An INS-1 832/13 𝛽-Cell Proteome Highlights the Rapid Regulation of Fatty Acid Biosynthesis in Glucose-Stimulated Insulin Secretion.

Pancreatic beta cells secrete insulin in response to rising glucose levels, a process known as glucose-stimulated insulin secretion (GSIS). Here, we acquire proteomes of rat pancreatic INS-1 832/13 beta cells that were short-term stimulated with 11 different glucose concentrations from 0 to 20 mM, quantifying the response of 3703 proteins. Ensemble clustering of proteome profiles revealed unique response patterns of proteins expressed by INS-1 832/13 cells.

Extensive modulation of the circulating blood proteome by hormonal contraceptive use across two population studies.

Background: The study of circulating blood proteins in population cohorts offers new avenues to explore lifestyle-related and genetic influences describing and shaping human health.

Methods: Utilizing high-throughput mass spectrometry, we quantified 148 highly abundant proteins, functioning in the innate and adaptive immune system, coagulation and nutrient transport in 3632 blood plasma, and 500 serum samples from the CHRIS and BASE-II cross-sectional population studies, respectively. Through multiple regression analyses, we aimed to identify the main factors influencing the circulating proteome at population level.

Semaphorin heterodimerization in cis regulates membrane targeting and neocortical wiring.

Disruption of neocortical circuitry and architecture in humans causes numerous neurodevelopmental disorders. Neocortical cytoarchitecture is orchestrated by various transcription factors such as Satb2 that control target genes during strict time windows. In humans, mutations of SATB2 cause SATB2 Associated Syndrome (SAS), a multisymptomatic syndrome involving epilepsy, intellectual disability, speech delay, and craniofacial defects.

High-throughput proteomics of nanogram-scale samples with Zeno SWATH MS.

The possibility to record proteomes in high throughput and at high quality has opened new avenues for biomedical research, drug discovery, systems biology, and clinical translation. However, high-throughput proteomic experiments often require high sample amounts and can be less sensitive compared to conventional proteomic experiments. Here, we introduce and benchmark Zeno SWATH MS, a data-independent acquisition technique that employs a linear ion trap pulsing (Zeno trap pulsing) to increase the sensitivity in high-throughput proteomic experiments.

Adhesion dynamics in the neocortex determine the start of migration and the post-migratory orientation of neurons.

The neocortex is stereotypically organized into layers of excitatory neurons arranged in a precise parallel orientation. Here we show that dynamic adhesion both preceding and following radial migration is essential for this organization. Neuronal adhesion is regulated by the Mowat-Wilson syndrome-associated transcription factor Zeb2 (Sip1/Zfhx1b) through direct repression of independent adhesion pathways controlled by Neuropilin-1 (Nrp1) and Cadherin-6 (Cdh6).

Ultra-High-Throughput Clinical Proteomics Reveals Classifiers of COVID-19 Infection.

The COVID-19 pandemic is an unprecedented global challenge, and point-of-care diagnostic classifiers are urgently required. Here, we present a platform for ultra-high-throughput serum and plasma proteomics that builds on ISO13485 standardization to facilitate simple implementation in regulated clinical laboratories. Our low-cost workflow handles up to 180 samples per day, enables high precision quantification, and reduces batch effects for large-scale and longitudinal studies.

Large database for the analysis and prediction of spliced and non-spliced peptide generation by proteasomes.

Proteasomes are the main producers of antigenic peptides presented to CD8 T cells. They can cut proteins and release their fragments or recombine non-contiguous fragments thereby generating novel sequences, i.e.

Parkin contributes to synaptic vesicle autophagy in Bassoon-deficient mice.

Mechanisms regulating the turnover of synaptic vesicle (SV) proteins are not well understood. They are thought to require poly-ubiquitination and degradation through proteasome, endo-lysosomal or autophagy-related pathways. Bassoon was shown to negatively regulate presynaptic autophagy in part by scaffolding Atg5.

ER-aminopeptidase 1 determines the processing and presentation of an immunotherapy-relevant melanoma epitope.

Dissecting the different steps of the processing and presentation of tumor-associated antigens is a key aspect of immunotherapies enabling to tackle the immune response evasion attempts of cancer cells. The immunodominant glycoprotein gp100 epitope, which is liberated from the melanoma differentiation antigen gp100 , is part of immunotherapy trials. By analyzing different human melanoma cell lines, we here demonstrate that a pool of N-terminal extended peptides sharing the common minimal epitope is generated by melanoma proteasome subtypes.

