The N-terminal Tails of Histones H2A and H2B Adopt Two Distinct Conformations in the Nucleosome with Contact and Reduced Contact to DNA

The nucleosome comprises two histone dimers of H2A-H2B and one histone tetramer of (H3-H4)₂, wrapped around by ~145 bp of DNA. Detailed core structures of nucleosomes have been established by X-ray and cryo-EM, however, histone tails have not been visualized. Here, we have examined the dynamic structures of the H2A and H2B tails in 145-bp and 193-bp nucleosomes using NMR, and have compared them with those of the H2A and H2B tail peptides unbound and bound to DNA. Whereas the H2A C-tail adopts a single but different conformation in both nucleosomes, the N-tails of H2A and H2B adopt two distinct conformations in each nucleosome. To clarify these conformations, we conducted molecular dynamics (MD) simulations, which suggest that the H2A N-tail can locate stably in either the major or minor grooves of nucleosomal DNA. While the H2B N-tail, which sticks out between two DNA gyres in the nucleosome, was considered to adopt two different orientations, one toward the entry/exit side and one on the opposite side. Then, the H2A N-tail minor groove conformation was obtained in the H2B opposite side and the H2B N-tail interacts with DNA similarly in both sides, though more varied conformations are obtained in the entry/exit side. Collectively, the NMR findings and MD simulations suggest that the minor groove conformer of the H2A N-tail is likely to contact DNA more strongly than the major groove conformer, and the H2A N-tail reduces contact with DNA in the major groove when the H2B N-tail is located in the entry/exit side.     

Temporal Analysis of Protein Ubiquitylation and Phosphorylation During Parkin-Dependent Mitophagy

Mitophagy, the selective degradation of mitochondria by autophagy, affects defective mitochondria following damage or stress. At the onset of mitophagy, parkin ubiquitylates proteins on the mitochondrial outer membrane. While the role of parkin at the onset of mitophagy is well understood, less is known about its activity during later stages in the process. Here, we used HeLa cells expressing catalytically active or inactive parkin to perform temporal analysis of the proteome, ubiquitylome, and phosphoproteome during 18 h after induction of mitophagy by mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazine. Abundance profiles of proteins downregulated in parkin-dependent manner revealed a stepwise and “outside–in” directed degradation of mitochondrial subcompartments. While ubiquitylation of mitochondrial outer membrane proteins was enriched among early parkin-dependent targets, numerous mitochondrial inner membrane, matrix, and cytosolic proteins were also found ubiquitylated at later stages of mitophagy. Phosphoproteome analysis revealed a possible crosstalk between phosphorylation and ubiquitylation during mitophagy on key parkin targets, such as voltage-dependent anion channel 2.     

Linker DNA Length is a Key to Tri-nucleosome Folding

The folding of a nucleosome array has long been one of the fundamental and unsolved problems in chromatin biology. In this study, we address how nucleosome array folding depends on the length of linker DNA. We performed molecular dynamics simulations of a tri-nucleosome, a minimal model of chromatin folding, with various linker lengths (LLs) ranging from 20 to 40 base pairs (bps). We found that the tri-nucleosome folding strongly depends on LLs, and classified the structure ensemble into five classes, named from trinuc-1 to trinuc-5. As a function of LL, the different classes appear, on average, every 2 bps with a period of 10 bps, and are characterized by distinct inter-nucleosome interactions. The trinuc-1 conformation corresponds to LL ~ 10n, where n is an integer, and is stabilized by the tight packing between the first and the third nucleosomes, consistent with a zigzag fiber form. Structures of the other four classes are more diverse and distributed continuously in the space of possible configurations. Histone-DNA electrostatic interactions in the tri-nucleosome are further analyzed.     

Proteins in Tumor-Derived Plasma Extracellular Vesicles Indicate Tumor Origin

Cancer-derived extracellular vesicles (EVs) promote tumorigenesis, premetastatic niche formation, and metastasis via their protein cargo. However, the proteins packaged by patient tumors into EVs cannot be determined in vivo because of the presence of EVs derived from other tissues. We therefore developed a cross-species proteomic method to quantify the human tumor-derived proteome of plasma EVs produced by patient-derived xenografts of four cancer types. Proteomic profiling revealed individualized packaging of novel protein cargo, and machine learning accurately classified the type of the underlying tumor.     

