Detection of Anti-RuvBL1/2 Autoantibodies by Targeted Liquid Chromatography-Tandem Mass Spectrometry.

Background: Anti-RuvBL1/2 autoantibodies are found in patients with systemic sclerosis and systemic sclerosis-myositis overlap syndrome. Anti-RuvBL1/2 antibodies recognize conformational epitopes of the RuvBL1/2 complex, which complicates detection by solid phase assays. Here, we propose a method for detection of anti-RuvBL1/2 autoantibodies based on liquid phase immunoprecipitation combined with quantification of the precipitated RuvBL1/2 by LC-MS/MS.

The phosphatase activity of the PPP2R5D-PP2A holoenzyme modulates liprin-α1 liquid-liquid phase separation.

Liprin-α1 is a widely expressed scaffolding protein known to regulate cellular processes such as cell motility and synaptic transmission through assembly of localized higher-order molecular complexes. However, the dynamic regulation of these complexes remains poorly understood. Liquid-liquid phase separation (LLPS) is a process that concentrates proteins into cellular nanodomains, facilitating efficient spatiotemporal signaling.

Proteome selectivity profiling of photoaffinity probes derived from imidazopyrazine-kinase inhibitors.

Kinases are attractive drug targets, but the design of highly selective kinase inhibitors remains challenging. Selectivity may be evaluated against a panel of kinases, or - preferred - in a complex proteome. Probes that allow photoaffinity-labeling of their targets can facilitate this process.

PPP1R2 stimulates protein phosphatase-1 through stabilisation of dynamic subunit interactions.

Protein Ser/Thr phosphatase PP1 is always associated with one or two regulatory subunits or RIPPOs. One of the earliest evolved RIPPOs is PPP1R2, also known as Inhibitor-2. Since its discovery nearly 5 decades ago, PPP1R2 has been variously described as an inhibitor, activator or (metal) chaperone of PP1, but it is still unknown how PPP1R2 affects the function of PP1 in intact cells.

The Effect of FTY720 on Sphingolipid Imbalance and Cognitive Decline in Aged EFAD Mice.

Background: During Alzheimer's disease (AD) progression, there is a decline in the bioactive sphingolipid sphingosine-1-phosphate (S1P). Previous research showed that FTY720, an S1P mimetic, prevented cognitive decline and reduced ceramide levels in transgenic mice with familial AD carrying the human APOE4 gene (E4FAD) at 6-7 months of age.

Objective: The objective of this study is to explore the protective effects of FTY720 at late-stage AD.

The long non-coding RNA ROSALIND protects the mitochondrial translational machinery from oxidative damage.

Upregulation of mitochondrial respiration coupled with high ROS-scavenging capacity is a characteristic shared by drug-tolerant cells in several cancers. As translational fidelity is essential for cell fitness, protection of the mitochondrial and cytosolic ribosomes from oxidative damage is pivotal. While mechanisms for recognising and repairing such damage exist in the cytoplasm, the corresponding process in the mitochondria remains unclear.

PP2A phosphatase regulatory subunit PPP2R3C is a new positive regulator of the hedgehog signaling pathway.

Cellular signaling pathways rely on posttranslational modifications (PTMs) to finely regulate protein functions, particularly transcription factors. The Hedgehog (Hh) signaling cascade, crucial for embryonic development and tissue homeostasis, is susceptible to aberrations that lead to developmental anomalies and various cancers. At the core of Hh signaling are Gli proteins, whose dynamic balance between activator (GliA) and repressor (GliR) states shapes cellular outcomes.

The role of liprin-α1 phosphorylation in its liquid-liquid phase separation: regulation by PPP2R5D/PP2A holoenzyme.

Liprin-α1 is a widely expressed scaffolding protein responsible for regulating cellular processes such as focal adhesion, cell motility, and synaptic transmission. Liprin-α1 interacts with many proteins including ELKS, GIT1, liprin-β, and LAR-family receptor tyrosine protein phosphatase. Through these protein-protein interactions, liprin-α1 assembles large higher-order molecular complexes; however, the regulation of this complex assembly/disassembly is unknown.