Cocoa-specific flavor components and their peptide precursors.

Essential precursors of the cocoa-specific roasting-flavor notes were formed during proteolysis of the cocoa vicilin-class(7S) globulin by a mixture of cocoa aspartic protease and carboxypeptidase. These could be partially purified by ligand-exchange chromatography. Many constituents of this peptide fraction were destroyed by post-treatment with pepsin, but the cocoa-specific flavor-precursor peptides were largely resistant against pepsin treatment.

Analysis of Proteasome-Generated Antigenic Peptides by Mass Spectrometry.

Mass spectrometry (MS) is today one of the most important analytical techniques in biosciences. The development of electro spray ionization (ESI) as a gentle method, in which molecules are not destroyed, has revolutionized the analytic of peptides. MS is an ideal technique for detection and analysis of peptides generated by purified 20S proteasomes in in vitro experiments.

Proteolytic dynamics of human 20S thymoproteasome.

An efficient immunosurveillance of CD8 T cells in the periphery depends on positive/negative selection of thymocytes and thus on the dynamics of antigen degradation and epitope production by thymoproteasome and immunoproteasome in the thymus. Although studies in mouse systems have shown how thymoproteasome activity differs from that of immunoproteasome and strongly impacts the T cell repertoire, the proteolytic dynamics and the regulation of human thymoproteasome are unknown. By combining biochemical and computational modeling approaches, we show here that human 20S thymoproteasome and immunoproteasome differ not only in the proteolytic activity of the catalytic sites but also in the peptide transport.

Structural Visualization of the Formation and Activation of the 50S Ribosomal Subunit during In Vitro Reconstitution.

The assembly of ribosomal subunits is an essential prerequisite for protein biosynthesis in all domains of life. Although biochemical and biophysical approaches have advanced our understanding of ribosome assembly, our mechanistic comprehension of this process is still limited. Here, we perform an in vitro reconstitution of the Escherichia coli 50S ribosomal subunit.

pH-Dependency of the proteolytic formation of cocoa- and nutty-specific aroma precursors.

Sensory evaluation of roasted cocoa not only revealed cocoa-specific but also more or less pronounced nutty-specific aroma notes. Essential precursors of the corresponding volatile compounds could be generated in vitro by proteolysis of the cocoa vicilin-class(7S) globulin using a mixture of cocoa aspartic protease and cocoa carboxypeptidase. Since both proteases have rather different pH optima (pH 3.

Multi-level Strategy for Identifying Proteasome-Catalyzed Spliced Epitopes Targeted by CD8 T Cells during Bacterial Infection.

Proteasome-catalyzed peptide splicing (PCPS) generates peptides that are presented by MHC class I molecules, but because their identification is challenging, the immunological relevance of spliced peptides remains unclear. Here, we developed a reverse immunology-based multi-level approach to identify proteasome-generated spliced epitopes. Applying this strategy to a murine Listeria monocytogenes infection model, we identified two spliced epitopes within the secreted bacterial phospholipase PlcB that primed antigen-specific CD8 T cells in L.

Immunoproteasome induction is suppressed in hepatitis C virus-infected cells in a protein kinase R-dependent manner.

By changing the relative abundance of generated antigenic peptides through alterations in the proteolytic activity, interferon (IFN)-γ-induced immunoproteasomes influence the outcome of CD8 cytotoxic T lymphocyte responses. In the present study, we investigated the effects of hepatitis C virus (HCV) infection on IFN-γ-induced immunoproteasome expression using a HCV infection cell culture system. We found that, although IFN-γ induced the transcriptional expression of mRNAs encoding the β1i/LMP2, β2i/MECL-1 and β5i/LMP7 immunoproteasome subunits, the formation of immunoproteasomes was significantly suppressed in HCV-infected cells.

CD8(+) T cells of Listeria monocytogenes-infected mice recognize both linear and spliced proteasome products.

