Proteasome Composition Research Articles

Altered Proteasome Composition in Aging Brains, Genetic Proteasome Augmentation Mitigates Age-Related Cognitive Declines, and Acute Proteasome Agonist Treatment Rescues Age-Related Cognitive Deficits in Mice.

The aging brain experiences a significant decline in proteasome function, The proteasome is critical for many key neuronal functions including neuronal plasticity, and memory formation/retention. Treatment with proteasome inhibitors impairs these processes. Our study reveals a marked reduction in 20S and 26S proteasome activities in aged mice brains driven by reduced functionality of aged proteasome. This is matched by a decline in 20S proteasome but an increase in 26S proteasome. Our data suggests this may be a compensatory response to reduced functionality. By overexpressing the proteasome subunit PSMB5 in the neurons of mice, enhancing proteasome function, we slowed age-related declines in spatial learning and memory as well neuromuscular declines. We then showed acute treatment with a proteasome activator to rescue spatial learning and memory deficits in aged mice. These findings highlight the potential of proteasome augmentation as a therapeutic strategy to mitigate age-related cognitive declines.

Quantitative mapping of proteasome interactomes and substrates using ProteasomeID.

Proteasomes are essential molecular machines responsible for the degradation of proteins in eukaryotic cells. Altered proteasome activity has been linked to neurodegeneration, auto-immune disorders and cancer. Despite the relevance for human disease and drug development, no method currently exists to monitor proteasome composition and interactions in vivo in animal models. To fill this gap, we developed a strategy based on tagging of proteasomes with promiscuous biotin ligases and generated a new mouse model enabling the quantification of proteasome interactions by mass spectrometry. We show that biotin ligases can be incorporated in fully assembled proteasomes without negative impact on their activity. We demonstrate the utility of our method by identifying novel proteasome-interacting proteins, charting interactomes across mouse organs, and showing that proximity-labeling enables the identification of both endogenous and small-molecule-induced proteasome substrates.

Proteasome composition in immune cells implies special immune‐cell‐specific immunoproteasome function

Immunoproteasomes are a special class of proteasomes, which can be induced with IFN-γ in an inflammatory environment. In recent years, it became evident that certain immune cell types constitutively express high levels of immunoproteasomes. However, information regarding the basal expression of proteolytically active immunoproteasome subunits in different types of immune cells is still rare. Hence, we quantified standard proteasome subunits (β1c, β2c, β5c) and immunoproteasome subunits (LMP2, MECL-1, LMP7) in the major murine (CD4+ Tcells, CD8+ Tcells, CD19+ B cells, CD11c+ dendritic cells, CD49d+ natural killer cells, Ly-6G+ neutrophils) and human immune cell (CD4+ Tcells, CD8+ Tcells, CD19+ B cells, CD1c+CD141+ myeloid dendritic cells, CD56+ natural killer cells, granulocytes) subsets. The different human immune cell types were isolated from peripheral blood and the murine immune cell subsets from spleen. We found that proteasomes of most immune cell subsets mainly consist of immunoproteasome subunits. Our data will serve as a reference and guideline for immunoproteasome expression and imply a special role of immunoproteasomes in immune cells.

Degrade to stay healthy-Proteolytic interplay during inflammation.

Proteasomes and autophagy constitute the 2 main proteolytic machineries for cytoplasmic content. A new study in PLOS Biology now demonstrates that autophagy stimulation alters proteasome composition, degrading hyperactive immunoproteasomes and thereby limiting inflammation.

