Aberrant Protein Research Articles

Two New Mutations in the CEL Gene Causing Diabetes and Hereditary Pancreatitis: How to Correctly Identify MODY8 Cases

ContextMaturity onset diabetes of the young, type 8 (MODY8) is associated with mutations in the CEL gene, which encodes the digestive enzyme carboxyl ester lipase. Several diabetes cases and families have in recent years been attributed to mutations in CEL without any functional or clinical evidence provided.ObjectiveTo facilitate correct MODY8 diagnostics, we screened 2 cohorts of diabetes patients and delineated the phenotype.MethodsYoung, lean Swedish and Finnish patients with a diagnosis of type 2 diabetes (352 cases, 406 controls) were screened for mutations in the CEL gene. We also screened 58 Czech MODY cases who had tested negative for common MODY genes. For CEL mutation-positive subjects, family history was recorded, and clinical investigations and pancreatic imaging performed.ResultsTwo cases (1 Swedish and 1 Czech) with germline mutation in CEL were identified. Clinical and radiological investigations of these 2 probands and their families revealed dominantly inherited insulin-dependent diabetes, pancreatic exocrine dysfunction, and atrophic pancreas with lipomatosis and cysts. Notably, hereditary pancreatitis was the predominant phenotype in 1 pedigree. Both families carried single-base pair deletions in the proximal part of the CEL variable number of tandem repeat (VNTR) region in exon 11. The mutations are predicted to lead to aberrant protein tails that make the CEL protein susceptible to aggregation.ConclusionThe diagnosis of MODY8 requires a pancreatic exocrine phenotype and a deletion in the CEL VNTR in addition to dominantly inherited diabetes. CEL screening may be warranted also in families with hereditary pancreatitis of unknown genetic etiology.

The Interplay of the Unfolded Protein Response in Neurodegenerative Diseases: A Therapeutic Role of Curcumin.

Abnormal accumulation of misfolded proteins in the endoplasmic reticulum and their aggregation causes inflammation and endoplasmic reticulum stress. This promotes accumulation of toxic proteins in the body tissues especially brain leading to manifestation of neurodegenerative diseases. The studies suggest that deregulation of proteostasis, particularly aberrant unfolded protein response (UPR) signaling, may be a common morbific process in the development of neurodegeneration. Curcumin, the mixture of low molecular weight polyphenolic compounds from turmeric, Curcuma longa has shown promising response to prevents many diseases including current global severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and neurodegenerative disorders. The UPR which correlates positively with neurodegenerative disorders were found affected by curcumin. In this review, we examine the evidence from many model systems illustrating how curcumin interacts with UPR and slows down the development of various neurodegenerative disorders (ND), e.g., Alzheimer’s and Parkinson’s diseases. The recent global increase in ND patients indicates that researchers and practitioners will need to develop a new pharmacological drug or treatment to manage and cure these neurodegenerative diseases.

Carrying Excess Baggage Can Slowdown Life: Protein Clearance Machineries That Go Awry During Aging and the Relevance of Maintaining Them.

Cellular homeostasis is maintained by rapid and systematic cleansing of aberrant and aggregated proteins within cells. Neurodegenerative diseases (NDs) especially Parkinson's and Alzheimer's disease are known to be associated with multiple factors, most important being impaired clearance of aggregates, resulting in the accumulation of specific aggregated protein in the brain. Protein quality control (PQC) of proteostasis network comprises proteolytic machineries and chaperones along with their regulators to ensure precise operation and maintenance of proteostasis. Such regulatory factors coordinate among each other multiple functional aspects related to proteins, including their synthesis, folding, transport, and degradation. During aging due to inevitable endogenous and external stresses, sustaining a proteome balance is a challenging task. Such stresses decline the capacity of the proteostasis network compromising the proteome integrity, affecting the fundamental physiological processes including reproductive fitness of the organism. This review focuses on highlighting proteome-wide changes during aging and the strategies for proteostasis improvements. The possibility of augmenting the proteostasis network either via genetic or pharmacological interventions may be a promising strategy towards delaying age-associated pathological consequences due to proteome disbalance, thus promoting healthy aging and prolonged longevity.

Exploration of Aberrant E3 Ligases Implicated in Alzheimer's Disease and Development of Chemical Tools to Modulate Their Function.

