Defective Proinsulin Handling Modulates the MHC I Bound Peptidome and Activates the Inflammasome in ß-Cells.

How immune tolerance is lost to pancreatic ß-cell peptides triggering autoimmune type 1 diabetes is enigmatic. We have shown that loss of the proinsulin chaperone glucose-regulated protein (GRP) 94 from the endoplasmic reticulum (ER) leads to mishandling of proinsulin, ER stress, and activation of the immunoproteasome. We hypothesize that inadequate ER proinsulin folding capacity relative to biosynthetic need may lead to an altered ß-cell major histocompatibility complex (MHC) class-I bound peptidome and inflammasome activation, sensitizing ß-cells to immune attack. We used INS-1E cells with or without GRP94 knockout (KO), or in the presence or absence of GRP94 inhibitor PU-WS13 (GRP94i, 20 µM), or exposed to proinflammatory cytokines interleukin (IL)-1ß or interferon gamma (IFNγ) (15 pg/mL and 10 ng/mL, respectively) for 24 h. RT1.A (rat MHC I) expression was evaluated using flow cytometry. The total RT1.A-bound peptidome analysis was performed on cell lysates fractionated by reverse-phase high-performance liquid chromatography (RP-HPLC), followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing protein (NLRP1), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα), and (pro) IL-1ß expression and secretion were investigated by Western blotting. GRP94 KO increased RT1.A expression in ß-cells, as did cytokine exposure compared to relevant controls. Immunopeptidome analysis showed increased RT1.A-bound peptide repertoire in GRP94 KO/i cells as well as in the cells exposed to cytokines. The GRP94 KO/cytokine exposure groups showed partial overlap in their peptide repertoire. Notably, proinsulin-derived peptide diversity increased among the total RT1.A peptidome in GRP94 KO/i along with cytokines exposure. NLRP1 expression was upregulated in GRP94 deficient cells along with decreased IκBα content while proIL-1ß cellular levels declined, coupled with increased secretion of mature IL-1ß. Our results suggest that limiting ß-cell proinsulin chaperoning enhances RT1.A expression alters the MHC-I peptidome including proinsulin peptides and activates inflammatory pathways, suggesting that stress associated with impeding proinsulin handling may sensitize ß-cells to immune-attack.

Spliced Peptides and Cytokine-Driven Changes in the Immunopeptidome of Melanoma.

Antigen recognition by CD8+ T cells is governed by the pool of peptide antigens presented on the cell surface in the context of HLA class I complexes. Studies have shown not only a high degree of plasticity in the immunopeptidome, but also that a considerable fraction of all presented peptides is generated through proteasome-mediated splicing of noncontiguous regions of proteins to form novel peptide antigens. Here, we used high-resolution mass spectrometry combined with new bioinformatic approaches to characterize the immunopeptidome of melanoma cells in the presence or absence of IFNγ. In total, we identified more than 60,000 peptides from a single patient-derived cell line (LM-MEL-44) and demonstrated that IFNγ induced changes in the peptidome, with an overlap of only approximately 50% between basal and treated cells. Around 6% to 8% of the peptides were identified as cis-spliced peptides, and 2,213 peptides (1,827 linear and 386 cis-spliced peptides) were derived from known melanoma-associated antigens. These peptide antigens were equally distributed between the constitutive- and IFNγ-induced peptidome. We next examined additional HLA-matched patient-derived cell lines to investigate how frequently these peptides were identified and found that a high proportion of both linear and spliced peptides was conserved between individual patient tumors, drawing on data amassing to more than 100,000 peptide sequences. Several of these peptides showed in vitro immunogenicity across multiple patients with melanoma. These observations highlight the breadth and complexity of the repertoire of immunogenic peptides that can be exploited therapeutically and suggest that spliced peptides are a major class of tumor antigens.

Human Plasma Protein Corona of Aß Amyloid and Its Impact on Islet Amyloid Polypeptide Cross-Seeding.

Alzheimer's disease (AD) is the most severe form of neurological disorder, characterized by the presence of extracellular amyloid-ß (Aß) plaques and intracellular tau tangles. For decades, therapeutic strategies against the pathological symptoms of AD have often relied on the delivery of monoclonal antibodies to target specifically Aß amyloid or oligomers, largely to no avail. Aß can be traced in the brain as well as in cerebrospinal fluid and the circulation, giving rise to abundant opportunities to interact with their environmental proteins. Using liquid chromatography tandem-mass spectrometry, here we identified for the first time the protein coronae of the two major amyloid forms of Aß-Aß1-42 and Aß1-40-exposed to human blood plasma. Out of the proteins identified in all groups, 58 proteins were unique to the Aß1-42 samples and 31 proteins unique to the Aß1-40 samples. Both fibrillar coronae consisted of proteins significant in complement activation, inflammation, and protein metabolic pathways involved in the pathology of AD. Structure-wise, the coronal proteins often possessed multidomains of high flexibility to maximize their association with the amyloid fibrils. The protein corona hindered recognition of Aß1-42 fibrils by their structurally specific antibodies and accelerated the aggregation but not the ß-cell toxicity of human islet amyloid polypeptide, the peptide associated with type 2 diabetes. This study highlights the importance of understanding the structural, functional, and pathological implications of the amyloid protein corona for the development of therapeutics against AD and a range of amyloid diseases.