Co-expression analysis of transcriptomic data from cancer and healthy specimens reveals rewiring of proteasome genes and an interaction with the XPO1 gene across several tumour types

BackgroundThe 26S proteasome is a large intracellular multiprotein complex, that plays a homeostatic role by degrading proteins that have been tagged by ubiquitin. It is composed of 64 subunits assembled according to a well-defined structure and stoichiometry. Several proteasome subunits have been found to be overexpressed in tumours. However, comprehensive data are lacking on the relative abundance of each subunit and the impact on proteasome composition or stoichiometry. In cancer treatment, proteasome inhibitors and inhibitors of XPO1 (Exportin-1) have unexpectedly a similar range of activity, but the interaction between the two pathways has not been studied.MethodsWe performed gene co-expression analysis of 38 genes encoding proteasome subunits and 38 genes encoding proteins involved in nucleocytoplasmic transport in specimens from the Cancer Genome Atlas (33 tumour types) and from the Gene Tissue Expression database (32 healthy tissue types). We obtained 65 matrices, each containing Pearson correlation factors for 2964 gene pairs. We applied cluster analysis to the correlation matrices and compared the distribution of Pearson correlation coefficients of thirteen tumour types with their healthy tissue counterpart.ResultsStrong positive correlation (R Pearson correlation > 0.8) was observed for pairs of proteasome genes in the majority of healthy tissues, whereas the correlation for co-expression was significantly lower (R ≤ 0.50) for most gene pairs in the majority of cancer types. Cluster analysis based on gene co-expression allowed to distinguish cancers from healthy tissues in a clear-cut manner, and to identify the genes that contributed most to the separation. The crossed analysis between proteasome and nucleocytoplasmic transport genes showed that the expression of XPO1 and a subset of proteasome genes, including in particular PSMD14, is correlated in several cancer types and not in their healthy counterpart.ConclusionsThis analysis reveals that in cancer the co-expression of proteasome genes is significantly altered, highlighting the genes that are more often deregulated. In addition, it finds that XPO1 expression is often correlated with the expression of proteasome genes. From a therapeutic perspective, these findings support the investigation of novel targets within the proteasome and strategies of co-targeting of the proteasome and nucleocytoplasmic transport.

Proteomic Analysis of Human iPSC-Derived Neural Stem Cells and Motor Neurons Identifies Proteasome Structural Alterations.

Proteins targeted by the ubiquitin proteasome system (UPS) are identified for degradation by the proteasome, which has been implicated in the development of neurodegenerative diseases. Major histocompatibility complex (MHC) molecules present peptides broken down by the proteasome and are involved in neuronal plasticity, regulating the synapse number and axon regeneration in the central or peripheral nervous system during development and in brain diseases. The mechanisms governing these effects are mostly unknown, but evidence from different compartments of the cerebral cortex indicates the presence of immune-like MHC receptors in the central nervous system. We used human induced pluripotent stem cells (iPSCs) differentiated into neural stem cells and then into motor neurons as a developmental model to better understand the structure of the proteasome in developing motor neurons. We performed a proteomic analysis of starting human skin fibroblasts, their matching iPSCs, differentiated neural stem cells and motor neurons that highlighted significant differences in the constitutive proteasome and immunoproteasome subunits during development toward motor neurons from iPSCs. The proteomic analysis showed that the catalytic proteasome subunits expressed in fibroblasts differed from those in the neural stem cells and motor neurons. Western blot analysis confirmed the proteomic data, particularly the decreased expression of the β5i (PSMB8) subunit immunoproteasome in MNs compared to HFFs and increased β5 (PSMB5) in MNs compared to HFFs. The constitutive proteasome subunits are upregulated in iPSCs and NSCs from HFFs. Immunoproteasome subunit β5i expression is higher in MNs than NSCs; however, overall, there is more of a constitutive proteasome structure in MNs when comparing HFFs to MNs. The proteasome composition may have implications for motor neuron development and neurodevelopmental diseases that warrant further investigation.

Effects of Hydrogen Sulfide Donor GYY4137 on the Proteasome Pool of Colon Cancer Cells