The Ubiquitin Proteasome System (UPS) is responsible for the degradation of misfolded or aggregated proteins via a multistep ATP-dependent proteolytic mechanism. This process involves a cascade of ubiquitin (Ub) transfer steps from E1 to E2 to E3 ligase. The E3 ligase transfers Ub to a targeted protein that is brought to the proteasome for degradation. The inability of the UPS to remove misfolded or aggregated proteins due to UPS dysfunction is commonly observed in neurodegenerative diseases, such as Alzheimer’s disease (AD). UPS dysfunction in AD drives disease pathology and is associated with the common hallmarks such as amyloid-β (Aβ) accumulation and tau hyperphosphorylation, among others. E3 ligases are key members of the UPS machinery and dysfunction or changes in their expression can propagate other aberrant processes that accelerate AD pathology. The upregulation or downregulation of expression or activity of E3 ligases responsible for these processes results in changes in protein levels of E3 ligase substrates, many of which represent key proteins that propagate AD. A powerful way to better characterize UPS dysfunction in AD and the role of individual E3 ligases is via the use of high-quality chemical tools that bind and modulate specific E3 ligases. Furthermore, through combining gene editing with recent advances in 3D cell culture, in vitro modeling of AD in a dish has become more relevant and possible. These cell-based models of AD allow for study of specific pathways and mechanisms as well as characterization of the role E3 ligases play in driving AD. In this review, we outline the key mechanisms of UPS dysregulation linked to E3 ligases in AD and highlight the currently available chemical modulators. We present several key approaches for E3 ligase ligand discovery being employed with respect to distinct classes of E3 ligases. Where possible, specific examples of the use of cultured neurons to delineate E3 ligase biology have been captured. Finally, utilizing the available ligands for E3 ligases in the design of proteolysis targeting chimeras (PROTACs) to degrade aberrant proteins is a novel strategy for AD, and we explore the prospects of PROTACs as AD therapeutics.

Abstract 10240: Key Pathomechanisms of Cardiomyopathy Due to TTN Truncation

Introduction: Heterozygous truncating variants in TTN (TTNtv) coding for titin cause dilated cardiomyopathy (DCM) but the underlying pathomechanisms are unclear and disease management remains uncertain. Here, we aim to elucidate the key pathomechanisms of TTNtv-DCM. Hypothesis: Stable expression of truncated titin proteins and titin haploinsufficiency characterize TTNtv-DCM and represent potential targets for therapy. Methods and Results: We studied left ventricular tissues from 14 nonfailing donor hearts and 113 endstage failing DCM patients and identified a TTNtv in 22 DCM patients (19.5%) by next generation sequencing. Using titin protein gel electrophoresis and western blot we demonstrate, for the first time, titin haploinsufficiency in TTNtv-DCM hearts. Strikingly, all 21 adult TTNtv-DCM hearts of our cohort showed stable expression of truncated titin proteins. Expression was variable, up to one-half of the total titin protein pool, and negatively correlated to patient age at heart transplantation. Truncated titin proteins were not detected in sarcomeres but were present in intracellular aggregates. Deregulated ubiquitin-dependent protein quality control was apparent. Next, we produced human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), comparing wildtype controls to cells with a patient-derived, prototypical A-band TTNtv or a CRISPR/Cas9-edited M-band TTNtv. TTNtv hiPSC-CMs showed reduced wildtype titin expression and contained truncated titin proteins, whose proportion increased upon inhibition of proteasomal activity. In engineered heart muscle generated from hiPSC-CMs, depressed contractility caused by TTNtv could be reversed by correction of the mutation using CRISPR/Cas9, thereby eliminating truncated titin proteins and raising wildtype titin content. Functional improvement also occurred when titin protein content was increased by proteasome inhibition. Conclusions: Our study reveals the major pathomechanisms of TTNtv-DCM, which include titin haploinsufficiency as a life-long condition and truncated titin protein enrichment with aggregate formation, along with aberrant protein quality control. Results can be exploited for new therapies of TTNtv cardiomyopathies.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Recent advances in PTP1B signaling in metabolism and cancer.