Graphene quantum dots rescue protein dysregulation of pancreatic ß-cells exposed to human islet amyloid polypeptide.

The amyloid aggregation of peptides and proteins is a hallmark of neurological disorders and type 2 diabetes. Human islet amyloid polypeptide (IAPP), co-secreted with insulin by pancreatic ß-cells, plays dual roles in both glycemic control and the pathology of type 2 diabetes. While IAPP can activate the NLRP3 inflammasome and modulate cellular autophagy, apoptosis and extracellular matrix metabolism, no data is available concerning intracellular protein expression upon exposure to the polypeptide. More surprisingly, how intracellular protein expression is modulated by nanoparticle inhibitors of protein aggregation remains entirely unknown. In this study, we first examined the changing proteomes of ßTC6, a pancreatic ß-cell line, upon exposure to monomeric, oligomeric and fibrillar IAPP, and detailed cellular protein expression rescued by graphene quantum dots (GQDs), an IAPP inhibitor. We found that 29 proteins were significantly dysregulated by the IAPP species, while majority of these proteins were nucleotide-binding proteins. Collectively, our liquid chromatography tandem-mass spectrometry, fluorescence quenching, helium ion microscopy, cytotoxicity and discreet molecular dynamics simulations data revealed a remarkable capacity of GQDs in regulating aberrant protein expression through H-bonding and hydrophobic interactions, pointing to nanomedicine as a new frontier against human amyloid diseases.

Incidence, prevalence, and mortality associated with head and neck cancer in India: Protocol for a systematic review.

BACKGROUND: Head and neck cancer (HNC) is a heterogeneous tumor at various anatomic sites and one of the most common cancers in India. Published and existing reports and studies highlight an alarming increase in the incidence, prevalence, and mortality of HNC. Despite its high incidence, there is a dearth of more precise estimates of reliable epidemiological data pertaining to HNC in India. AIM: This protocol aims to conduct a full-scale systematic review and meta-analysis on the HNC epidemiology (incidence, prevalence, and mortality) in 29 states and 7 union territories of India. METHODS AND ANALYSIS: We will search for eligible published studies through PubMed, Scopus, Science Direct, MEDLINE, Web of Science, and Cochrane Review. Cancer registries such as (but not limited to) World Health Organization, International Agency for Research on Cancer, and the National Centre for Disease Informatics and Research-National Cancer Registry Program, which is maintained by the Indian Council of Medical Research, will be used for extracting relevant data using a standardized data collection form. The random-effects model of meta-analysis will be employed to aggregate the pooled estimates of relative ratios with 95% confidence intervals. Publication bias will be assessed using a funnel plot, and Egger's regression will be applied to test the symmetry of the funnel plot. DISCUSSION: This review will provide updated evidence of the current burden of HNC in India. This will guide future studies and cancer registry reports to provide holistically representative epidemiological data. SYSTEMATIC REVIEW REGISTRATION: In accordance with the guidelines, our systematic review protocol was registered with the International Prospective Register of Systematic Reviews and was assigned the registration number, CRD42017077482.

Head and neck cancer risk factors in India: protocol for systematic review and meta-analysis.

INTRODUCTION: Demographic, behavioural and environmental factors have been associated with increased risk of head and neck cancer (HNC). We will review published reports and explore connections between risk factors and HNC incidence. This protocol aims to provide strategies for a systematic review and meta-analysis of HNC risk factor analysis in India. It also provides guidelines in order to visualise obtained HNC risk factor data in the form of a heat-map highlighting variations across gender, age and geographical location. METHODS AND ANALYSIS: We will identify well-established HNC risk factors and perform a comprehensive systematic review and meta-analysis to quantify each risk factor's impact on HNC incidence. A systematic search will be performed to identify the studies and published reports of HNC risk factors in India. Meta-analysis will be conducted to estimate the proportional contribution of the most prevalent risk factor in HNC on a city-wide basis in Indian states and territories. ETHICS AND DISSEMINATION: The review protocol draws on publicly available anonymised data without directly involving human participants and therefore requires neither formal human ethical review nor approval by a human research ethics committee. We published an outline of the protocol in the International Prospective Register of Systematic Reviews (PROSPERO) in 2017. The results will provide an updated analysis of HNC risk factor prevalence in India, and we will discuss the applicability of rehabilitation care. We plan to disseminate the findings of this systematic review through publication in a peer-reviewed journal and presentation at relevant conference proceedings. PROSPERO REGISTRATION NUMBER: CRD42017077758.