Cancer cells are characterized by an increased level of metabolism and are highly dependent on the correct functioning of the processes that ensure homeostasis. Reactive sulfur species (RSS) are important molecular modulators of metabolic processes in both healthy and tumor cells. The effect of RSS and, in particular, H2S, on key cellular systems, including the ubiquitin-proteasome system (UPS), which ensures the destruction of most intracellular proteins, has been shown. The main components of the UPS are proteasomes ‒ multisubunit protein complexes, within which protein proteolysis occurs. At the same time, data on the effect of H2S directly on the pool of proteasomes in tumor cells are insufficient. Here, we studied the effect of incubation of SW620B8-mCherry colon adenocarcinoma cells expressing a fluorescently labeled proteasome subunit with 50, 100, and 200 µM of hydrogen sulfide donor GYY4137. The effect of the substance on the proteasome pool was assessed 6, 24, 48, and 72 h after administration. It was shown that the chymotrypsin-like and caspase-like proteasome activity decreases in cells incubated with 200 µM of the GYY4137 for 24 h. This coincided with an increase in the expression of proteasome subunit genes. In lysates of cells incubated with 200 µM GYY4137 for 48 h an increase in the content of the constitutive β5 subunit was observed. Against this background, the activity of proteasomes in cells levels off. Following prolonged incubation with GYY4137 (72h), an increase in the expression levels of some proteasome genes was also observed, though this did not have a significant effect on the activity and subunit composition of proteasomes. Thus, the data obtained indicate the modulation of proteasome activity by the hydrogen sulfide donor, as well as the effect of GYY4137 on the levels of transcription and translation of proteasome genes.

IFNɣ but not IFNα increases recognition of insulin defective ribosomal product-derived antigen to amplify islet autoimmunity.

The inflammatory milieu characteristic of insulitis affects translation fidelity and generates defective ribosomal products (DRiPs) that participate in autoimmune beta cell destruction in type 1 diabetes. Here, we studied the role of early innate cytokines (IFNα) and late immune adaptive events (IFNɣ) in insulin DRiP-derived peptide presentation to diabetogenic CD8+ T cells. Single-cell transcriptomics of human pancreatic islets was used to study the composition of the (immuno)proteasome. Specific inhibition of the immunoproteasome catalytic subunits was achieved using siRNA, and antigenic peptide presentation at the cell surface of the human beta cell line EndoC-βH1 was monitored using peptide-specific CD8 T cells. We found that IFNγ induces the expression of the PSMB10 transcript encoding the β2i catalytic subunit of the immunoproteasome in endocrine beta cells, revealing a critical role in insulin DRiP-derived peptide presentation to T cells. Moreover, we showed that PSMB10 is upregulated in a beta cell subset that is preferentially destroyed in the pancreases of individuals with type 1 diabetes. Our data highlight the role of the degradation machinery in beta cell immunogenicity and emphasise the need for evaluation of targeted immunoproteasome inhibitors to limit beta cell destruction in type 1 diabetes. The single-cell RNA-seq dataset is available from the Gene Expression Omnibus (GEO) using the accession number GSE218316 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE218316 ).

Immunoproteasome-specific subunit PSMB9 induction is required to regulate cellular proteostasis upon mitochondrial dysfunction

Perturbed cellular protein homeostasis (proteostasis) and mitochondrial dysfunction play an important role in neurodegenerative diseases, however, the interplay between these two phenomena remains unclear. Mitochondrial dysfunction leads to a delay in mitochondrial protein import, causing accumulation of non-imported mitochondrial proteins in the cytosol and challenging proteostasis. Cells respond by increasing proteasome activity and molecular chaperones in yeast and C. elegans. Here, we demonstrate that in human cells mitochondrial dysfunction leads to the upregulation of a chaperone HSPB1 and, interestingly, an immunoproteasome-specific subunit PSMB9. Moreover, PSMB9 expression is dependent on the translation elongation factor EEF1A2. These mechanisms constitute a defense response to preserve cellular proteostasis under mitochondrial stress. Our findings define a mode of proteasomal activation through the change in proteasome composition driven by EEF1A2 and its spatial regulation, and are useful to formulate therapies to prevent neurodegenerative diseases.

The proteasome regulator PSME4 modulates proteasome activity and antigen diversity to abrogate antitumor immunity in NSCLC.