Protein tyrosine phosphorylation is one of the major post-translational modifications in eukaryotic cells and represents a critical regulatory mechanism of a wide variety of signaling pathways. Aberrant protein tyrosine phosphorylation has been linked to various diseases, including metabolic disorders and cancer. Few years ago, protein tyrosine phosphatases (PTPs) were considered as tumor suppressors, able to block the signals emanating from receptor tyrosine kinases. However, recent evidence demonstrates that misregulation of PTPs activity plays a critical role in cancer development and progression. Here, we will focus on PTP1B, an enzyme that has been linked to the development of type 2 diabetes and obesity through the regulation of insulin and leptin signaling, and with a promoting role in the development of different types of cancer through the activation of several pro-survival signaling pathways. In this review, we discuss the molecular aspects that support the crucial role of PTP1B in different cellular processes underlying diabetes, obesity and cancer progression, and its visualization as a promising therapeutic target.

Dissecting GATA1 Protein Interactions in Normal and Malignant Human Erythroblasts

Acute erythroid leukemia (AEL) is characterized by uncontrolled accumulation of transformed erythroblasts. Previous analysis of murine and human AEL revealed aberrant regulation of the master regulator GATA1, which controls terminal erythroid differentiation in multi-protein complexes acting as activators or repressors of gene expression. Although most malignant erythroblasts constitutively express abundant GATA1 protein, terminal erythroid differentiation is impaired. Notably, overexpression of GATA1 significantly induced partial or complete terminal erythroid differentiation of the human AEL cell line K562 or immortalised HUDEP2 human erythroblasts, respectively. These observations led us to hypothesize that blocked terminal erythroid differentiation in AEL might be the consequence of titratable dose-dependent aberrant GATA1 protein interactions. We comparatively analysed nuclear extracts from three human AEL cell lines (F36P, K562, KMOE2) and primary cells from an AEL patient. In addition, we analysed HUDEP2 and primary human erythroblasts (hEBST) from healthy donors that retain the potential for complete in vitro terminal erythroid differentiation. We quantified protein expression using a tandem mass tag (TMT) based approach (n=3/cell type) and we compared putative GATA1 interactions by immunoprecipitation (IP) followed by liquid chromatography mass spectrometry (MS) (n=3/cell type). Quantitative proteomics identified 6774 commonly expressed proteins in AEL and “normal” erythroblasts with a high reproducibility (mean coefficients of variation <10%) for all six different cell types. Unsupervised hierarchical clustering displayed a clear separation of the AEL cells from the “normal erythroblasts” (hEBST, HUDEP2). 386 proteins were higher expressed in the AEL group (logFC>=2; q<0.05), whereas 623 were more abundant in normal erythroblasts (logFC>=2; q<0.05). IP-MS analysis of nuclear lysates from the AEL cell lines, the AEL primary sample, HUDEP2 and hEBST resulted in a matrix containing 1616 proteins from which 126 proteins seem to significantly differentially interact with GATA1. 54 proteins were more enriched in the AEL group, whereas 72 proteins were more enriched in “normal” erythroblasts (q<0.5). Principal component analysis (PCA) showed for all cell lines a similar clustering pattern, accounting for 24% and 32% of the variance. Pulled-down proteins in hEBST and HUDEP2 clustered together and were closer to F36P and KMOE, than LAM49 and K562. Notably, we found significant enrichment (validated by immunoblotting) of the SKI protooncogene in AEL cells (logFC=1.82; q=0.013), a finding which not only confirmed previous findings in murine AEL models (MEL cells, erythroblasts from Nsd1 -/- mice) but also speaks for the functionality of our approach. Similarly, the LRPPRC leucine-rich PPR-motif-containing protein overexpressed in several cancers, as well the lactate dehydrogenases A and B (LDHA, LDHB) were significantly enriched in malignant erythroblasts (logFC>2; q<0.05). Furthermore, the ZEB2 zinc finger E-box-binding homeobox 2 protein, was significantly enriched in AEL cells (logFC=2.02; q=0.005). In contrast, the hematopoietic master transcription factor Runt-related transcription factor 1 (RUNX1) (logFC=2.48; q=0.0018) as well as DNA binding protein Ikaros (IKZF1) (logFC=1.71; q=0.13) were significantly enriched (validated by immunoblotting) in HUDEP2 and hEBST. Moreover, the MCM6 DNA binding mini-chromosome maintenance complex component 6 critical for proper DNA replication was enriched in normal erythroblasts (logFC=1.99; q=0.0001). Interestingly, one of the most strongly enriched (and validated by immunoblotting) proteins in normal erythroblasts was the nuclear pore complex protein NUP155 (logFC=6.1; q=0.0000001). Integration of the quantitative proteomics and the IP-MS analysis identified 118 proteins differentially expressed and differentially pulled-down by GATA1-IP, of which 49 were enriched in malignant and 69 proteins in normal erythroblasts (q<0.5). This shows that we reproducibly identified proteins that are differentially associated with GATA1 which are also differentially expressed in AEL cells versus normal erythroblasts. A targeted CRISPR/Cas9 screen is under way to identify GATA1-interacting proteins responsible for impaired erythroid differentiation of AEL cells. DisclosuresValent: Novartis: Honoraria; Pfizer: Honoraria, Research Funding; Celgene/BMS: Honoraria, Research Funding; Incyte: Honoraria, Research Funding; OAP Orphan Pharmaceuticals: Honoraria.

