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.     

Identification and characterization of LPLAT7 as an sn-1-specific lysophospholipid acyltransferase

The main fatty acids at the sn-1 position of phospholipids (PLs) are saturated or monounsaturated fatty acids such as palmitic acid (C16:0), stearic acid (C18:0), and oleic acid (C18:1) and are constantly replaced, like unsaturated fatty acids at the sn-2 position. However, little is known about the molecular mechanism underlying the replacement of fatty acids at the sn-1 position, i.e., the sn-1 remodeling. Previously, we established a method to evaluate the incorporation of fatty acids into the sn-1 position of lysophospholipids (lyso-PLs). Here, we used this method to identify the enzymes capable of incorporating fatty acids into the sn-1 position of lyso-PLs (sn-1 lysophospholipid acyltransferase [LPLAT]). Screenings using siRNA knockdown and recombinant proteins for 14 LPLATs identified LPLAT7/lysophosphatidylglycerol acyltransferase 1 (LPGAT1) as a candidate. In vitro, we found LPLAT7 mainly incorporated several fatty acids into the sn-1 position of lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE), with weak activities toward other lyso-PLs. Interestingly, however, only C18:0-containing phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were specifically reduced in the LPLAT7-mutant cells and tissues from knockout mice, with a concomitant increase in the level of C16:0- and C18:1-containing PC and PE. Consistent with this, the incorporation of deuterium-labeled C18:0 into PLs dramatically decreased in the mutant cells, while deuterium-labeled C16:0 and C18:1 showed the opposite dynamic. Identifying LPLAT7 as an sn-1 LPLAT facilitates understanding the biological significance of sn-1 fatty acid remodeling of PLs. We also propose to use the new nomenclature, LPLAT7, for LPGAT1 since the newly assigned enzymatic activities are quite different from the LPGAT1s previously reported.     

Identification of a Feed-Forward Loop Between 15(S)-HETE and PGE2 in Human Amnion at Parturition

Human parturition is associated with massive arachidonic acid (AA) mobilization in the amnion, indicating that large amounts of AA-derived eicosanoids are required for parturition. Prostaglandin E2 (PGE2) synthesized from the cyclooxygenase (COX) pathway is the best characterized AA-derived eicosanoid in the amnion which plays a pivotal role in parturition. The existence of any other pivotal AA-derived eicosanoids involved in parturition remains elusive. Here, we screened such eicosanoids in human amnion tissue with AA-targeted metabolomics and studied their role and synthesis in parturition by using human amnion fibroblasts and a mouse model. We found that lipoxygenase (ALOX) pathway-derived 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) and its synthetic enzymes ALOX15 and ALOX15B were significantly increased in human amnion at parturition. Although 15(S)-HETE is ineffective on its own, it potently potentiated the activation of NF-κB by inflammatory mediators including lipopolysaccharide, interleukin-1β, and serum amyloid A1, resulting in the amplification of COX-2 expression and PGE2 production in amnion fibroblasts. In turn, we determined that PGE2 induced ALOX15/15B expression and 15(S)-HETE production through its EP2 receptor-coupled PKA pathway, thereby forming a feed-forward loop between 15(S)-HETE and PGE2 production in the amnion at parturition. Our studies in pregnant mice showed that 15(S)-HETE injection induced preterm birth with increased COX-2 and PGE2 abundance in the fetal membranes and placenta. Conclusively, 15(S)-HETE is identified as another crucial parturition-pertinent AA-derived eicosanoid in the amnion, which may form a feed-forward loop with PGE2 in parturition. Interruption of this feed-forward loop may be of therapeutic value for the treatment of preterm birth.     

The more the merrier: how homo-oligomerization alters the interactome and function of ribonucleotide reductase

Stereotyped as a nexus of dNTP synthesis, the dual-subunit enzyme — ribonucleotide reductase (RNR) — is coming into view as a paradigm of oligomerization and moonlighting behavior. In the present issue of ‘omics’, we discuss what makes the larger subunit of this enzyme (RNR-α) so interesting, highlighting its emerging cellular interactome based on its unique oligomeric dynamism that dictates its compartment-specific occupations. Linking the history of the field with the multivariable nature of this exceedingly sophisticated enzyme, we further discuss implications of new data pertaining to DNA-damage response, S-phase checkpoints, and ultimately tumor suppression. We hereby hope to provide ideas for those interested in these fields and exemplify conceptual frameworks and tools that are useful to study RNR's broader roles in biology.     