Immunotherapy revolutionized treatment options in cancer, yet the mechanisms underlying resistance in many patients remain poorly understood. Cellular proteasomes have been implicated in modulating antitumor immunity by regulating antigen processing, antigen presentation, inflammatory signaling and immune cell activation. However, whether and how proteasome complex heterogeneity may affect tumor progression and the response to immunotherapy has not been systematically examined. Here, we show that proteasome complex composition varies substantially across cancers and impacts tumor-immune interactions and the tumor microenvironment. Through profiling of the degradation landscape of patient-derived non-small-cell lung carcinoma samples, we find that the proteasome regulator PSME4 is upregulated in tumors, alters proteasome activity, attenuates presented antigenic diversity and associates with lack of response to immunotherapy. Collectively, our approach affords a paradigm by which proteasome composition heterogeneity and function should be examined across cancer types and targeted in the context of precision oncology.

Orthogonal approaches required to measure proteasome composition and activity in mammalian brain tissue

Proteasomes are large macromolecular complexes with multiple distinct catalytic activities that are each vital to human brain health and disease. Despite their importance, standardized approaches to investigate proteasomes have not been universally adapted. Here, we describe pitfalls and define straightforward orthogonal biochemical approaches essential to measure and understand changes in proteasome composition and activity in the mammalian central nervous system. Through our experimentation in the mammalian brain, we determined an abundance of catalytically active proteasomes exist with and without a 19S cap(s), the regulatory particle essential for ubiquitin-dependent degradation. Moreover, we learned that in-cell measurements using activity-based probes (ABPs) are more sensitive in determining the available activity of the 20S proteasome without the 19S cap and in measuring individual catalytic subunit activities of each β subunit within all neuronal proteasomes. Subsequently, applying these tools to human brain samples, we were surprised to find that post-mortem tissue retained little to no 19S-capped proteasome, regardless of age, sex, or disease state. In comparing brain tissues (parahippocampal gyrus) from patients with Alzheimer's disease (AD) and unaffected individuals, the available 20S proteasome activity was significantly elevated in severe cases of AD, an observation not previously noted. Taken together, our study establishes standardized approaches for the comprehensive investigation of proteasomes in mammalian brain tissue, and we reveal new insight into brain proteasome biology.

Application of Activity‐Based Probe, MV151, in the Mammalian Nervous System Reveals New Insights into Proteasome Changes in Human Alzheimer's Disease Brain

Proteasome complexes play a critical role in human brain health and disease. Despite enormous effort, proteasome composition, activity, and abundance in the human brain remains poorly understood. In this study, we describe a series of tools that can measure the catalytic activity and levels of individual proteasome β‐subunits and distinct proteasome complexes in mammalian tissue. Adapting these tools to brain tissue from human Alzheimer’s disease (AD) patients, we found that the human brain has a high abundance of catalytically active 20S proteasomes and that these 20S proteasomes exhibit higher total activity in AD when compared to unaffected controls. Additionally, we showed that 20S proteasome abundance is inversely correlated with the severity of the AD case. Taken together, our results indicate that while 20S proteasome abundance is decreasing in AD patient brains, this is counter‐balanced by an increase in proteasome activity. We now propose that homeostasis of 20S proteasome activity is a hallmark of human brain health, and changes in abundance and activity are correlated to the severity of AD disease. This discovery sets the stage for further investigation into 20S proteasome activity in neurodegenerative disease states.

Targeting an alternate Wilms' tumor antigen 1 peptide bypasses immunoproteasome dependency.

Designing effective antileukemic immunotherapy will require understanding mechanisms underlying tumor control or resistance. Here, we report a mechanism of escape from immunologic targeting in an acute myeloid leukemia (AML) patient, who relapsed 1 year after immunotherapy with engineered T cells expressing a human leukocyte antigen A*02 (HLA-A2)-restricted T cell receptor (TCR) specific for a Wilms' tumor antigen 1 epitope, WT1126-134 (TTCR-C4). Resistance occurred despite persistence of functional therapeutic T cells and continuous expression of WT1 and HLA-A2 by the patient's AML cells. Analysis of the recurrent AML revealed expression of the standard proteasome, but limited expression of the immunoproteasome, specifically the beta subunit 1i (β1i), which is required for presentation of WT1126-134. An analysis of a second patient treated with TTCR-C4 demonstrated specific loss of AML cells coexpressing β1i and WT1. To determine whether the WT1 protein continued to be processed and presented in the absence of immunoproteasome processing, we identified and tested a TCR targeting an alternative, HLA-A2-restricted WT137-45 epitope that was generated by immunoproteasome-deficient cells, including WT1-expressing solid tumor lines. T cells expressing this TCR (TTCR37-45) killed the first patients' relapsed AML resistant to WT1126-134 targeting, as well as other primary AML, in vitro. TTCR37-45 controlled solid tumor lines lacking immunoproteasome subunits both in vitro and in an NSG mouse model. As proteasome composition can vary in AML, defining and preferentially targeting these proteasome-independent epitopes may maximize therapeutic efficacy and potentially circumvent AML immune evasion by proteasome-related immunoediting.