Proteogenomic Characterization of Highly Enriched Viable Leukemic Blasts in Acute Myeloid Leukemia: A SWOG Report

Acknowledgements: This work was funded by the following NIH/NCI/NCTN grant awards: RO1CA190661, R01CA160872, U10CA180888, U10CA180819, U24CA196175. The authors wish to gratefully acknowledge the importantcontributions of the late Dr. Stephen H. Petersdorf to SWOG and to the study S0106. The authors would like to acknowledge that a portion of the specimens were obtained from the Fred Hutch/University of Washington Hematopoietic Disorder Repository.AML is a molecularly heterogenous disease that harbors multiple genomic, epigenomic, and transcriptomic abnormalities. Despite the use of newer therapeutic agents and identification of multiple prognostic markers, most patients with AML still relapse or succumb to their disease. Understanding biological factors that determine relapse is of major clinical interest in AML. Proteins are responsible for much of the functional biology of the cells. However, few studies have examined the global proteome in AML, and, to our knowledge, studies have not previously examined the proteome in highly enriched undifferentiated AML blasts. Therefore, we have developed an integrated approach utilizing mass spectrometry-based proteomics and leveraging next generation RNA sequencing (RNAseq) to identify novel protein biomarkers associated with clinical outcome in a homogeneous population of undifferentiated viable leukemic blasts (uVLBs) from AML patients. Cryopreserved specimens from previously untreated de novo AML patients (n=27) were obtained from the SWOG Leukemia Repository. Patients were treated with dose intensive induction and consolidation as part of SWOG-9031, SWOG-9333, S0106, and S0112 studies. uVLBs were isolated from specimens using fluorescence-activated cell sorting (FACS). Targeted genome sequencing examined for genomic mutations in uVLBs, while global RNA sequencing (RNAseq) and tandem mass tag labelling followed by liquid chromatograph with tandem mass spectrometry (TMT-LC-MS/MS) were used to quantify transcript and protein expression, respectively. Analyses identified 6761 unique proteins, with 238 and 460 proteins significantly associated with complete response (CR) and overall survival (OS), respectively. There was modest overlap between the prognostically significant transcript and protein biomarkers (Figure 1). We also were able to identify and quantify aberrant proteins arising from genomic mutations . Examples of the “neoproteins” detected included proteins harboring NPM1 mutations (Figure 2a) and those harboring frameshift insertions/deletions in the terminal region of RUNX1 (Figure 2b).For validation of prognostic associations, we examined the proteome in an independent population of de novo AML patients (N=27) treated at the Fred Hutch and University of Washington. As with the SWOG patients, pre-treatment specimens were obtained from patients receiving intensive induction and consolidation with curative intent; however, OS data was not available for many of these patients. As such, the validation studies focused on CR. Patients were randomized by CR response into 9 pools (CR=6 and no CR=3 pools), and specimens were sorted for uVLBs. TMT-based LC-MS/MS quantified protein expression across pools, and analyses examined the prognostic significance of the 238 proteins from the SWOG analyses associated with CR. Thirteen of the most promising candidates were significantly associated with CR prognosis (Table 1), many of which are associated with cancer biology.Together, these studies show the feasibility and biological importance of examining the proteome in uVLBs. Studies examining for biomarkers in the proteome may be a powerful tool to uncover novel prognostic biomarkers that would otherwise not be identified by examining the genome or transcriptome. Furthermore, the multi-omics approach can be used to confirm the translation of potential neoantigens into actionable protein targets, which may lead to more cost-effective mechanisms for the development of innovative adoptive immunotherapies. [Display omitted] DisclosuresOthus: Biosight: Consultancy; Merck: Consultancy; Celgene: Other: Data safety monitoring board; Glycomimetics: Other: Data safety monitoring board; Daiichi Sankyo: Consultancy. Moseley: BioSight Ltd: Consultancy. Erba: AbbVie Inc; Agios Pharmaceuticals Inc; Bristol Myers Squibb; Celgene, a Bristol Myers Squibb company; Incyte Corporation; Jazz Pharmaceuticals Inc; Novartis: Speakers Bureau; AbbVie Inc; Agios Pharmaceuticals Inc; Astellas; Bristol Myers Squibb; Celgene, a Bristol Myers Squibb company; Daiichi Sankyo Inc; Genentech, a member of the Roche Group; GlycoMimetics Inc; Incyte Corporation; Jazz Pharmaceuticals Inc; Kura Oncology; Nov: Other: Advisory Committee; AbbVie Inc: Other: Independent review committee; AbbVie Inc; Agios Pharmaceuticals Inc; ALX Oncology; Amgen Inc; Daiichi Sankyo Inc; FORMA Therapeutics; Forty Seven Inc; Gilead Sciences Inc; GlycoMimetics Inc; ImmunoGen Inc; Jazz Pharmaceuticals Inc; MacroGenics Inc; Novartis; PTC Therapeutics: Research Funding. Radich: Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees.