The PPP2R1A cancer hotspot mutant p.R183W increases clofarabine resistance in uterine serous carcinoma cells by a gain-of-function mechanism.

Purpose: Uterine serous carcinoma (USC) is generally associated with poor prognosis due to a high recurrence rate and frequent treatment resistance; hence, there is a need for improved therapeutic strategies. Molecular analysis of USC identified several molecular markers, useful to improve current treatments or identify new druggable targets. PPP2R1A, encoding the Aα subunit of the tumor suppressive Ser/Thr phosphatase PP2A, is mutated in up to 40% of USCs.

CD4+ T Cells From Individuals With Type 1 Diabetes Respond to a Novel Class of Deamidated Peptides Formed in Pancreatic Islets.

The β-cell plays a crucial role in the pathogenesis of type 1 diabetes, in part through the posttranslational modification of self-proteins by biochemical processes such as deamidation. These neoantigens are potential triggers for breaking immune tolerance. We report the detection by LC-MS/MS of 16 novel Gln and 27 novel Asn deamidations in 14 disease-related proteins within inflammatory cytokine-stressed human islets of Langerhans.

TIPRL1 and its ATM-dependent phosphorylation promote radiotherapy resistance in head and neck cancer.

Purpose: TIPRL1 (target of rapamycin signaling pathway regulator-like 1) is a known interactor and inhibitor of protein phosphatases PP2A, PP4 and PP6 - all pleiotropic modulators of the DNA Damage Response (DDR). Here, we investigated the role of TIPRL1 in the radiotherapy (RT) response of Head and Neck Squamous Cell Carcinoma (HNSCC).

Methods: TIPRL1 mRNA (cBioportal) and protein expression (immunohistochemistry) in HNSCC samples were linked with clinical patient data.

PME-1 sensitizes glioblastoma cells to oxidative stress-induced cell death by attenuating PP2A-B55α-mediated inactivation of MAPKAPK2-RIPK1 signaling.

Glioblastoma (GBM) is the most common primary brain tumor in adults. Current standard therapy is surgery followed by radiotherapy, with concurrent and adjuvant temozolomide chemotherapy. GBM is characterized by almost uniformly fatal outcomes, highlighting the unmet clinical need for more efficient, biomarker-guided treatments.

Autoantibodies against the NineTeen complex and U5 RNP in systemic sclerosis.

ObjectiveMultiple spliceosome components are known autoantigens in systemic sclerosis (SSc). Here we aim to identify new and characterize rare anti-spliceosomal autoantibodies in patients with SSc without known autoantibody specificity. MethodsSera that precipitated spliceosome subcomplexes, as detected by immunoprecipitation-mass spectrometry (IP-MS), were identified from a database of 106 patients with SSc without known autoantibody specificity.

NET Proteome in Established Type 1 Diabetes Is Enriched in Metabolic Proteins.

Background And Aims: Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by a T-cell-mediated destruction of the pancreatic insulin-producing beta cells. A growing body of evidence suggests that abnormalities in neutrophils and neutrophil extracellular trap (NET) formation (NETosis) are associated with T1D pathophysiology. However, little information is available on whether these changes are primary neutrophil defects or related to the environmental signals encountered during active disease.

Human pancreatic cancer patients with Epithelial-to-Mesenchymal Transition and an aggressive phenotype show a disturbed balance in Protein Phosphatase Type 2A expression and functionality.

Background: Pancreatic ductal adenocarcinoma (PDAC) has a low survival, its incidence is rising and little therapeutic improvements are expected in the near future. It has been observed that Epithelial-to-Mesenchymal transition (EMT) contributes (including in PDAC) to a more aggressive cancer phenotype. Additionally, largely unexplored, studies indicate a mechanistic interplay between Protein Phosphatase Type 2A (PP2A) enzymes and EMT that could offer treatment opportunities.

Identification of Peroxisome-Derived Hydrogen Peroxide-Sensitive Target Proteins Using a YAP1C-Based Genetic Probe.

