Methylmalonic acidemia triggers lysosomal-autophagy dysfunctions.

BACKGROUND: Methylmalonic acidemia (MMA) is a rare inborn error of propionate metabolism caused by deficiency of the mitochondrial methylmalonyl-CoA mutase (MUT) enzyme. As matter of fact, MMA patients manifest impairment of the primary metabolic network with profound damages that involve several cell components, many of which have not been discovered yet. We employed cellular models and patients-derived fibroblasts to refine and uncover new pathologic mechanisms connected with MUT deficiency through the combination of multi-proteomics and bioinformatics approaches. RESULTS: Our data show that MUT deficiency is connected with profound proteome dysregulations, revealing molecular actors involved in lysosome and autophagy functioning. To elucidate the effects of defective MUT on lysosomal and autophagy regulation, we analyzed the morphology and functionality of MMA-lysosomes that showed deep alterations, thus corroborating omics data. Lysosomes of MMA cells present as enlarged vacuoles with low degradative capabilities. Notwithstanding, treatment with an anti-propionigenic drug is capable of totally rescuing lysosomal morphology and functional activity in MUT-deficient cells. These results indicate a strict connection between MUT deficiency and lysosomal-autophagy dysfunction, providing promising therapeutic perspectives for MMA. CONCLUSIONS: Defective homeostatic mechanisms in the regulation of autophagy and lysosome functions have been demonstrated in MUT-deficient cells. Our data prove that MMA triggers such dysfunctions impacting on autophagosome-lysosome fusion and lysosomal activity.

Multi-phased Kinetics and Interaction of Protein Kinase Signaling in Glycoprotein VI-Induced Platelet αIIbß3 Integrin Activation and Degranulation.

BACKGROUND: Platelet glycoprotein VI (GPVI) stimulation activates the tyrosine kinases Syk and Btk, and the effector proteins phospholipase Cγ 2 (PLCγ2) and protein kinase C (PKC). Here, the activation sequence, crosstalk, and downstream effects of this Syk-Btk-PKC signalosome in human platelets were analyzed. METHODS AND RESULTS: Using immunoblotting, we quantified 14 regulated phospho-sites in platelets stimulated by convulxin with and without inhibition of Syk, Btk, or PKC. Convulxin induced fast, reversible tyrosine phosphorylation (pY) of Syk, Btk, LAT, and PLCγ2, followed by reversible serine/threonine phosphorylation (pS/T) of Syk, Btk, and downstream kinases MEK1/2, Erk1/2, p38, and Akt. Syk inhibition by PRT-060318 abolished all phosphorylations, except Syk pY352. Btk inhibition by acalabrutinib strongly decreased Btk pY223/pS180, Syk pS297, PLCγ2 pY759/Y1217, MEK1/2 pS217/221, Erk1/2 pT202/Y204, p38 pT180/Y182, and Akt pT308/S473. PKC inhibition by GF109203X abolished most pS/T phosphorylations except p38 pT180/Y182 and Akt pT308, but enhanced most Y-phosphorylations. Acalabrutinib, but not GF109203X, suppressed convulxin-induced intracellular Ca2+ mobilization, whereas all three protein kinase inhibitors abolished degranulation and αIIbß3 integrin activation assessed by flow cytometry. Inhibition of autocrine ADP effects by AR-C669931 partly diminished convulxin-triggered degranulation. CONCLUSION: Kinetic analysis of GPVI-initiated multisite protein phosphorylation in human platelets demonstrates multiple phases and interactions of tyrosine and serine/threonine kinases with activation-altering feedforward and feedback loops partly involving PKC. The protein kinase inhibitor effects on multisite protein phosphorylation and functional readouts reveal that the signaling network of Syk, Btk, and PKC controls platelet granule exocytosis and αIIbß3 integrin activation.

Proteomic studies in VWA1-related neuromyopathy allowed new pathophysiological insights and the definition of blood biomarkers.