α-Synuclein Decreases the Abundance of Proteasome Subunits and Alters Ubiquitin Conjugates in Yeast.

Parkinson’s disease (PD) is the most prevalent movement disorder characterized with loss of dopaminergic neurons in the brain. One of the pathological hallmarks of the disease is accumulation of aggregated α-synuclein (αSyn) in cytoplasmic Lewy body inclusions that indicates significant dysfunction of protein homeostasis in PD. Accumulation is accompanied with highly elevated S129 phosphorylation, suggesting that this posttranslational modification is linked to pathogenicity and altered αSyn inclusion dynamics. To address the role of S129 phosphorylation on protein dynamics further we investigated the wild type and S129A variants using yeast and a tandem fluorescent timer protein reporter approach to monitor protein turnover and stability. Overexpression of both variants leads to inhibited yeast growth. Soluble S129A is more stable and additional Y133F substitution permits αSyn degradation in a phosphorylation-independent manner. Quantitative cellular proteomics revealed significant αSyn-dependent disturbances of the cellular protein homeostasis, which are increased upon S129 phosphorylation. Disturbances are characterized by decreased abundance of the ubiquitin-dependent protein degradation machinery. Biotin proximity labelling revealed that αSyn interacts with the Rpt2 base subunit. Proteasome subunit depletion by reducing the expression of the corresponding genes enhances αSyn toxicity. Our studies demonstrate that turnover of αSyn and depletion of the proteasome pool correlate in a complex relationship between altered proteasome composition and increased αSyn toxicity.

Proteasome Composition and Activity Changes in Cultured Fibroblasts Derived From Mucopolysaccharidoses Patients and Their Modulation by Genistein.

In this study, we have asked whether proteasome composition and function are affected in cells derived from patients suffering from all types of mucopolysaccharidosis (MPS), an inherited metabolic disease caused by accumulation of undegraded glycosaminoglycans (GAGs). Moreover, we have tested if genistein, a small molecule proposed previously as a potential therapeutic agent in MPS, can modulate proteasomes, which might shed a new light on the molecular mechanisms of action of this isoflavone as a potential drug for macromolecule storage diseases. Significant changes in expression of various proteasome-linked genes have been detected during transcriptomic (RNA-seq) analyses in vast majority of MPS types. These results were corroborated by demonstration of increased proteasomal activities in MPS cells. However, GAGs were not able to stimulate the 26S proteasome in vitro, suggesting that the observed activation in cells is indirect rather than arising from direct GAG-proteasome interactions. Genistein significantly reduced proteasomal activities in fibroblasts derived from patients suffering from all MPS types, while its effects on in vitro 26S proteasome activity were negligible. Unexpectedly, levels of many proteasomal subunits were increased in genistein-treated MPS cells. On the other hand, this ostensible discrepancy between results of experiments designed for estimation of effects of genistein on proteasome activities and abundance of proteasomal subunits can be explained by demonstration that in the presence of this isoflavone, levels of ubiquitinated proteins were decreased. The genistein-mediated reduction of proteasomal activities might have beneficial effects in cells of MPS patients due to potential increasing of residual activities of defective lysosomal enzymes which would otherwise be subjected to efficient ubiquitination and proteasomal degradation as misfolded proteins. These results indicate another activity of genistein (apart from previously demonstrated reduction of GAG synthesis efficiency, stimulation of lysosomal biogenesis, and activation of the autophagy process) which can be beneficial in the use of this small molecule in treatment of MPS.