As the reversible oxidation of protein cysteine thiols is an important mechanism in signal transduction, it is essential to have access to experimental approaches that allow for spatiotemporal indexing of the cellular sulfenome in response to local changes in HO levels. Here, we provide a step-by-step guide for enriching and identifying the sulfenome of mammalian cells at the subcellular level in response to peroxisome-derived HO by the combined use of (i) a previously developed cell line in which peroxisomal HO production can be induced in a time- and dose-dependent manner; (ii) YAP1C, a genetically encoded yeast AP-1-like transcription factor-based probe that specifically reacts with S-sulfenylated cysteines and traps them through mixed disulfide bonds; and (iii) mass spectrometry. Given that this approach includes differential labeling of reduced and reversibly oxidized cysteine residues, it can also provide additional information on the positions of the modified cysteines.

Structural mechanism for inhibition of PP2A-B56α and oncogenicity by CIP2A.

The protein phosphatase 2A (PP2A) heterotrimer PP2A-B56α is a human tumour suppressor. However, the molecular mechanisms inhibiting PP2A-B56α in cancer are poorly understood. Here, we report molecular level details and structural mechanisms of PP2A-B56α inhibition by an oncoprotein CIP2A.

Antinuclear antibodies in individuals with COVID-19 reflect underlying disease: Identification of new autoantibodies in systemic sclerosis (CDK9) and malignancy (RNF20, RCC1, TRIP13).

A high prevalence of antinuclear antibodies (ANA) in COVID-19 has been insinuated, but the nature of the target antigens is poorly understood. We studied ANA by indirect immunofluorescence in 229 individuals with COVID-19. The target antigens of high titer ANA (≥1:320) were determined by immunoprecipitation (IP) combined with liquid-chromatography-mass spectrometry (MS).

Identification of new telomere- and telomerase-associated autoantigens in systemic sclerosis.

Purpose: In up to 20% of patients with systemic sclerosis (SSc) no known autoantibody specificity can be identified. Recently discovered autoantigens, such as telomeric repeat binding factor 1 (TERF1), as well as established autoantigens, like RuvBL1/2, are associated with telomere and telomerase biology. We aimed to identify new telomere- and telomerase-associated autoantigens in patients with SSc without known autoantibody specificity.

Mass spectrometry-based identification of new anti-Ly and known antisynthetase autoantibodies.

Objectives: To discover new and detect known antisynthetase autoantibodies (ASAs) through protein immunoprecipitation combined with gel-free liquid chromatography-tandem mass spectrometry (IP-MS).

Methods: IP-MS was performed using sera of individuals showing features of antisynthetase syndrome (ASyS) without (n=5) and with (n=12) previously detected ASAs, and healthy controls (n=4). New candidate aminoacyl-tRNA-synthetase (ARS) autoantigens identified through unbiased IP-MS were confirmed by IP-western blot.

Extracellular vesicle-derived miRNAs improve stem cell-based therapeutic approaches in muscle wasting conditions.

Skeletal muscle holds an intrinsic capability of growth and regeneration both in physiological conditions and in case of injury. Chronic muscle illnesses, generally caused by genetic and acquired factors, lead to deconditioning of the skeletal muscle structure and function, and are associated with a significant loss in muscle mass. At the same time, progressive muscle wasting is a hallmark of aging.

Anti-Ki/anti-PA28γ autoantibodies contribute to the HEp-2 indirect immunofluorescence nuclear speckled pattern.

Objectives: Antinuclear antibodies (ANAs) are associated with several autoimmune diseases. Indirect immunofluorescence (IIF) on human epithelial type 2 (HEp-2) cells is the golden standard for ANA detection in the clinic. In case of a positive HEp-2 IIF test result, follow-up tests are done to determine autoantibody specificity.

2D-DIGE Analysis of Liver Disease in Mice.

Hepatocellular carcinoma (HCC) is the major type of primary liver cancer. In this chapter, we describe our routine two-dimensional difference gel electrophoresis (2D-DIGE) workflow for analysis of mouse liver tissue in physiological conditions, as well as of mouse HCC. 2D-DIGE still constitutes a valuable comparative proteomics technique, not only providing information on global protein expression in a sample but also on potential posttranslational protein modifications, occurrence of protein degradation fragments, and the existence of protein isoforms.