An INS‐1 832/13 ‐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. Three hundred and fourteen proteins, amongst them proteins associated with vesicular SNARE interactions, protein export, and pancreatic secretion, increased in abundance upon glucose stimulation. In contrast, many proteins implicated in metabolic glucose sensing processes such as glycolysis, the TCA cycle, and the respiratory chain, did not respond. Interestingly, we observe that enzymes participating in fatty acid metabolism showed a “switch‐on” response upon release of complete glucose starvation with no further changes in abundance upon increasing glucose levels. We speculate that increased activity of fatty acid metabolic activity might either be part of GSIS by replenishing membrane lipids required for vesicle‐mediated exocytosis and/or by providing an electron sink to compensate for the increase in glucose catabolism. These findings offer new insights into beta cell function and may inform future strategies for targeting metabolic pathways in diabetes treatment. Summary: We used high‐throughput proteomics to capture comprehensive proteome changes 30 min post stimulation in the INS‐1 832/13 beta cell line, a commonly used cell model in studying glucose‐induced insulin secretion. Our results show that specific parts of the proteome respond promptly upon glucose exposure in this cell line. Furthermore, while many proteins canonically associated with GSIS did not change in abundance in the time frame and cell line investigated, our results attribute a specific role to fatty acid biosynthesis in the early steps of insulin secretion. By documenting protein abundance alterations in the initial phase of GSIS in the INS‐1 832/13 beta cell line, our study highlights the necessity of sampling early time points, well‐controlled study design and biological replicates in the study of beta cell function

Driving Forces of Proteasome-catalyzed Peptide Splicing in Yeast and Humans

Proteasome-catalyzed peptide splicing (PCPS) represents an additional activity of mammalian 20S proteasomes recently identified in connection with antigen presentation. We show here that PCPS is not restricted to mammalians but that it is also a feature of yeast 20S proteasomes catalyzed by all three active site β subunits. No major differences in splicing efficiency exist between human 20S standard- and immuno-proteasome or yeast 20S proteasome. Using H(2)(18)O to monitor the splicing reaction we also demonstrate that PCPS occurs via direct transpeptidation that slightly favors the generation of peptides spliced in cis over peptides spliced in trans. Splicing efficiency itself is shown to be controlled by proteasomal cleavage site preference as well as by the sequence characteristics of the spliced peptides. By use of kinetic data and quantitative analyses of PCPS obtained by mass spectrometry we developed a structural model with two PCPS binding sites in the neighborhood of the active Thr1.</p

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. monocytogenes-infected mice. While reacting to the spliced epitopes, these CD8+ T cells failed to recognize the non-spliced peptide parts in the context of their natural flanking sequences. Thus, we here show that PCPS expands the CD8+ T cell response against L. monocytogenes by exposing spliced epitopes on the cell surface. Moreover, our multi-level strategy opens up opportunities to systematically investigate proteins for spliced epitope candidates and thus strategies for immunotherapies or vaccine design

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

peer reviewedThe 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). We reveal that to initiate radial migration, neurons must first suppress adhesion to the extracellular matrix. Zeb2 regulates the multipolar stage by transcriptional repression of Nrp1 and thereby downstream inhibition of integrin signaling. Upon completion of migration, neurons undergo an orientation process that is independent of migration. The parallel organization of neurons within the neocortex is controlled by Cdh6 through atypical regulation of integrin signaling via its RGD motif. Our data shed light on the mechanisms that regulate initiation of radial migration and the postmigratory orientation of neurons during neocortical development

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. Results: Many demographic covariates and common medications affect the concentration of high-abundant plasma proteins, but the most significant changes are linked to the use of hormonal contraceptives (HCU). HCU particularly alters amongst others the levels of Angiotensinogen and Transcortin. We robustly replicated these findings in the BASE-II cohort. Furthermore, our results indicate that combined hormonal contraceptives with ethinylestradiol have a stronger effect compared to bioidentical estrogens. Our analysis detects no lasting impact of hormonal contraceptives on the plasma proteome. Conclusions: HCU is the dominant factor reshaping the high-abundant circulating blood proteome in two population studies. Given the high prevalence of HCU among young women, it is essential to account for this treatment in human proteome studies to avoid misinterpreting its impact as sex- or age-related effects. Although we did not investigate the influence of HCU-induced proteomic changes on human health, our data suggest that future studies on this topic are warranted