Identification of a novel compound that simultaneously impairs the ubiquitin-proteasome system and autophagy

ABSTRACT The ubiquitin-proteasome system (UPS) and macroautophagy/autophagy are the main proteolytic systems in eukaryotic cells for preserving protein homeostasis, i.e., proteostasis. By facilitating the timely destruction of aberrant proteins, these complementary pathways keep the intracellular environment free of inherently toxic protein aggregates. Chemical interference with the UPS or autophagy has emerged as a viable strategy for therapeutically targeting malignant cells which, owing to their hyperactive state, heavily rely on the sanitizing activity of these proteolytic systems. Here, we report on the discovery of CBK79, a novel compound that impairs both protein degradation by the UPS and autophagy. While CBK79 was identified in a high-content screen for drug-like molecules that inhibit the UPS, subsequent analysis revealed that this compound also compromises autophagic degradation of long-lived proteins. We show that CBK79 induces non-canonical lipidation of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 beta) that requires ATG16L1 but is independent of the ULK1 (unc-51 like autophagy activating kinase 1) and class III phosphatidylinositol 3-kinase (PtdIns3K) complexes. Thermal preconditioning of cells prevented CBK79-induced UPS impairment but failed to restore autophagy, indicating that activation of stress responses does not allow cells to bypass the inhibitory effect of CBK79 on autophagy. The identification of a small molecule that simultaneously impairs the two main proteolytic systems for protein quality control provides a starting point for the development of a novel class of proteostasis-targeting drugs.

Current Approaches in Supersecondary Structures Investigation.

Proteins expressed during the cell cycle determine cell function, topology, and responses to environmental influences. The development and improvement of experimental methods in the field of structural biology provide valuable information about the structure and functions of individual proteins. This work is devoted to the study of supersecondary structures of proteins and determination of their structural motifs, description of experimental methods for their detection, databases, and repositories for storage, as well as methods of molecular dynamics research. The interest in the study of supersecondary structures in proteins is due to their autonomous stability outside the protein globule, which makes it possible to study folding processes, conformational changes in protein isoforms, and aberrant proteins with high productivity.

Profiling Tryptophan Catabolites of Human Gut Microbiota and Acute-Phase Protein Levels in Neonatal Dried Blood Specimens.

National screening programs use dried blood specimens to detect metabolic disorders or aberrant protein functions that are not clinically evident in the neonatal period. Similarly, gut microbiota metabolites and immunological acute-phase proteins may reveal latent immune aberrations. Microbial metabolites interact with xenobiotic receptors (i.e., aryl hydrocarbon and pregnane-X) to maintain gastrointestinal tissue health, supported by acute-phase proteins, functioning as sensors of microbial immunomodulation and homeostasis. The delivery (vaginal or cesarean section) shapes the microbial colonization, which substantially modulates both the immune system’s response and mucosal homeostasis. This study profiled microbial metabolites of the kynurenine and tryptophan pathway and acute-phase proteins in 134 neonatal dried blood specimens. We newly established neonatal blood levels of microbial xenobiotic receptors ligands (i.e., indole-3-aldehyde, indole-3-butyric acid, and indole-3-acetamide) on the second day of life. Furthermore, we observed diverse microbial metabolic profiles in neonates born vaginally and via cesarean section, potentially due to microbial immunomodulatory influence. In summary, these findings suggest the supportive role of human gut microbiota in developing and maintaining immune system homeostasis.