Identification of compounds in red wine that effectively upregulate aquaporin-3 as a potential mechanism of enhancement of skin moisturizing

In a previous clinical study, the moisture content in the stratum corneum of healthy Japanese women who consumed a beverage rich in oligomeric proanthocyanidins (OPCs) made from red wine extract was found to be higher than that in the control group. This finding suggested that OPCs can increase skin moisture content. In this study, we determined the expression level of aquaporin-3 (AQP3) in keratinocytes to elucidate the mechanism by which compounds in red wine grape increase moisture content in stratum corneum. Through in vitro studies, we confirmed that normal human epidermal keratinocytes (NHEK) incubated with red wine induced AQP3 expression. Furthermore, the supplementation of red wine fractions enriched in OPC was shown to increase AQP3 expression. Besides, the component of OPC-rich fractions that upregulated AQP3 expression was found to be a gallic acid (GA)-binding flavan-3-ol, particularly oligomeric compounds. We found that GA-binding OPC were able to upregulate AQP3 expression and that these compounds were enriched in red wine. Our findings might suggest that the mechanism of enhancement of moisture content in stratum corneum by red wine might be via the upregulation of AQP3 expression in the epidermal keratinocytes.     

Biochemical characterization of TRIM72 E3 ligase and its interaction with the insulin receptor substrate 1

TRIM family of E3 ubiquitin ligases have an amino-terminal conserved tripartite motif consisting of RING, B-Box, coiled-coil domain and different C-terminal domain leading it to classification into 11 subclasses. TRIM72 is an E3 ligase of class IV and subclass 1 with its role in a multitude of cellular processes. Despite being crucial in multiple cellular processes, TRIM72 still hasn't been biochemically characterized. In the present study, we have characterized the oligomeric status of TRIM72 and found that it forms both monomers, dimers, and tetramers. We have screened a set of 12 E2s and identified two novel E2 enzymes (Ubch5c and Ubch10) that work in cooperation with TRIM72. Nevertheless, E3 ligase activity is minimal and we propose that additional regulation is required to enhance its E3 ligase activity. We have also used surface plasmon resonance to study interaction with one of its substrate proteins, IRS1, and identified the PH domain of IRS1 is mediating interaction with the TRIM72 E3 ligase while the PTB domain of IRS1, does not show any interaction.     

Bipartite mechanism for laminin-integrin interactions: Identification of the integrin-binding site in LG domains of the laminin α chain

Laminins are major cell-adhesive proteins consisting of α, β, and γ chains, in which the three C-terminal globular domains of the α chain (LMα/LG1–3) and the C-terminal tail region of the γ1 chain (LMγ1-tail) are required for binding to integrin. Despite the recent progress on the role of LMγ1-tail in coordinating the metal ion-dependent adhesion site of the integrin β subunit, the mechanism by which LMα/LG1–3 interacts with integrin remains to be elucidated. We found that basic residues on the bottom face of LMα5/LG2 are required for binding laminin-511 to α6β1 integrin. Intermolecular cysteine scanning assays demonstrated that the basic residues in LMα5/LG2 were in contact with the acidic residues within the laminin-binding X1 region of the integrin α subunit in the laminin-integrin complex. These results indicate that LMα5/LG2 interacts directly with the integrin α subunit and comprises a bipartite integrin binding site of laminin-511 with the LMγ1-tail.     

Apolipoprotein F concentration,activity, and the properties of LDL controlling ApoF activation in hyperlipidemic plasma

Apolipoprotein F (ApoF) modulates lipoprotein metabolism by selectively inhibiting cholesteryl ester transfer protein activity on LDL. This ApoF activity requires that it is bound to LDL. How hyperlipidemia alters total plasma ApoF and its binding to LDL are poorly understood. In this study, total plasma ApoF and LDL-bound ApoF were quantified by ELISA (n = 200). Plasma ApoF was increased 31% in hypercholesterolemic plasma but decreased 20% in hypertriglyceridemia. However, in donors with combined hypercholesterolemia and hypertriglyceridemia, the elevated triglyceride ameliorated the rise in ApoF caused by hypercholesterolemia alone. Compared with normolipidemic LDL, hypercholesterolemic LDL contained ～2-fold more ApoF per LDL particle, whereas ApoF bound to LDL in hypertriglyceridemia plasma was <20% of control. To understand the basis for altered association of ApoF with hyperlipidemic LDL, the physiochemical properties of LDL were modified in vitro by cholesteryl ester transfer protein ± LCAT activities. The time-dependent change in LDL lipid composition, proteome, core and surface lipid packing, LDL surface charge, and LDL size caused by these factors were compared with the ApoF binding capacity of these LDLs. Only LDL particle size correlated with ApoF binding capacity. This positive association between LDL size and ApoF content was confirmed in hyperlipidemic plasmas. Similarly, when in vitro produced and enlarged LDLs with elevated ApoF binding capacity were incubated with LPL to reduce their size, ApoF binding was reduced by 90%. Thus, plasma ApoF levels and the activation status of this ApoF are differentially altered by hypercholesterolemia and hypertriglyceridemia. LDL size is a key determinate of ApoF binding and activation.     