Bi-allelic variants in VWA1, encoding Von Willebrand Factor A domain containing 1 protein localized to the extracellular matrix (ECM), were linked to a neuromuscular disorder with manifestation in child- or adulthood. Clinical findings indicate a neuromyopathy presenting with muscle weakness. Given that pathophysiological processes are still incompletely understood, and biomarkers are still missing, we aimed to identify blood biomarkers of pathophysiological relevance: white blood cells (WBC) and plasma derived from six VWA1-patients were investigated by proteomics. Four proteins, BET1, HNRNPDL, NEFM and PHGDH, known to be involved in neurological diseases and dysregulated in WBC were further validated by muscle-immunostainings unravelling HNRNPDL as a protein showing differences between VWA1-patients, healthy controls and patients suffering from neurogenic muscular atrophy and BICD2-related neuromyopathy. Immunostaining studies of PHGDH indicate its involvement in apoptotic processes via co-localisation with caspase-3. NEFM showed an increase in cells within the ECM in biopsies of all patients studied. Plasma proteomics unravelled dysregulation of 15 proteins serving as biomarker candidates among which a profound proportion of increased ones (6/11) are mostly related to antioxidative processes and have even partially been described as blood biomarkers for other entities of neuromuscular disorders before. CRP elevated in plasma also showed an increase in the extracellular space of VWA1-mutant muscle. Results of our combined studies for the first time describe pathophysiologically relevant biomarkers for VWA1-related neuromyopathy and suggest that VWA1-patient derived blood might hold the potential to study disease processes of clinical relevance, an important aspect for further preclinical studies.

Highly sensitive therapeutic drug monitoring of infliximab in serum by targeted mass spectrometry in comparison to ELISA data.

BACKGROUND: Presently, antibody concentration measurements for patients undergoing treatment are predominantly determined by ELISA, which still comes with known disadvantages. Therefore, our aim was to establish a targeted mass-spectrometric assay enabling the reproducible absolute quantification of peptides from the hypervariable and interaction regions of infliximab. METHODS: Peptides of infliximab were measured post-trypsin digestion and subsequent separation on a Vanquish Horizon UHPLC coupled to a TSQ Altis Triple-Quad mass spectrometer. Normalization and absolute quantification were conducted using stable isotope-synthesized peptides. Calibration curves covering a range of 0.25-50 µg/ml were employed for quantitation. RESULTS: We demonstrated the substantial influence of peptide selection, choice of hydrolase for digestion, and digestion time on absolute peptide yield (28-44% for peptide 1 and 64-97% for peptide 2). Furthermore, we showed that the generated calibration curves for absolute quantification were highly reproducible and robust (LLOQ1 0.72 µg/ml and LLOQ2 1.00 µg/ml) over several months. In comparison to ELISA values, the absolute values obtained by mass spectrometry often yielded lower results for both targeted peptides. CONCLUSIONS: In this study, a semi-automated workflow was employed and tested with 8 patients and corresponding replicates (n = 3-4). We demonstrated the robust implementation of calibration curves for the absolute quantification of infliximab in patient samples, with coefficients of variation ranging from 0.5 to 9%. Taken together, we have developed a platform enabling the rapid (2 days of sample preparation and 30 min of measurement time per sample) and robust quantification of Infliximab antibody concentration in patients. The use of mass spectrometry also facilitates the straightforward expansion of the method to include additional antibody peptides.

Endothelium-mediated regulation of platelet activation: Involvement of multiple protein kinases.

The endothelial regulation of platelet activity is incompletely understood. Here we describe novel approaches to find molecular pathways implicated on the platelet-endothelium interaction. Using high-shear whole-blood microfluidics, employing coagulant or non-coagulant conditions at physiological temperature, we observed that the presence of human umbilical vein endothelial cells (HUVEC) strongly suppressed platelet adhesion and activation, via the collagen receptor glycoprotein VI (GPVI) and the PAR receptors for thrombin. Real-time monitoring of the cytosolic Ca2+ rises in the platelets indicated no major improvement of inhibition by prostacyclin or nitric oxide. Similarly under stasis, exposure of isolated platelets to HUVEC reduced the Ca2+ responses by collagen-related peptide (CRP-XL, GPVI agonist) and thrombin (PAR agonist). We then analyzed the label-free phosphoproteome of platelets (three donors), exposed to HUVEC, CRP-XL, and/or thrombin. High-resolution mass spectrometry gave 5463 phosphopeptides, corresponding to 1472 proteins, with good correlation between biological and technical replicates (R > .86). Stringent filtering steps revealed 26 regulatory pathways (Reactome) and 143 regulated kinase substrates (PhosphoSitePlus), giving a set of protein phosphorylation sites that was differentially (44) or similarly (110) regulated by HUVEC or agonist exposure. The differential regulation was confirmed by stable-isotope analysis of platelets from two additional donors. Substrate analysis indicated major roles of poorly studied protein kinase classes (MAPK, CDK, DYRK, STK, PKC members). Collectively, these results reveal a resetting of the protein phosphorylation profile in platelets exposed to endothelium or to conventional agonists and to endothelium-promoted activity of a multi-kinase network, beyond classical prostacyclin and nitric oxide actors, that may contribute to platelet inhibition.