Aberrant Expression of β-Catenin Correlates with Infiltrating Immune Cells and Prognosis in NSCLC.

Aims: β-catenin is a critical regulating factor of the Wnt pathway, which is closely linked to tumorigenesis, tumor growth, metastasis, and tumor immunity. Our study focused on exploring the relationship between β-catenin and clinicopathological features, prognosis, as well as infiltrating immune cells and immune scores, so as to illustrate its clinical significance in NSCLC. Materials and Methods: The β-catenin mRNA (CTNNB1) and protein expression data were downloaded from the UALCAN and the UCSC Xena website, respectively. All tumor-immune infiltrating cells’ data were downloaded from the TIMER platform and immune scores were downloaded from ESTIMATE website. The expression of β-catenin protein in our cohort was measured by immunohistochemistry. Results: β-catenin mRNA level was higher in lung adenocarcinoma (LUAD) compared to normal tissues (p < 0.001) and was related to overall survival (OS) (p < 0.001) and post-progression survival (PPS) (both p = 0.049) in LUAD. Aberrant β-catenin protein expression was higher in male and lung squamous cell carcinoma (LUSC) patients (both p = 0.001). Also, it was considered to be a prognosis factor independently (p = 0.034). In addition, β-catenin protein was negatively correlated with CD8+T cells (r = −0.128, p = 0.008), neutrophils (r = −0.198, p < 0.001), immune score (r = −0.109, p = 0.024), stromal score (r = −0.097, p = 0.045), and ESTIMATE score (r = −0.113, p = 0.020). Conclusions: Aberrant β-catenin protein expression was evidently higher in NSCLC and might serve as a biomarker for poor prognosis. Most importantly, β-catenin protein might play an important part in tumor immunity and the tumor microenvironment by inhibiting the infiltration of CD8+ T cells and neutrophils.

Histone deacetylase 3 (HDAC3) as an important epigenetic regulator of kidney diseases.

Development and progression of many kidney diseases are substantially influenced by aberrant protein acetylation modifications of gene expression crucial for kidney functions. Histone deacetylase (HDAC) expression alterations are detected from renal samples of patients and animal models of various kidney diseases, and the administrations of HDAC inhibitors display impressive renal protective effects in vitro and in vivo. However, when the expression alterations of multiple HDACs occur, not all the HDACs causally affect the disease onset or progression. Identification of a single HDAC as a disease-causing factor will allow subtype-targeted intervention with less side effect. HDAC3 is a unique HDAC with distinct structural and subcellular distribution features and co-repressor dependency. HDAC3 is required for kidney development and its aberrations actively participate in many pathological processes, such as cancer, cardiovascular diseases, diabetes, and neurodegenerative disorders, and contribute significantly to the pathogenesis of kidney diseases. This review will discuss the recent studies that investigate the critical roles of HDAC3 aberrations in kidney development, renal aging, renal cell carcinoma, renal fibrosis, chronic kidney disease, polycystic kidney disease, glomerular podocyte injury, and diabetic nephropathy. These studies reveal the distinct characters of HDAC3 aberrations that act on different molecules/signaling pathways under various renal pathological conditions, which might shed lights into the epigenetic mechanisms of renal diseases and the potentially therapeutic strategies.

Upstream open reading frame with NOTCH2NLC GGC expansion generates polyglycine aggregates and disrupts nucleocytoplasmic transport: implications for polyglycine diseases.

Neuronal intranuclear inclusion disease (NIID) is neurodegenerative disease characterized by widespread inclusions. Despite the identification of GGC repeat expansion in 5'UTR of NOTCH2NLC gene in adult-onset NIIDs, its pathogenic mechanism remains unclear. Gain-of-function poly-amino-acid proteins generated by unconventional translation have been revealed in nucleotide repeat expansion disorders, inspiring us to explore the possibility of unconventional translation in NIID. Here we demonstrated that NOTCH2NLC 5'UTR triggers the translation of a polyglycine (polyG)-containing protein, N2NLCpolyG. N2NLCpolyG accumulates in p62-positive inclusions in cultured cells, mouse models, and NIID patient tissues with NOTCH2NLC GGC expansion. Translation of N2NLCpolyG is initiated by an upstream open reading frame (uORF) embedding the GGC repeats. N2NLCpolyG tends to aggregate with the increase of GGC repeat units, and displays phase separation properties. N2NLCpolyG aggregation impairs nuclear lamina and nucleocytoplasmic transport but does not necessarily cause acute death on neuronal cells. Our study suggests a similarity of pathogenic mechanisms between NIID and another GGC-repeat disease, fragile X-associated tremor ataxia syndrome. These findings expand our knowledge of protein gain-of-function in NIID, and further highlight evidence for a novel spectrum of diseases caused by aberrant polyG protein aggregation, namely the polyG diseases.