Natural Proteome Diversity Links Aneuploidy Tolerance to Protein Turnover

Accessing the natural genetic diversity of species unveils hidden genetic traits, clarifies gene functions and allows the generalizability of laboratory findings to be assessed. One notable discovery made in natural isolates ofSaccharomyces cerevisiaeis that aneuploidy—an imbalance in chromosome copy numbers—is frequent$^{1,2}$(around 20%), which seems to contradict the substantial fitness costs and transient nature of aneuploidy when it is engineered in the laboratory$^{3–5}$. Here we generate a proteomic resource and merge it with genomic$^{1}$and transcriptomic$^{6}$data for 796 euploid and aneuploid natural isolates. We find that natural and lab-generated aneuploids differ specifically at the proteome. In lab-generated aneuploids, some proteins—especially subunits of protein complexes—show reduced expression, but the overall protein levels correspond to the aneuploid gene dosage. By contrast, in natural isolates, more than 70% of proteins encoded on aneuploid chromosomes are dosage compensated, and average protein levels are shifted towards the euploid state chromosome-wide. At the molecular level, we detect an induction of structural components of the proteasome, increased levels of ubiquitination, and reveal an interdependency of protein turnover rates and attenuation. Our study thus highlights the role of protein turnover in mediating aneuploidy tolerance, and shows the utility of exploiting the natural diversity of species to attain generalizable molecular insights into complex biological processes

The 20S Proteasome Splicing Activity Discovered by SpliceMet

The identification of proteasome-generated spliced peptides (PSP) revealed a new unpredicted activity of the major cellular protease. However, so far characterization of PSP was entirely dependent on the availability of patient-derived cytotoxic CD8+ T lymphocytes (CTL) thus preventing a systematic investigation of proteasome-catalyzed peptide splicing (PCPS). For an unrestricted PSP identification we here developed SpliceMet, combining the computer-based algorithm ProteaJ with in vitro proteasomal degradation assays and mass spectrometry. By applying SpliceMet for the analysis of proteasomal processing products of four different substrate polypeptides, derived from human tumor as well as viral antigens, we identified fifteen new spliced peptides generated by PCPS either by cis or from two separate substrate molecules, i.e., by trans splicing. Our data suggest that 20S proteasomes represent a molecular machine that, due to its catalytic and structural properties, facilitates the generation of spliced peptides, thereby providing a pool of qualitatively new peptides from which functionally relevant products may be selected

Natural proteome diversity links aneuploidy tolerance to protein turnover

Accessing the natural genetic diversity of species unveils hidden genetic traits, clarifies gene functions and allows the generalizability of laboratory findings to be assessed. One notable discovery made in natural isolates of Saccharomyces cerevisiae is that aneuploidy—an imbalance in chromosome copy numbers—is frequent1, 2 (around 20%), which seems to contradict the substantial fitness costs and transient nature of aneuploidy when it is engineered in the laboratory3–5. Here we generate a proteomic resource and merge it with genomic1 and transcriptomic6 data for 796 euploid and aneuploid natural isolates. We find that natural and lab-generated aneuploids differ specifically at the proteome. In lab-generated aneuploids, some proteins—especially subunits of protein complexes—show reduced expression, but the overall protein levels correspond to the aneuploid gene dosage. By contrast, in natural isolates, more than 70% of proteins encoded on aneuploid chromosomes are dosage compensated, and average protein levels are shifted towards the euploid state chromosome-wide. At the molecular level, we detect an induction of structural components of the proteasome, increased levels of ubiquitination, and reveal an interdependency of protein turnover rates and attenuation. Our study thus highlights the role of protein turnover in mediating aneuploidy tolerance, and shows the utility of exploiting the natural diversity of species to attain generalizable molecular insights into complex biological processes

Peptide generation by the proteasome: the influence of a T210M substitution in the gp100209-217 tumour epitope on antigen presentation