Semaphorin 3A potentiates the profibrotic effects of transforming growth factor-β1 in the cornea

Corneal scarring is a major cause of blindness worldwide with few effective therapeutic options. Finding a treatment would be of tremendous public health benefit, but requires a thorough understanding of the complex interactions that underlie this phenomenon. Here, we tested the hypothesis that the large increase in expression of Semaphorin 3A (SEMA3A) in corneal wounds contributes to the development of stromal fibrosis. We first verified this increased expression in vivo, in a cat model of photorefractive keratectomy-induced corneal wounding. We then examined the impact of adding exogenous SEMA3A to cultured corneal fibroblasts, and assessed how this affected the ability of transforming growth factor-beta1 (TGF-β1) to induce their differentiation into myofibroblasts. Finally, we examined how siRNA knockdown of endogenous SEMA3A affected these same phenomena. We found exogenous SEMA3A to significantly potentiate TGF-β1’s profibrotic effects, with only a minimal contribution from cell-intrinsic SEMA3A. Our results suggest a previously unrecognized interaction between SEMA3A and TGF-β1 in the wounded cornea, and a possible contribution of SEMA3A to the regulation of tissue fibrosis and remodeling in this transparent organ.     

Genotypic and phenotypic characterization of industrial autochthonous Saccharomyces cerevisiae for the selection of well-adapted bioethanol-producing strains

In northwestern Argentina, sugarcane-derived industrial fermentation is being extensively used for bioethanol production, where highly adaptive native strains compete with the baker's yeast Saccharomyces cerevisiae traditionally used as starter culture. Yeast populations of 10 distilleries from Tucumán (Argentina) were genotypic and phenotypic characterized to select well-adapted bioethanol-producing autochthonous strains to be used as starter cultures for the industrial production of bioethanol fuel. From the 192 isolates, 69.8% were identified as S. cerevisiae, 25.5% as non-Saccharomyces, and 4.7% as Saccharomyces sp. wild yeasts. The majority of S. cerevisiae isolates (68.5%) were non-flocculating yeasts, while the flocculating strains were all obtained from the only continuous fermentation process included in the study. Simple Sequence Repeat analysis revealed a high genetic diversity among S. cerevisiae genotypes, where all of them were very different from the original baker's strain used as starter. Among these, 38 strains multi-tolerant to stress by ethanol (8%), temperature (42.5 °C) and pH (2.0) were obtained. No major differences were found among these strains in terms of ethanol production and residual sugars in batch fermentation experiments with cell recycling. However, only 10 autochthonous strains maintained their viability (more than 80%) throughout five consecutive cycles of sugarcane-based fermentations. In summary, 10 autochthonous isolates were found to be superior to baker's yeast used as starter culture (S. cerevisiae Calsa) in terms of optimal technological, physiological and ecological properties. The knowledge generated on the indigenous yeast populations in industrial fermentation processes of bioethanol-producing distilleries allowed the selection of well-adapted bioethanol-producing strains.     

Structural analysis revealed a novel conformation of the NTRC reductase domain from Chlamydomonas reinhardtii

In plant chloroplasts, thiol regulation is driven by two systems. One relies on the activity of thioredoxins through their light dependent reduction by ferredoxin via a ferredoxin-thioredoxin reductase (FTR). In the other system, a NADPH-dependent redox regulation is driven by a NADPH-thioredoxin reductase C (NTRC). While the thioredoxin system has been deeply studied, a more thorough understanding of the function of this plant specific NTRC is desirable. NTRC is a single polypeptide harbouring a thioredoxin domain (Trx) at the C-terminus of a NADPH-dependent Thioredoxin reductase (TrxR). To provide functional and structural insights, we studied the crystal structure of the TrxR domain of the NTRC from Chlamydomonas reinhardtii (CrNTRC, Cre01.g054150.t1.2) and its Cys136Ser (C136S) mutant, which is characterized by the mutation of the resolving cysteine in the active site of the TrxR domain. Furthermore, we confirmed the role of NTRC as electron donor for 2-Cys peroxiredoxin (PRX) also in C. reinhardtii. The structural data of TrxR were employed to develop a scheme of action which addresses electron transfer between TrxR and Trx of NTRC and between NTRC and its substrates.     