Distinct roles in phagocytosis of the early and late increases of cell surface calreticulin induced by oxaliplatin

Calreticulin (CRT), a chaperone typically located in the endoplasmic reticulum (ER), is known to translocate to the cell surface in response to anticancer drugs. Cell surface CRT (ecto-CRT) on apoptotic or pre-apoptotic cells serves as an “eat me” signal that can promote phagocytosis. In this study, we observed the biphasic (early transient and late sustained) increase of ecto-CRT on HT-29 cells after treatment with oxaliplatin (L-OHP). To investigate the role of ecto-CRT that accumulates in the early and late phases as “eat me” signals, we examined the phagocytosis of HT-29 cells by macrophage-like cells and dendritic cell (DC) -like cells prepared from THP-1 cells. The results indicated that the early ecto-CRT-expressed cells were phagocytosed by immature DC-like cells, and the late ecto-CRT-expressed cells were phagocytosed primarily by macrophage-like cells, while mature DC-like cells did not respond to the either class of ecto-CRT-expressed cells. Both types of phagocytotic events were inhibited by CRT Blocking Peptide, suggesting that such events depended on the ecto-CRT. Our results suggested that the early increase of ecto-CRT is related to phagocytosis as part of immunogenic cell death (ICD), while the late increase of ecto-CRT is related to the removal of apoptotic cells by macrophages.     

Widespread Arginine Phosphorylation in Staphylococcus aureus

Arginine phosphorylation was only recently discovered to play a significant and relevant role in the Gram-positive bacterium Bacillus subtilis. In addition, arginine phosphorylation was also detected in Staphylococcus aureus, suggesting a widespread role in bacteria. However, the large-scale analysis of protein phosphorylation, and especially those that involve a phosphoramidate bond, comes along with several challenges. The substoichiometric nature of protein phosphorylation requires proper enrichment strategies prior to LC-MS/MS analysis, and the acid instability of phosphoramidates was long thought to impede those enrichments. Furthermore, good spectral quality is required, which can be impeded by the presence of neutral losses of phosphoric acid upon higher energy collision–induced dissociation. Here we show that pArg is stable enough for commonly used Fe³⁺-IMAC enrichment followed by LC-MS/MS and that HCD is still the gold standard for the analysis of phosphopeptides. By profiling a serine/threonine kinase (Stk1) and phosphatase (Stp1) mutant from a methicillin-resistant S. aureus mutant library, we identified 1062 pArg sites and thus the most comprehensive arginine phosphoproteome to date. Using synthetic arginine phosphorylated peptides, we validated the presence and localization of arginine phosphorylation in S. aureus. Finally, we could show that the knockdown of Stp1 significantly increases the overall amount of arginine phosphorylation in S. aureus. However, our analysis also shows that Stp1 is not a direct protein-arginine phosphatase but only indirectly influences the arginine phosphoproteome.     

New Insights on Effects of Glucocorticoids in Patients With SARS-CoV-2 Infection

ObjectiveSince January 2020, the highly contagious novel coronavirus SARS-CoV-2 has caused a global pandemic. Severe COVID-19 leads to a massive release of proinflammatory mediators, leading to diffuse damage to the lung parenchyma, and the development of acute respiratory distress syndrome. Treatment with the highly potent glucocorticoid (GC) dexamethasone was found to be effective in reducing mortality in severely affected patients.MethodsTo review the effects of glucocorticoids in the context of COVID-19 we performed a literature search in the PubMed database using the terms COVID-19 and glucocorticoid treatment. We identified 1429 article publications related to COVID-19 and glucocorticoid published from 1.1.2020 to the present including 238 review articles and 36 Randomized Controlled Trials. From these studies, we retrieved 13 Randomized Controlled Trials and 86 review articles that were relevant to our review topics. We focused on the recent literature dealing with glucocorticoid metabolism in critically ill patients and investigating the effects of glucocorticoid therapy on the immune system in COVID-19 patients with severe lung injury.ResultsIn our review, we have discussed the regulation of the hypothalamic-pituitary-adrenal axis in patients with critical illness, selection of a specific GC for critical illness-related GC insufficiency, and recent studies that investigated hypothalamic-pituitary-adrenal dysfunction in patients with COVID-19. We have also addressed the specific activation of the immune system with chronic endogenous glucocorticoid excess, as seen in patients with Cushing syndrome, and, finally, we have discussed immune activation due to coronavirus infection and the possible mechanisms leading to improved outcomes in patients with COVID-19 treated with GCs.ConclusionFor clinical endocrinologists prescribing GCs for their patients, a precise understanding of both the molecular- and cellular-level mechanisms of endogenous and exogenous GCs is imperative, including timing of administration, dosage, duration of treatment, and specific formulations of GCs.     