Features of the Proteasome Pool in Spontaneously Occurring Malignant Tumors of the Mammary Gland in Mice

The aim of this work is to investigate the features of the proteasome pool in malignant tumors of mammary glands on a unique model of inbred BLRB/BYRB mice characterized by a high probability of spontaneous development of this type of cancer. It was found that the proteasome pool of mammary glands was relatively stable and it did not change in mice of various physiological states: normal females of different age and a tumor carrier. However, with the formation of malignant tumors, this stability is violated, and the proteasome pool undergoes complex changes. In general, it increases in the number of proteolytic subparticles. At the same time, its chymotrypsin-like activity increases disproportionately more intensively, which indicates additional mechanisms for its regulation. These mechanisms include the detected increased expression of activators PA28αβ and 19S RC as well as changes in the subunit composition of proteasomes due to increased expression of the immune subunits LMP2 and LMP7. The amount of each of the studied components in the proteasome pool increased in the tumor in different ways. The most significant increase was observed in the amount of two components, 19S RC activator and LMP2 subunit, which indicates the prospects of their use as targets for breast cancer therapy. However, we must not forget that these components are also important for the vital functions of normal cells. Therefore, when creating new drugs, we should simultaneously develop methods for their targeted delivery to the tumor or other ways of safe use.

Proteasome Composition in Cytokine-Treated Neurons and Astrocytes is Determined Mainly by Subunit Displacement.

In this study, we investigated if subunit displacement and/or alterations in proteasome biosynthesis are responsible for the changes in the levels of constitutive proteasomes (c-20S), immunoproteasomes (i-20S) and the activators PA28 and PA700 in neurons and astrocytes cultured with a cytokine mixture (IFN-γ/TNF-α/IL-1β). Exposure of both cell types to cytokines for 24h increases mRNA and protein expression of the i-20S-specific subunit β5i and PA28α/β, and leads to a decline in the amount of the c-20S-specific subunit β5. Since β5 mRNA levels are unchanged by the cytokine treatment, it is fair to conclude that displacement of constitutive β-subunits with inducible β5i subunits is likely the mechanism underlying the decrease in c-20S. As expected, the increase in the amount of the IFN-γ-inducible subunits coincides with elevated expression of phospho-STAT-1 and interferon regulatory factor-1 (IRF-1). However, inhibition of NF-κB signaling in cytokine-treated astrocytes reduces IRF-1 expression without affecting that of i-20S, c-20S and PA28. This suggests that STAT-1 is capable of increasing the transcription of i20S-specific subunits and PA28α/β by itself. The lack of a decrease in proteasome β5 mRNA expression is consistent with the fact that Nrf1 (Nfe2l1) and Nrf2 (Nfe2l2) levels are not reduced by pro-inflammatory cytokines. In contrast, we previously found that there is a significant Nrf1 dysregulation and reduced β5 mRNA expression in the spinal cords of mice with experimental autoimmune encephalomyelitis (EAE). Thus, there are stressors in EAE, other than a pro-inflammatory environment, that are not present in cytokine-treated cells.

Proteasome subunit \u03b11 overexpression preferentially drives canonical proteasome biogenesis and enhances stress tolerance in yeast

The 26S proteasome conducts the majority of regulated protein catabolism in eukaryotes. At the heart of the proteasome is the barrel-shaped 20S core particle (CP), which contains two β-rings sandwiched between two α-rings. Whereas canonical CPs contain α-rings with seven subunits arranged α1-α7, a non-canonical CP in which a second copy of the α4 subunit replaces the α3 subunit occurs in both yeast and humans. The mechanisms that control canonical versus non-canonical CP biogenesis remain poorly understood. Here, we have repurposed a split-protein reporter to identify genes that can enhance canonical proteasome assembly in mutant yeast producing non-canonical α4-α4 CPs. We identified the proteasome subunit α1 as an enhancer of α3 incorporation, and find that elevating α1 protein levels preferentially drives canonical CP assembly under conditions that normally favor α4-α4 CP formation. Further, we demonstrate that α1 is stoichiometrically limiting for α-ring assembly, and that enhancing α1 levels is sufficient to increase proteasome abundance and enhance stress tolerance in yeast. Together, our data indicate that the abundance of α1 exerts multiple impacts on proteasome assembly and composition, and we propose that the limited α1 levels observed in yeast may prime cells for alternative proteasome assembly following environmental stimuli.