In-vivo tracking of deuterium metabolism in mouse organs using LC-MS/MS.

Mass spectrometry-based metabolomics with stable isotope labeling (SIL) is an established tool for sensitive and precise analyses of tissue metabolism, its flux, and pathway activities in diverse models of physiology and disease. Despite the simplicity and broad applicability of deuterium (2H)-labeled precursors for tracing metabolic pathways with minimal biological perturbations, they are rarely employed in LC-MS/MS-guided metabolomics. In this study, we have developed a LC-MS/MS-guided workflow to trace deuterium metabolism in mouse organs following 2H7 -glucose infusion. The workflow includes isotopically labeled glucose infusion, mouse organ isolation and metabolite extraction, zwitterion-based hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution tandem mass spectrometry, targeted data acquisition for sensitive detection of deuterated metabolites, a spectral library of over 400 metabolite standards, and multivariate data analysis with pathway mapping. The optimized method was validated for matrix effects, normalization, and quantification to provide both tissue metabolomics and tracking the in-vivo metabolic fate of deuterated glucose through key metabolic pathways. We quantified more than 100 metabolites in five major mouse organ tissues (liver, kidney, brain, brown adipose tissue, and heart). Furthermore, we mapped isotopologues of deuterated metabolites from glycolysis, tricarboxylic acid (TCA) cycle, and amino acid pathways, which are significant for studying both health and various diseases. This study will open new avenues in LC-MS based analysis of 2H-labeled tissue metabolism research in animal models and clinical settings.

A Homozygous NDUFS6 Variant Associated with Neuropathy and Optic Atrophy.

Background: The NADH dehydrogenase [ubiquinone] iron-sulfur protein 6 (NDUFS6) gene encodes for an accessory subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (complex I). Bi-allelic NDUFS6 variants have been linked with a severe disorder mostly reported as a lethal infantile mitochondrial disease (LMID) or Leigh syndrome (LS). Objective: Here, we identified a homozygous variant (c.309 + 5 G > A) in NDUFS6 in one male patient with axonal neuropathy accompanied by loss of small fibers in skin biopsy and further complicated by optic atrophy and borderline intellectual disability. Methods: To address the pathogenicity of the variant, biochemical studies (mtDNA copy number quantification, ELISA, Proteomic profiling) of patient-derived leukocytes were performed. Results: The analyses revealed loss of NDUFS6 protein associated with a decrease of three further mitochondrial NADH dehydrogenase subunit/assembly proteins (NDUFA12, NDUFS4 and NDUFV1). Mitochondrial copy number is not altered in leukocytes and the mitochondrial biomarker GDF15 is not significantly changed in serum. Conclusions: Hence, our combined clinical and biochemical data strengthen the concept of NDUFS6 being causative for a very rare form of axonal neuropathy associated with optic atrophy and borderline intellectual disability, and thus expand (i) the molecular genetic landscape of neuropathies and (ii) the clinical spectrum of NDUFS6-associated phenotypes.

Multi-level profiling unravels mitochondrial dysfunction in myotonic dystrophy type 2.