Reduction in O-glycome induces differentially glycosylated CD44 to promote stemness and metastasis in pancreatic cancer.

Aberrant protein glycosylation has been shown to contribute to aggressive cancer, including pancreatic cancer (PC). Emerging evidence has implicated the involvement of cancer stem cells (CSCs) in PC aggressiveness; however, the contribution of glycosylation on self-renewal properties and maintenance of CSC is understudied. Here, using several in vitro and in vivo models lacking C1GALT1 expression, we identified the role of aberrant O-glycosylation in stemness properties and aggressive PC metastasis. A loss in C1GALT1 was found to result in the truncation of O-glycosylation on several glycoproteins with enrichment of Tn carbohydrate antigen. Mapping of Tn-bearing glycoproteins in C1GALT1 KO cells identified significant Tn enrichment on CSC glycoprotein CD44. Notably, a loss of C1GALT1 in PC cells was found to enhance CSC features (side population-SP, ALDH1+, and tumorspheres) and self-renewal markers NANOG, SOX9, and KLF4. Furthermore, a loss of CD44 in existing C1GALT1 KO cells decreased NANOG expression and CSC features. We determined that O-glycosylation of CD44 activates ERK/NF-kB signaling, which results in increased NANOG expression in PC cells, which facilitated the alteration of CSC features, suggesting that NANOG is essential for PC stemness. Finally, we identified that loss of C1GALT1 expression was found to augment tumorigenic and metastatic potential, while an additional loss of CD44 in these cells reversed the effects. Overall, our results identified that truncation of O-glycans on CD44 increases NANOG activation that mediates increased CSC activation.

Protein carbonylation in food and nutrition: a concise update

Protein oxidation is a topic of indisputable scientific interest given the impact of oxidized proteins on food quality and safety. Carbonylation is regarded as one of the most notable post-translational modifications in proteins and yet, this reaction and its consequences are poorly understood. From a mechanistic perspective, primary protein carbonyls (i.e. α-aminoadipic and γ-glutamic semialdehydes) have been linked to radical-mediated oxidative stress, but recent studies emphasize the role alternative carbonylation pathways linked to the Maillard reaction. Secondary protein carbonyls are introduced in proteins via covalent linkage of lipid carbonyls (i.e. protein-bound malondialdehyde). The high reactivity of protein carbonyls in foods and other biological systems indicates the intricate chemistry of these species and urges further research to provide insight into these molecular mechanisms and pathways. In particular, protein carbonyls are involved in the formation of aberrant and dysfunctional protein aggregates, undergo further oxidation to yield carboxylic acids of biological relevance and establish interactions with other biomolecules such as oxidizing lipids and phytochemicals. From a methodological perspective, the routine dinitrophenylhydrazine (DNPH) method is criticized not only for the lack of accuracy and consistency but also authors typically perform a poor interpretation of DNPH results, which leads to misleading conclusions. From a practical perspective, the biological relevance of protein carbonyls in the field of food science and nutrition is still a topic of debate. Though the implication of carbonylation on impaired protein functionality and poor protein digestibility is generally recognized, the underlying mechanism of such connections requires further clarification. From a medical perspective, protein carbonyls are highlighted as markers of protein oxidation, oxidative stress and disease. Yet, the specific role of specific protein carbonyls in the onset of particular biological impairments needs further investigations. Recent studies indicates that regardless of the origin (in vivo or dietary) protein carbonyls may act as signalling molecules which activate not only the endogenous antioxidant defences but also implicate the immune system. The present paper concisely reviews the most recent advances in this topic to identify, when applicable, potential fields of interest for future studies.

Tackling the Biological Meaning of the Human Olfactory Bulb Dyshomeostatic Proteome across Neurological Disorders: An Integrative Bioinformatic Approach.