Abstract 528: Restricted Proteasome Heterogeneity Promotes Premature Heart Failure and Deterioration of Associated Protein Levels Upon Continuous β-Adrenergic Stimulation

Introduction: The ubiquitin proteasome-system is a main contributor to cellular proteostasis. The dynamic regulation of proteasome composition and function during cardiac remodeling has been of high interest. To what extend discrete proteasome subunits in turn modify cardiac remodeling, is poorly understood. Objective: Aim of the study was to investigate the contribution of the non-essential proteasome subunit low molecular mass peptide 2 (Lmp2) on cardiac function, tissue and proteome upon induced hypertrophic remodeling. Methods & Results: We found induction of cardiac Lmp2 during isoproterenol (Iso)- and TAC-induced remodeling and hypertrophic cardiomyopathy. Unchallenged adult mice congenitally lacking Lmp2 (KO) were indistinguishable from their WT littermates. After 7 days of continuous Iso administration (30 mg/kg/d) though, cardiac function was significantly reduced in Lmp2 KO mice as compared to WT (fractional shortening: -32% vs. WT, p<0.01, n≥9). Interestingly, reduced cardiac function was accompanied by augmented cardiac remodeling as shown by heart weight to body weight ratio, wall thickness, cardiomyocyte cross-sectional area and interstitial collagen content. Cardiac-restricted re-expression of Lmp2 utilizing AAV9-mediated gene transfer rescued cardiac function and restricted remodeling to WT levels, attributing those findings to cardiac Lmp2. Hence, we analyzed the cardiac proteome utilizing 2-D DIGE. Out of approximately 1500 detected protein spots, 114 were showing significant differences upon cardiac remodeling depending on absolute Lmp2 protein expression (p≤0.05). All spots were manually inspected for correct matching across 10 gels. Subsequently, spots were picked with priority on those with the most prominent changes in abundance and identified by mass spectrometry. For example, we found significantly less carbonic anhydrase 2 and manganese superoxide dismutase in the remodeled myocardium of WT compared to Lmp2 KO mice. Conclusions: Cardiac lack of Lmp2 exacerbates cardiac remodeling, which is reflected in the cardiac proteome by deteriorated levels of proteins with a known pivotal role in human cardiac disease.

Impact of Leishmania donovani infection on the HLA I self peptide repertoire of human macrophages.

Macrophages are specialized antigen-presenting cells that process and present self-antigens for induction of tolerance, and foreign antigens to initiate T cell-mediated immunity. Despite this, Leishmania donovani (LD) are able to parasitize the macrophages and persist. The impact of this parasitizing and persistence on antigen processing and presentation by macrophages remains poorly defined. To gain insight into this, we analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) and compared the HLA-I self-peptidomes, proteasome compositions, HLA expression and activation states of non-infected and LD-infected THP1-derived macrophages. We found that, though both HLA-I peptidomes were dominated by nonapeptides, they were heterogeneous and individualized, with differences in HLA binding affinities and anchor residues. Non-infected and LD-infected THP1-derived macrophages were able to sample peptides from source proteins of almost all subcellular locations and involved in various cellular functions, but in different proportions. In the infected macrophages, there was increased sampling of plasma membrane and extracellular proteins, and those involved in immune responses, cell communication/signal transduction and metabolism/energy pathways, and decreased sampling of nuclear and cytoplasmic proteins and those involved in protein metabolism, RNA binding and cell growth and/or maintenance. Though the activation state of infected macrophages was unchanged, their proteasome composition was altered.