A new era of cysteine proteomics - Technological advances in thiol biology.

Cysteines are amenable to a diverse set of modifications that exhibit critical regulatory functions over the proteome and thereby control a wide range of cellular processes. Proteomic technologies have emerged as a powerful strategy to interrogate cysteine modifications across the proteome. Recent advancements in enrichment strategies, multiplexing capabilities and increased analytical sensitivity have enabled deeper quantitative cysteine profiling, capturing a substantial proportion of the cysteine proteome. This is complemented by a rapidly growing repertoire of analytical strategies illuminating the diverse landscape of cysteine modifications. Cysteine chemoproteomics technologies have evolved into a powerful strategy to facilitate the development of covalent drugs, opening unprecedented opportunities to target the extensive undrugged proteome. Herein we review recent technological and scientific advances that shape the cysteine proteomics field.

A comprehensive landscape of the zinc-regulated human proteome.

Zinc is an essential micronutrient that regulates a wide range of physiological processes, principally through Zn 2+ binding to protein cysteine residues. Despite being critical for modulation of protein function, for the vast majority of the human proteome the cysteine sites subject to regulation by Zn 2+ binding remain undefined. Here we develop ZnCPT, a comprehensive and quantitative mapping of the zinc-regulated cysteine proteome. We define 4807 zinc-regulated protein cysteines, uncovering protein families across major domains of biology that are subject to either constitutive or inducible modification by zinc. ZnCPT enables systematic discovery of zinc-regulated structural, enzymatic, and allosteric functional domains. On this basis, we identify 52 cancer genetic dependencies subject to zinc regulation, and nominate malignancies sensitive to zinc-induced cytotoxicity. In doing so, we discover a mechanism of zinc regulation over Glutathione Reductase (GSR) that drives cell death in GSR-dependent lung cancers. We provide ZnCPT as a resource for understanding mechanisms of zinc regulation over protein function.

Once-Daily Oxycodone Prolonged-Release Tablets Are Resistant to Alcohol-Induced Dose Dumping: Results From a Randomized Trial in Healthy Volunteers.

The objective of this study was to determine the effect of concomitant alcohol intake on the bioavailability of oxycodone from an oxycodone once-daily (OOD) formulation and an oxycodone twice-daily (OTD) formulation. A phase I, open-label, randomized, crossover alcohol interaction study in 20 healthy volunteers under fasting conditions was conducted. Participants received five treatments, OOD with 240 mL of 0%, 20%, or 40% alcohol; and OTD with 240 mL of 0% or 40% alcohol. Pharmacokinetic parameters did not differ between participants taking OOD with water or with 240 mL of 20% alcohol. There was a slight increase in overall oxycodone absorption from OOD with 40% alcohol but no increase in peak absorption. Oxycodone absorption from OTD showed peak and overall increases with 40% alcohol but maintained a prolonged-release profile. Although it is recommended that alcohol be avoided while taking opioids, there was no evidence of alcohol-induced dose dumping in these oxycodone formulations.

Mutations in PMPCB Encoding the Catalytic Subunit of the Mitochondrial Presequence Protease Cause Neurodegeneration in Early Childhood.

Mitochondrial disorders causing neurodegeneration in childhood are genetically heterogeneous, and the underlying genetic etiology remains unknown in many affected individuals. We identified biallelic variants in PMPCB in individuals of four families including one family with two affected siblings with neurodegeneration and cerebellar atrophy. PMPCB encodes the catalytic subunit of the essential mitochondrial processing protease (MPP), which is required for maturation of the majority of mitochondrial precursor proteins. Mitochondria isolated from two fibroblast cell lines and induced pluripotent stem cells derived from one affected individual and differentiated neuroepithelial stem cells showed reduced PMPCB levels and accumulation of the processing intermediate of frataxin, a sensitive substrate for MPP dysfunction. Introduction of the identified PMPCB variants into the homologous S. cerevisiae Mas1 protein resulted in a severe growth and MPP processing defect leading to the accumulation of mitochondrial precursor proteins and early impairment of the biogenesis of iron-sulfur clusters, which are indispensable for a broad range of crucial cellular functions. Analysis of biopsy materials of an affected individual revealed changes and decreased activity in iron-sulfur cluster-containing respiratory chain complexes and dysfunction of mitochondrial and cytosolic Fe-S cluster-dependent enzymes. We conclude that biallelic mutations in PMPCB cause defects in MPP proteolytic activity leading to dysregulation of iron-sulfur cluster biogenesis and triggering a complex neurological phenotype of neurodegeneration in early childhood.

The novel mitochondrial matrix protease Ste23 is required for efficient presequence degradation and processing.

Approximately 70% of mitochondrial precursor proteins are imported from the cytosol via N-terminal presequences, which are cleaved upon exposure to the mitochondrial processing protease MPP in the matrix. Cleaved presequence peptides then need to be efficiently degraded, and impairment of this clearance step, for example, by amyloid ß peptides, causes feedback inhibition of MPP, leading ultimately to accumulation of immature precursor proteins within mitochondria. Degradation of mitochondrial peptides is performed by Cym1 in yeast and its homologue, PreP, in humans. Here we identify the novel mitochondrial matrix protease Ste23 in yeast, a homologue of human insulin-degrading enzyme, which is required for efficient peptide degradation. Ste23 and Cym1 tightly cooperate to ensure the correct functioning of the essential presequence processing machinery.

Isolated syndesmotic injuries in acute ankle sprains: diagnostic significance of clinical examination and MRI.