Myotonic dystrophy type 2 (DM2) is an autosomal-dominant multisystemic disease with a core manifestation of proximal muscle weakness, muscle atrophy, myotonia, and myalgia. The disease-causing CCTG tetranucleotide expansion within the CNBP gene on chromosome 3 leads to an RNA-dominated spliceopathy, which is currently untreatable. Research exploring the pathophysiological mechanisms in myotonic dystrophy type 1 has resulted in new insights into disease mechanisms and identified mitochondrial dysfunction as a promising therapeutic target. It remains unclear whether similar mechanisms underlie DM2 and, if so, whether these might also serve as potential therapeutic targets. In this cross-sectional study, we studied DM2 skeletal muscle biopsy specimens on proteomic, molecular, and morphological, including ultrastructural levels in two separate patient cohorts consisting of 8 (explorative cohort) and 40 (confirmatory cohort) patients. Seven muscle biopsy specimens from four female and three male DM2 patients underwent proteomic analysis and respiratory chain enzymology. We performed bulk RNA sequencing, immunoblotting of respiratory chain complexes, mitochondrial DNA copy number determination, and long-range PCR (LR-PCR) to study mitochondrial DNA deletions on six biopsies. Proteomic and transcriptomic analyses revealed a downregulation of essential mitochondrial proteins and their respective RNA transcripts, namely of subunits of respiratory chain complexes I, III, and IV (e.g., mt-CO1, mt-ND1, mt-CYB, NDUFB6) and associated translation factors (TACO1). Light microscopy showed mitochondrial abnormalities (e.g., an age-inappropriate amount of COX-deficient fibers, subsarcolemmal accumulation) in most biopsy specimens. Electron microscopy revealed widespread ultrastructural mitochondrial abnormalities, including dysmorphic mitochondria with paracrystalline inclusions. Immunofluorescence studies with co-localization of autophagy (p62, LC-3) and mitochondrial marker proteins (TOM20, COX-IV), as well as immunohistochemistry for mitophagy marker BNIP3 indicated impaired mitophagic flux. Immunoblotting and LR-PCR did not reveal significant differences between patients and controls. In contrast, mtDNA copy number measurement showed a reduction of mtDNA copy numbers in the patient group compared to controls. This first multi-level study of DM2 unravels thus far undescribed functional and structural mitochondrial abnormalities. However, the molecular link between the tetranucleotide expansion and mitochondrial dysfunction needs to be further elucidated.

Multiple reaction monitoring assays for large-scale quantitation of proteins from 20 mouse organs and tissues.

Mouse is the mammalian model of choice to study human health and disease due to its size, ease of breeding and the natural occurrence of conditions mimicking human pathology. Here we design and validate multiple reaction monitoring mass spectrometry (MRM-MS) assays for quantitation of 2118 unique proteins in 20 murine tissues and organs. We provide open access to technical aspects of these assays to enable their implementation in other laboratories, and demonstrate their suitability for proteomic profiling in mice by measuring normal protein abundances in tissues from three mouse strains: C57BL/6NCrl, NOD/SCID, and BALB/cAnNCrl. Sex- and strain-specific differences in protein abundances are identified and described, and the measured values are freely accessible via our MouseQuaPro database: http://mousequapro.proteincentre.com . Together, this large library of quantitative MRM-MS assays established in mice and the measured baseline protein abundances represent an important resource for research involving mouse models.

Blood-Derived Lipid and Metabolite Biomarkers in Cardiovascular Research from Clinical Studies: A Recent Update.

The primary prevention, early detection, and treatment of cardiovascular disease (CVD) have been long-standing scientific research goals worldwide. In the past decades, traditional blood lipid profiles have been routinely used in clinical practice to estimate the risk of CVDs such as atherosclerotic cardiovascular disease (ASCVD) and as treatment targets for the primary prevention of adverse cardiac events. These blood lipid panel tests often fail to fully predict all CVD risks and thus need to be improved. A comprehensive analysis of molecular species of lipids and metabolites (defined as lipidomics and metabolomics, respectively) can provide molecular insights into the pathophysiology of the disease and could serve as diagnostic and prognostic indicators of disease. Mass spectrometry (MS) and nuclear magnetic resonance (NMR)-based lipidomics and metabolomics analysis have been increasingly used to study the metabolic changes that occur during CVD pathogenesis. In this review, we provide an overview of various MS-based platforms and approaches that are commonly used in lipidomics and metabolomics workflows. This review summarizes the lipids and metabolites in human plasma/serum that have recently (from 2018 to December 2022) been identified as promising CVD biomarkers. In addition, this review describes the potential pathophysiological mechanisms associated with candidate CVD biomarkers. Future studies focused on these potential biomarkers and pathways will provide mechanistic clues of CVD pathogenesis and thus help with the risk assessment, diagnosis, and treatment of CVD.