Olfactory dysfunction is considered an early prodromal marker of many neurodegenerative diseases. Neuropathological changes and aberrant protein aggregates occur in the olfactory bulb (OB), triggering a tangled cascade of molecular events that is not completely understood across neurological disorders. This study aims to analyze commonalities and differences in the olfactory protein homeostasis across neurological backgrounds with different spectrums of smell dysfunction. For that, an integrative analysis was performed using OB proteomics datasets derived from subjects with Alzheimer’s disease (AD), Parkinson’s disease (PD), mixed dementia (mixD), dementia with Lewy bodies (DLB), frontotemporal lobar degeneration (FTLD-TDP43), progressive supranuclear palsy (PSP) and amyotrophic lateral sclerosis (ALS) with respect to OB proteome data from neurologically intact controls. A total of 80% of the differential expressed protein products were potentially disease-specific whereas the remaining 20% were commonly altered across two, three or four neurological phenotypes. A multi-level bioinformatic characterization revealed a subset of potential disease-specific transcription factors responsible for the downstream effects detected at the proteome level as well as specific densely connected protein complexes targeted by several neurological phenotypes. Interestingly, common or unique pathways and biofunctions were also identified, providing novel mechanistic clues about each neurological disease at olfactory level. The analysis of olfactory epithelium, olfactory tract and primary olfactory cortical proteotypes in a multi-disease format will functionally complement the OB dyshomeostasis, increasing our knowledge about the neurodegenerative process across the olfactory axis.

Alpha-Synuclein Aggregation in Parkinson's Disease.

Parkinson's disease (PD), a neurodegenerative disorder characterized by distinct aging-independent loss of dopaminergic neurons in substantia nigra pars compacta (SNpc) region urging toward neuronal loss. Over the decade, various key findings from clinical perspective to molecular pathogenesis have aided in understanding the genetics with assorted genes related with PD. Subsequently, several pathways have been incriminated in the pathogenesis of PD, involving mitochondrial dysfunction, protein aggregation, and misfolding. On the other hand, the sporadic form of PD cases is found with no genetic linkage, which still remain an unanswered question? The exertion in ascertaining vulnerability factors in PD considering the genetic factors are to be further dissevered in the forthcoming decades with advancement in research studies. One of the major proponents behind the prognosis of PD is the pathogenic transmutation of aberrant alpha-synuclein protein into amyloid fibrillar structures, which actuates neurodegeneration. Alpha-synuclein, transcribed by SNCA gene is a neuroprotein found predominantly in brain. It is implicated in the modulation of synaptic vesicle transport and eventual release of neurotransmitters. Due to genetic mutations and other elusive factors, the alpha-synuclein misfolds into its amyloid form. Therefore, this review aims in briefing the molecular understanding of the alpha-synuclein associated with PD.

Trends in oligomannosylation and α1,2-mannosidase expression in human cancers.

Aberrant protein glycosylation is a prominent cancer feature. While many tumour-associated glycoepitopes have been reported, advances in glycoanalytics continue to uncover new associations between glycosylation and cancer. Guided by a comprehensive literature survey suggesting that oligomannosylation (Man5–9 GlcNAc2) is a widespread and often regulated glycosignature in human cancers, we here revisit a valuable compilation of nearly 500 porous graphitized carbon LC-MS/MS N-glycomics datasets acquired across 11 human cancer types to systematically test for oligomannose-cancer associations. Firstly, the quantitative glycomics data obtained across 34 cancerous cell lines demonstrated that oligomannosylation is a pan-cancer feature spanning in a wide abundance range. In keeping with literature, our quantitative glycomics data of tumour and matching control tissues and new MALDI-MS imaging data of tissue microarrays showed a strong cancer-associated elevation of oligomannosylation in both basal cell (p = 1.78 × 10–12) and squamous cell (p = 1.23 × 10–11) skin cancer and colorectal cancer (p = 8.0 × 10–4). The glycomics data also indicated that some cancer types including gastric and liver cancer exhibit unchanged or reduced oligomannose levels, observations also supported by literature and MALDI-MS imaging data. Finally, expression data from public cancer repositories indicated that several α1,2-mannosidases are regulated in tumour tissues suggesting that these glycan-processing enzymes may contribute to the cancer-associated modulation of oligomannosylation. This omics-centric study has compiled robust glycomics and enzyme expression data revealing interesting molecular trends that open avenues to better understand the role of oligomannosylation in human cancers.