by poor clinical outcomes and a shortage of targeted treatment options. To discover molecular features of triple-negative breast cancer, we performed quantitative proteomics analysis of twenty human-derived breast cell lines and four primary breast tumors to a depth of more than 12,000 distinct proteins. We used this data to identify breast cancer subtypes at the protein level and demonstrate the precise quantification of biomarkers, signaling proteins, and biological pathways by mass spectrometry. We integrated proteomics data with exome sequence resources to identify genomic aberrations that affect protein expression. We performed a high-throughput drug screen to identify protein markers of drug sensitivity and understand the mechanisms of drug resistance. The genome and proteome provide complementary information that, when combined, yield a powerful engine for therapeutic discovery. This resource is available to the cancer research community to catalyze further analysis and investigation.

Abstract The coordinated regulation of protein kinases is a rapid mechanism that integrates diverse cues and swiftly determines appropriate cellular responses. However, our understanding of cellular decision‐making has been limited by the small number of simultaneously monitored phospho‐regulatory events. Here, we have estimated changes in activity in 215 human kinases in 399 conditions derived from a large compilation of phosphopeptide quantifications. This atlas identifies commonly regulated kinases as those that are central in the signaling network and defines the logic relationships between kinase pairs. Co‐regulation along the conditions predicts kinase–complex and kinase–substrate associations. Additionally, the kinase regulation profile acts as a molecular fingerprint to identify related and opposing signaling states. Using this atlas, we identified essential mediators of stem cell differentiation, modulators of Salmonella infection, and new targets of AKT1. This provides a global view of human phosphorylation‐based signaling and the necessary context to better understand kinase‐driven decision‐making.

Activating mutations of FMS-like tyrosine kinase-3 (FLT3) are found in approximately 30% of patients with acute myeloid leukemia (AML). FLT3 is therefore an attractive drug target. However, the molecular mechanisms by which FLT3 mutations lead to cell transformation in AML remain unclear. To develop a better understanding of FLT3 signaling as well as its downstream effectors, we performed detailed phosphoproteomic analysis of FLT3 signaling in human leukemia cells. We identified over 1000 tyrosine phosphorylation sites from about 750 proteins in both AML (wild type and mutant FLT3) and B cell acute lymphoblastic leukemia (normal and amplification of FLT3) cell lines. Furthermore, using stable isotope labeling by amino acids in cell culture (SILAC), we were able to quantified over 400 phosphorylation sites (pTyr, pSer, and pThr) that were responsive to FLT3 inhibition in FLT3 driven human leukemia cell lines. We also extended this phosphoproteomic analysis on bone marrow from primary AML patient samples, and identify over 200 tyrosine and 800 serine/threonine phosphorylation sites in vivo. This study showed that oncogenic FLT3 regulates proteins involving diverse cellular processes and affects multiple signaling pathways in human leukemia that we previously appreciated, such as Fc epsilon RI-mediated signaling, BCR, and CD40 signaling pathways. It provides a valuable resource for investigation of oncogenic FLT3 signaling in human leukemia.

The production of a functional mRNA is regulated at every step of transcription. An area not well-understood is the transition of RNA polymerase II from elongation to termination. The S. cerevisiae SR-like protein Npl3 functions to negatively regulate transcription termination by antagonizing the binding of polyA/termination proteins to the mRNA. In this study, Npl3 is shown to interact with the CTD and have a direct stimulatory effect on the elongation activity of the polymerase. The interaction is inhibited by phosphorylation of Npl3. In addition, Casein Kinase 2 was found to be required for the phosphorylation of Npl3 and affect its ability to compete against Rna15 (Cleavage Factor I) for binding to polyA signals. Our results suggest that phosphorylation of Npl3 promotes its dissociation from the mRNA/RNAP II, and contributes to the association of the polyA/termination factor Rna15. This work defines a novel role for Npl3 in elongation and its regulation by phosphorylation.

The septins are a conserved family of proteins that have been proposed to carry out diverse functions. In budding yeast, the septins become localized to the site of bud emergence in G1 but have not been thought to carry out important functions at this stage of the cell cycle. We show here that the septins function in redundant mechanisms that are required for formation of the bud neck and for the normal pattern of cell growth early in the cell cycle. The Shs1 septin shows strong genetic interactions with G1 cyclins and is directly phosphorylated by G1 cyclin-dependent kinases, consistent with a role in early cell cycle events. However, Shs1 phosphorylation site mutants do not show genetic interactions with the G1 cyclins or obvious defects early in the cell cycle. Rather, they cause an increased cell size and aberrant cell morphology that are dependent upon inhibitory phosphorylation of Cdk1 at the G2/M transition. Shs1 phosphorylation mutants also show defects in interaction with the Gin4 kinase, which associates with the septins during G2/M and plays a role in regulating inhibitory phosphorylation of Cdk1. Phosphorylation of Shs1 by G1 cyclin-dependent kinases plays a role in events that influence Cdk1 inhibitory phosphorylation.