CD8(+) T cells responding to infection recognize pathogen-derived epitopes presented by MHC class-I molecules. While most of such epitopes are generated by proteasome-mediated antigen cleavage, analysis of tumor antigen processing has revealed that epitopes may also derive from proteasome-catalyzed peptide splicing (PCPS). To determine whether PCPS contributes to epitope processing during infection, we analyzed the fragments produced by purified proteasomes from a Listeria monocytogenes polypeptide.

The T210M Substitution in the HLA-a*02:01 gp100 Epitope Strongly Affects Overall Proteasomal Cleavage Site Usage and Antigen Processing.

MHC class I-restricted epitopes, which carry a tumor-specific mutation resulting in improved MHC binding affinity, are preferred T cell receptor targets in innovative adoptive T cell therapies. However, T cell therapy requires efficient generation of the selected epitope. How such mutations may affect proteasome-mediated antigen processing has so far not been studied.

The proteasome immunosubunits, PA28 and ER-aminopeptidase 1 protect melanoma cells from efficient MART-126-35 -specific T-cell recognition.

The immunodominant MART-1(26(27)-35) epitope, liberated from the differentiation antigen melanoma antigen recognized by T cells/melanoma antigen A (MART-1/Melan-A), has been frequently targeted in melanoma immunotherapy, but with limited clinical success. Previous studies suggested that this is in part due to an insufficient peptide supply and epitope presentation, since proteasomes containing the immunosubunits β5i/LMP7 (LMP, low molecular weight protein) or β1i/LMP2 and β5i/LMP7 interfere with MART-1(26-35) epitope generation in tumor cells. Here, we demonstrate that in addition the IFN-γ-inducible proteasome subunit β2i/MECL-1 (multicatalytic endopeptidase complex-like 1), proteasome activator 28 (PA28), and ER-resident aminopeptidase 1 (ERAP1) impair MART-1(26-35) epitope generation.

Partial purification and characterisation of the peptide precursors of the cocoa-specific aroma components.

Essential precursors of the cocoa-specific aroma notes are formed during fermentation of the cocoa beans by acid-induced proteolysis. It has been shown that, in addition to free amino acids, hydrophilic peptides derived from the vicilin-class(7S) globular storage protein are required for the generation of the cocoa-specific aroma notes during the roasting process. To identify those peptides responsible for the generation of the cocoa-specific aroma components, we have developed a procedure for the fractionation of the aroma precursor extract from well-fermented cocoa beans by ligand-exchange and subsequent Sephadex-LH20 chromatography.

Proteasome isoforms exhibit only quantitative differences in cleavage and epitope generation.

Immunoproteasomes are considered to be optimised to process Ags and to alter the peptide repertoire by generating a qualitatively different set of MHC class I epitopes. Whether the immunoproteasome at the biochemical level, influence the quality rather than the quantity of the immuno-genic peptide pool is still unclear. Here, we quantified the cleavage-site usage by human standard- and immunoproteasomes, and proteasomes from immuno-subunit-deficient mice, as well as the peptides generated from model polypeptides.

Adult human liver contains intermediate-type proteasomes with different enzymatic properties.

Background: The 20S proteasome is the proteolytic core of the major intracellular protein degradative system, the ubiquitin-proteasome system. Since little is known about proteasomes of human liver, we have investigated the proteasome spectrum in adult human liver.

Material And Methods: 20S proteasomes were chromatographically purified from adult human liver and from HuH7 cells.

Molecular alterations in proteasomes of rat liver during aging result in altered proteolytic activities.

Aging induces alterations of tissue protein homoeostasis. To investigate one of the major systems catalysing intracellular protein degradation we have purified 20S proteasomes from rat liver of young (2 months) and aged (23 months) animals and separated them into three subpopulations containing different types of intermediate proteasomes with standard- and immuno-subunits. The smallest subpopulation ΙΙΙ and the major subpopulation Ι comprised proteasomes containing immuno-subunits β1i and β5i beside small amounts of standard-subunits, whereas proteasomes of subpopulation ΙΙ contained only β5i beside standard-subunits.

Analysis of proteasome generated antigenic peptides by mass spectrometry.

Mass spectrometry (MS) is today one of the most important analytical techniques in biosciences. The development of electro spray ionization (ESI) as a gentle ionization method, in which molecules are not destroyed, has revolutionized the analytic of peptides. MS is an ideal technique for detection and analysis of peptides generated by in vitro experiments using purified 20S proteasomes.