Das 20S Proteasom ist als multikatalytische Protease für die Degradation von zellulären Proteinen verantwortlich und entscheidend an der Prozessierung MHC Klasse I-restringierter tumorspezifischer Peptide (Tumorepitope) beteiligt, die an der Zelloberfläche zytotoxischen T-Zellen (CTLs, cytotoxic T lymphocytes) präsentiert werden. Voraussetzung für die Aktivierung der CD8+ T-Zellen ist die spezifische Erkennung und Interaktion des T-Zell-Rezeptors (TCRs) an der Oberfläche der T-Zelle mit dem Peptid-MHC-Komplex auf Tumorzellen. Somatische tumorspezifische Mutationen können zu einer auf den Tumor beschränkten Veränderung der Aminosäuresequenz und infolgedessen zur Entstehung neuer, tumorspezifischer Epitope (Neoepitope) beitragen. Derartige somatische Mutationen sind insbesondere dann therapeutisch interessant, wenn sie die MHC Klasse I-Bindungsaffinität eines antigenen Peptids erhöhen und so die Erkennung durch TCRs optimieren. Der Erfolg einer adoptiven T-Zell- Therapie hängt dabei stark von der effizienten Generierung solcher Neoepitope durch das Proteasom ab. Die vorliegende Arbeit untersucht den Einfluss eines Aminosäureaustausches am Beispiel einer Substitution von Threonin gegen Methionin an Position 210 (T210M) im HLA-A*02:01-restringierten GlucoProtein(gp) 100209-217 Tumorepitops aus Melanomazellen auf die Antigenpräsentation. Zunächst wurde die Bedeutung dieses Aminosäureaustausches innerhalb des Tumorepitops auf das Spaltverhalten des Proteasoms untersucht. Bei der massenspektrometrischen Analyse der proteasomal generierten Spaltprodukte zeigten sich zwischen dem tumorassozierten Wildtyp (wt) und dem mutierten (mut) gp100201-230 Polypeptid qualitative als auch quantitative Unterschiede. Der T210M Austausch führte zur Generierung eines neuen, spezifischen Peptidpools, der neue, potenziell antigene Peptide mit einer guten theoretischen MHC Klasse I-Bindungsaffinität enthielt, und verbesserte die Menge an proteasomal prozessiertem Neoepitop bzw. dessen N-terminal verlängerten Vorläuferpeptiden. Untersuchungen zur Bindung des substituierten gp100209-217 (T210M) Epitops an die MHC Klasse I-Protein-Komplexe und Antigenpräsentationsanalysen mit antigenspezifischen CD8+ T-Zellen zeigten sowohl eine verbesserte Bindungsaffinität als auch eine erhöhte CD8+ T-Zell- Stimulation durch das T210M mut gp100209-217 im Vergleich zum wt gp100209-217 Epitop. Im Gegensatz dazu hatte der T210M Aminosäureaustausch im Tumorepitop keinen relevanten Einfluss auf das Trimmverhalten der Endoplasmatischen Retikulum residenten Aminopeptidase I (ERAP I), die für die N-terminale Verkürzung der proteasomal gebildeten Vorläuferpeptide zum minimalen Epitop verantwortlich ist. Zusammenfassend zeigen die in vitro Untersuchungen, dass es durch einen einzigen Aminosäureaustausch von Threonin zu Methionin innerhalb der Sequenz des HLA-A*02:01-restringierten gp100209-217 melanomen Tumorepitops zur Generierung eines veränderten, potenziell immunrelevanten Peptidpools durch das Proteasom kommt. Die Epitop-spezifische CD8+ T-Zellantwort wird aufgrund der verbesserten proteasomalen Prozessierung des Tumorepitops bzw. dessen N-terminal verlängerter Vorläuferpeptide verstärkt.The 20S proteasome a multi-catalytic protease, is responsible for degradation of most cellular proteins. It plays a key role in the generation of MHC class-I restricted tumour-specific epitopes that are presented on the cell surface to cytotoxic T cells (CTLs, cytotoxic T lymphocytes). A prerequisite for the activation of CD8+ T cells is the specific recognition and the interaction of T cell receptors (TCRs) on the cell surface with the MHC- peptide complex. Somatic tumour-specific mutations can contribute to alterations in the amino-acid sequence of an antigen and can result in the formation of new, tumour-specific epitopes (neo-epitopes). Such mutations are of therapeutic interest when they enhance the MHC class-I binding affinity of an epitope and in turn, potentially optimize the recognition by TCRs. The success of adoptive T cell therapy strongly depends on the efficient generation of neo-epitopes by the proteasome. This study examines the influence on antigen presentation of a threonine to methionine substitution at position 210 (T210M) in the HLA-A*02:01-restricted gp100209-217 melanoma tumour epitope. At first, the relevance of the T210M exchange was investigated with respect to its influence on the cleavage site usage of the proteasome. Mass-spectrometric analysis of the proteasome-generated fragments disclosed qualitative as well as quantitative differences between the tumour-associated wild type (wt) and the mutated (mut) gp100201-230 polypeptide. The T210M exchange resulted in the generation of a new, specific peptide pool that contained potentially antigenic peptides with good theoretical MHC class-I binding affinity. It also led to an enhancement of the amount of the proteasome-generated neoepitope and its N-terminally extended precursor- peptides. Analysing the binding of the neo-epitope to the MHC class-I protein complexes, and antigen presentation analyses using gp100209-217 specific CD8+ T cells, demonstrated an enhanced binding affinity of the mutant peptide as well as an increased CD8+ T cell stimulation by the mut gp100209-217 in comparison to the wt gp100209-217 epitope. In contrast, the T210M exchange in the tumour epitope had no relevant influence on the trimming behaviour of the endoplasmic reticulum-resident aminopeptidase I (ERAP I), responsible for N-terminal shortening of the proteasome-formed precursors to the minimal epitope