Molecular mechanisms in chloroquine-exposed muscle cells elucidated by combined proteomic and microscopic studies.

OBJECTIVES: Chloroquine (CQ) is an antimalarial drug with a growing number of applications as recently demonstrated in attempts to treat Covid-19. For decades, it has been well known that skeletal and cardiac muscle cells might display vulnerability against CQ exposure resulting in the clinical manifestation of a CQ-induced myopathy. In line with the known effect of CQ on inhibition of the lysosomal function and thus cellular protein clearance, the build-up of autophagic vacuoles along with protein aggregates is a histological hallmark of the disease. Given that protein targets of the perturbed proteostasis are still not fully discovered, we applied different proteomic and immunological-based studies to improve the current understanding of the biochemical nature of CQ-myopathy. METHODS: To gain a comprehensive understanding of the molecular pathogenesis of this acquired myopathy and to define proteins targets as well as pathophysiological processes beyond impaired proteolysis, utilising CQ-treated C2C12 cells and muscle biopsies derived from CQ-myopathy patients, we performed different proteomic approaches and Coherent Anti-Stokes Raman Scattering (CARS) microscopy, in addition to immunohistochemical studies. RESULTS: Our combined studies confirmed an impact of CQ-exposure on proper protein processing/folding and clearance, highlighted changes in the interactome of p62, a known aggregation marker and hereby identified the Rett syndrome protein MeCP2 as being affected. Moreover, our approach revealed-among others-a vulnerability of the extracellular matrix, cytoskeleton and lipid homeostasis. CONCLUSION: We demonstrated that CQ exposure (secondarily) impacts biological processes beyond lysosomal function and linked a variety of proteins with known roles in the manifestation of other neuromuscular diseases.

Simple, scalable, and ultrasensitive tip-based identification of protease substrates.

Proteases are in the center of many diseases, and consequently, proteases and their substrates are important drug targets as represented by an estimated 5-10% of all drugs under development. Mass spectrometry has been an indispensable tool for the discovery of novel protease substrates, particularly through the proteome-scale enrichment of so-called N-terminal peptides representing endogenous protein N termini. Methods such as combined fractional diagonal chromatography (COFRADIC)1 and, later, terminal amine isotopic labeling of substrates (TAILS) have revealed numerous insights into protease substrates and consensus motifs. We present an alternative and simple protocol for N-terminal peptide enrichment, based on charge-based fractional diagonal chromatography (ChaFRADIC) and requiring only well-established protein chemistry and a pipette tip. Using iTRAQ-8-plex, we quantified on average 2,073 ± 52 unique N-terminal peptides from only 4.3 µg per sample/channel, allowing the identification of proteolytic targets and consensus motifs. This high sensitivity may even allow working with clinical samples such as needle biopsies in the future. We applied our method to study the dynamics of staurosporine-induced apoptosis. Our data demonstrate an orchestrated regulation of specific pathways after 1.5 h, 3 h, and 6 h of treatment, with many important players of homeostasis targeted already after 1.5 h. We additionally observed an early multilevel modulation of the splicing machinery both by proteolysis and phosphorylation. This may reflect the known role of alternative splicing variants for a variety of apoptotic genes, which seems to be a driving force of staurosporine-induced apoptosis.

Protease Specificity Profiling in a Pipet Tip Using "Charge-Synchronized" Proteome-Derived Peptide Libraries.

About 2% of the genome of human and other organisms codes for proteases. An important step toward deciphering the biological function of a protease and designing inhibitors is the profiling of protease specificity. In this work we present a novel, label-free, proteomics-based protease specificity profiling method that only requires simple sample preparation steps. It uses proteome-derived peptide libraries and enriches the cleaved sequences using strong cation exchange chromatography (SCX) material in a pipet tip. As a demonstration of the method's versatility, we successfully determined the specificity of GluC, caspase-3, chymotrypsin, MMP-1 and cathepsin G from several hundreds to almost 2000 cleavage events per protease. Interestingly, we also found a novel intrinsic preference of cathepsin G for Asn at the P1 subsite, which we confirmed using synthetic peptides. Overall, this method is straightforward and requires so far the lowest investment in material and equipment for protease specificity profiling. Therefore, we think it will be applicable in any biochemistry laboratory and promote an increased understanding of protease specificity.

Detecting post-translational modification signatures as potential biomarkers in clinical mass spectrometry.

INTRODUCTION: Numerous diseases are caused by changes in post-translational modifications (PTMs). Therefore, the number of clinical proteomics studies that include the analysis of PTMs is increasing. Combining complementary information-for example changes in protein abundance, PTM levels, with the genome and transcriptome (proteogenomics)-holds great promise for discovering important drivers and markers of disease, as variations in copy number, expression levels, or mutations without spatial/functional/isoform information is often insufficient or even misleading. Areas covered: We discuss general considerations, requirements, pitfalls, and future perspectives in applying PTM-centric proteomics to clinical samples. This includes samples obtained from a human subject, for instance (i) bodily fluids such as plasma, urine, or cerebrospinal fluid, (ii) primary cells such as reproductive cells, blood cells, and (iii) tissue samples/biopsies. Expert commentary: PTM-centric discovery proteomics can substantially contribute to the understanding of disease mechanisms by identifying signatures with potential diagnostic or even therapeutic relevance but may require coordinated efforts of interdisciplinary and eventually multi-national consortia, such as initiated in the cancer moonshot program. Additionally, robust and standardized mass spectrometry (MS) assays-particularly targeted MS, MALDI imaging, and immuno-MALDI-may be transferred to the clinic to improve patient stratification for precision medicine, and guide therapies.

Mild proteasomal stress improves photosynthetic performance in Arabidopsis chloroplasts.

The proteasome is an essential protein-degradation machinery in eukaryotic cells that controls protein turnover and thereby the biogenesis and function of cell organelles. Chloroplasts import thousands of nuclear-encoded precursor proteins from the cytosol, suggesting that the bulk of plastid proteins is transiently exposed to the cytosolic proteasome complex. Therefore, there is a cytosolic equilibrium between chloroplast precursor protein import and proteasomal degradation. We show here that a shift in this equilibrium, induced by mild genetic proteasome impairment, results in elevated precursor protein abundance in the cytosol and significantly increased accumulation of functional photosynthetic complexes in protein import-deficient chloroplasts. Importantly, a proteasome lid mutant shows improved photosynthetic performance, even in the absence of an import defect, signifying that functional precursors are continuously degraded. Hence, turnover of plastid precursors in the cytosol represents a mechanism to constrain thylakoid membrane assembly and photosynthetic electron transport.