SP3-Enabled Rapid and High Coverage Chemoproteomic Identification of Cell-State–Dependent Redox-Sensitive Cysteines

Proteinaceous cysteine residues act as privileged sensors of oxidative stress. As reactive oxygen and nitrogen species have been implicated in numerous pathophysiological processes, deciphering which cysteines are sensitive to oxidative modification and the specific nature of these modifications is essential to understanding protein and cellular function in health and disease. While established mass spectrometry-based proteomic platforms have improved our understanding of the redox proteome, the widespread adoption of these methods is often hindered by complex sample preparation workflows, prohibitive cost of isotopic labeling reagents, and requirements for custom data analysis workflows. Here, we present the SP3-Rox redox proteomics method that combines tailored low cost isotopically labeled capture reagents with SP3 sample cleanup to achieve high throughput and high coverage proteome-wide identification of redox-sensitive cysteines. By implementing a customized workflow in the free FragPipe computational pipeline, we achieve accurate MS1-based quantitation, including for peptides containing multiple cysteine residues. Application of the SP3-Rox method to cellular proteomes identified cysteines sensitive to the oxidative stressor GSNO and cysteine oxidation state changes that occur during T cell activation.     

Phosphoproteome Profiling of the Receptor Tyrosine Kinase MuSK Identifies Tyrosine Phosphorylation of Rab GTPases

Muscle-specific receptor tyrosine kinase (MuSK) agonist antibodies were developed 2 decades ago to explore the benefits of receptor activation at the neuromuscular junction. Unlike agrin, the endogenous agonist of MuSK, agonist antibodies function independently of its coreceptor low-density lipoprotein receptor–related protein 4 to delay the onset of muscle denervation in mouse models of ALS. Here, we performed dose–response and time-course experiments on myotubes to systematically compare site-specific phosphorylation downstream of each agonist. Remarkably, both agonists elicited similar intracellular responses at known and newly identified MuSK signaling components. Among these was inducible tyrosine phosphorylation of multiple Rab GTPases that was blocked by MuSK inhibition. Importantly, mutation of this site in Rab10 disrupts association with its effector proteins, molecule interacting with CasL 1/3. Together, these data provide in-depth characterization of MuSK signaling, describe two novel MuSK inhibitors, and expose phosphorylation of Rab GTPases downstream of receptor tyrosine kinase activation in myotubes.     

Evaluation of Factors Associated With Fracture and Loss of Bone Mineral Density Within 1 Year After Liver Transplantation

ObjectiveOrthotopic liver transplant recipients are at high risk of fragility fractures both in pre-liver transplant (pre-LT) and in the immediate posttransplant (post-LT) period. The aims of this study were to identify risk factors associated with post-LT fracture and identify factors that contribute to changes in bone mineral density (BMD) in post-LT as they relate to the risk of fracture in the immediate post-LT period.MethodsWe conducted a retrospective cohort study of first-time LT recipients who had BMD testing within 2-year pre-LT and 1-year post-LT. We assessed factors associated with immediate post-LT fracture using logistic regression models and linear regression models.ResultsNew fractures occurred in 41/286 (14.3%) of LT recipients during the first year following LT. In multivariate analysis, we noted an increased odds of fracture for patients with prior history of fracture (P < .001), patients who were older (P = .03), patients with higher end-stage liver disease score (P = .03), and patients with lower BMD. After adjustment for multiple testing, only a history of prior fracture was statistically significant.ConclusionOur study demonstrated that prior fracture at any site was associated with developing a new fracture in the first year post-LT.     

Differential Proteome and Interactome Analysis Reveal the Basis of Pleiotropy Associated With the Histidine Methyltransferase Hpm1p

The methylation of histidine is a post-translational modification whose function is poorly understood. Methyltransferase histidine protein methyltransferase 1 (Hpm1p) monomethylates H243 in the ribosomal protein Rpl3p and represents the only known histidine methyltransferase in Saccharomyces cerevisiae. Interestingly, the hpm1 deletion strain is highly pleiotropic, with many extraribosomal phenotypes including improved growth rates in alternative carbon sources. Here, we investigate how the loss of histidine methyltransferase Hpm1p results in diverse phenotypes, through use of targeted mass spectrometry (MS), growth assays, quantitative proteomics, and differential crosslinking MS. We confirmed the localization and stoichiometry of the H243 methylation site, found unreported sensitivities of Δhpm1 yeast to nonribosomal stressors, and identified differentially abundant proteins upon hpm1 knockout with clear links to the coordination of sugar metabolism. We adapted the emerging technique of quantitative large-scale stable isotope labeling of amino acids in cell culture crosslinking MS for yeast, which resulted in the identification of 1267 unique in vivo lysine–lysine crosslinks. By reproducibly monitoring over 350 of these in WT and Δhpm1, we detected changes to protein structure or protein–protein interactions in the ribosome, membrane proteins, chromatin, and mitochondria. Importantly, these occurred independently of changes in protein abundance and could explain a number of phenotypes of Δhpm1, not addressed by expression analysis. Further to this, some phenotypes were predicted solely from changes in protein structure or interactions and could be validated by orthogonal techniques. Taken together, these studies reveal a broad role for Hpm1p in yeast and illustrate how crosslinking MS will be an essential tool for understanding complex phenotypes.