PURPOSE: Acute ankle sprains are frequently accompanied by syndesmotic injuries. These injuries are often overlooked in clinical examinations. The aim of this study was (1) to evaluate the incidence of syndesmotic injuries in acute ankle sprains using MRI, (2) to determine the accuracy of common clinical diagnostic tests, (3) to analyse their inter-rater reliability, and (4) to evaluate the role of clinical symptoms in the diagnosis of syndesmotic injuries. METHODS: A total of 100 patients with acute ankle sprain injury without associated fractures in plane radiographs were enrolled. The clinical assessment was performed by two independent examiners. Local findings, ankle ligament palpation, squeeze test, external rotation test, Drawer test, Cotton test, and the crossed-leg test (two examiners) were compared with MRI results (read by two blinded radiologists) as a reference standard. RESULTS: Ninety-six participants (57% male) met the inclusion criteria. MRI detected a ruptured anterior inferior tibiofibular ligament (AITFL) in 14 patients (15%); 9 partial tears and 5 complete tears were evident. Evidence of pain at rest was found to predict syndesmotic injuries most accurately (p = 0.039). The palpation test over the proximal fibula produced the highest inter-rater correlation (κ = 0.65), but the lowest sensitivity for syndesmotic injuries of 8%. All other clinical tests demonstrated moderate to fair inter-rater reliabilities (κ = 0.37-0.52). Low sensitivity values were found with all clinical tests (13.9-55.6%). CONCLUSION: In this study, clinical examination was insufficient to detect syndesmotic injuries in acute ankle sprains. MRI scanning revealed a syndesmotic lesion in 15% of patients. MRI scanning should be recommended in patients with ongoing pain at rest following ankle sprains. LEVEL OF EVIDENCE: I.

Singlet oxygen treatment of tumor cells triggers extracellular singlet oxygen generation, catalase inactivation and reactivation of intercellular apoptosis-inducing signaling.

Intracellular singlet oxygen generation in photofrin-loaded cells caused cell death without discrimination between nonmalignant and malignant cells. In contrast, extracellular singlet oxygen generation caused apoptosis induction selectively in tumor cells through singlet oxygen-mediated inactivation of tumor cell protective catalase and subsequent reactivation of intercellular ROS-mediated apoptosis signaling through the HOCl and the NO/peroxynitrite signaling pathway. Singlet oxygen generation by extracellular photofrin alone was, however, not sufficient for optimal direct inactivation of catalase, but needed to trigger the generation of cell-derived extracellular singlet oxygen through the interaction between H2O2 and peroxynitrite. Thereby, formation of peroxynitrous acid, generation of hydroxyl radicals and formation of perhydroxyl radicals (HO2(.)) through hydroxyl radical/H2O2 interaction seemed to be required as intermediate steps. This amplificatory mechanism led to the formation of singlet oxygen at a sufficiently high concentration for optimal inactivation of membrane-associated catalase. At low initial concentrations of singlet oxygen, an additional amplification step needed to be activated. It depended on singlet oxygen-dependent activation of the FAS receptor and caspase-8, followed by caspase-8-mediated enhancement of NOX activity. The biochemical mechanisms described here might be considered as promising principle for the development of novel approaches in tumor therapy that specifically direct membrane-associated catalase of tumor cells and thus utilize tumor cell-specific apoptosis-inducing ROS signaling.

Amyloid-ß peptide induces mitochondrial dysfunction by inhibition of preprotein maturation.

Most mitochondrial proteins possess N-terminal presequences that are required for targeting and import into the organelle. Upon import, presequences are cleaved off by matrix processing peptidases and subsequently degraded by the peptidasome Cym1/PreP, which also degrades Amyloid-beta peptides (Aß). Here we find that impaired turnover of presequence peptides results in feedback inhibition of presequence processing enzymes. Moreover, Aß inhibits degradation of presequence peptides by PreP, resulting in accumulation of mitochondrial preproteins and processing intermediates. Dysfunctional preprotein maturation leads to rapid protein degradation and an imbalanced organellar proteome. Our findings reveal a general mechanism by which Aß peptide can induce the multiple diverse mitochondrial dysfunctions accompanying Alzheimer's disease.

Protein X of Borna disease virus inhibits apoptosis and promotes viral persistence in the central nervous systems of newborn-infected rats.

Borna disease virus (BDV) is a neurotropic member of the order Mononegavirales with noncytolytic replication and obligatory persistence in cultured cells and animals. Here we show that the accessory protein X of BDV represents the first mitochondrion-localized protein of an RNA virus that inhibits rather than promotes apoptosis induction. Rat C6 astroglioma cells persistently infected with wild-type BDV were significantly more resistant to death receptor-dependent and -independent apoptotic stimuli than uninfected cells or cells infected with a BDV mutant expressing reduced amounts of X. Confocal microscopy demonstrated that X colocalizes with mitochondria and expression of X from plasmid DNA rendered human 293T and mouse L929 cells resistant to apoptosis induction. A recombinant virus encoding a mutant X protein unable to associate with mitochondria (BDV-X(A6A7)) failed to block apoptosis in C6 cells. Furthermore, Lewis rats neonatally infected with BDV-X(A6A7) developed severe neurological symptoms and died around day 30 postinfection, whereas all animals infected with wild-type BDV remained healthy and became persistently infected. TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) staining revealed a significant increase in the number of apoptotic cells in the brain of BDV-X(A6A7)-infected animals, whereas the numbers of CD3(+) T lymphocytes were comparable to those detected in animals infected with wild-type BDV. Our data thus indicate that inhibition of apoptosis by X promotes noncytolytic viral persistence and is required for the survival of cells in the central nervous system of BDV-infected animals.