Cardiac electrophysiology consultative experience at the epicenter of the COVID-19 pandemic in the United States

BackgroundThe COVID-19 pandemic has greatly altered the practice of cardiac electrophysiology around the world for the foreseeable future. Professional organizations have provided guidance for practitioners, but real-world examples of the consults and responsibilities cardiac electrophysiologists face during a surge of COVID-19 patients is lacking.MethodsIn this observational case series we report on 29 consecutive inpatient electrophysiology consultations at a major academic medical center in New York City, the epicenter of the pandemic in the United States, during a 2 week period from March 30-April 12, 2020, when 80% of hospital beds were occupied by COVID-19 patients, and the New York City metropolitan area accounted for 10% of COVID-19 cases worldwide.ResultsReasons for consultation included: Atrial tachyarrhythmia (31%), cardiac implantable electronic device management (28%), bradycardia (14%), QTc prolongation (10%), ventricular arrhythmia (7%), post-transcatheter aortic valve replacement conduction abnormality (3.5%), ventricular pre-excitation (3.5%), and paroxysmal supraventricular tachycardia (3.5%). Twenty-four patients (86%) were positive for COVID-19 by nasopharyngeal swab. All elective procedures were canceled, and only one urgent device implantation was performed. Thirteen patients (45%) required in-person evaluation and the remainder were managed remotely.ConclusionOur experience shows that the application of a massive alteration in workflow and personnel forced by the pandemic allowed our team to efficiently address the intersection of COVID-19 with a range of electrophysiology issues. This experience will prove useful as guidance for emerging hot spots or areas affected by future waves of the pandemic.     

Enhanced targeted DNA methylation of the CMV and endogenous promoters with dCas9-DNMT3A3L entails distinct subsequent histone modification changes in CHO cells

With the emergence of new CRISPR/dCas9 tools that enable site specific modulation of DNA methylation and histone modifications, more detailed investigations of the contribution of epigenetic regulation to the precise phenotype of cells in culture, including recombinant production subclones, is now possible. These also allow a wide range of applications in metabolic engineering once the impact of such epigenetic modifications on the chromatin state is available.In this study, enhanced DNA methylation tools were targeted to a recombinant viral promoter (CMV), an endogenous promoter that is silenced in its native state in CHO cells, but had been reactivated previously (β-galactoside α-2,6-sialyltransferase 1) and an active endogenous promoter (α-1,6-fucosyltransferase), respectively. Comparative ChIP-analysis of histone modifications revealed a general loss of active promoter histone marks and the acquisition of distinct repressive heterochromatin marks after targeted methylation. On the other hand, targeted demethylation resulted in autologous acquisition of active promoter histone marks and loss of repressive heterochromatin marks. These data suggest that DNA methylation directs the removal or deposition of specific histone marks associated with either active, poised or silenced chromatin. Moreover, we show that de novo methylation of the CMV promoter results in reduced transgene expression in CHO cells. Although targeted DNA methylation is not efficient, the transgene is repressed, thus offering an explanation for seemingly conflicting reports about the source of CMV promoter instability in CHO cells.Importantly, modulation of epigenetic marks enables to nudge the cell into a specific gene expression pattern or phenotype, which is stabilized in the cell by autologous addition of further epigenetic marks. Such engineering strategies have the added advantage of being reversible and potentially tunable to not only turn on or off a targeted gene, but also to achieve the setting of a desirable expression level.     

Simulating cellular galectin networks by mixing galectins in vitro reveals synergistic activity

BackgroundEven though members of the family of adhesion/growth-regulatory galectins are increasingly detected to be co-expressed, they are still being routinely tested separately. The recent discovery of heterodimer formation among galectins-1, -3, and -7 in mixtures prompts further study of their functional activities in mixtures.MethodsCell agglutination, galectin binding to cells, as well as effects on cell proliferation, onset of apoptosis and migration were determined in assays using various cell types and mixtures of galectins-1, -3, and -7.ResultsEvidence for a more than additive increases of experimental parameters was consistently obtained.ConclusionTesting galectins in mixtures simulates the situation of co-expression in situ and reveals unsuspected over-additive activities. This new insight is relevant for analyzing galectin functionality in (patho)physiological conditions.     

Identification of glycated and acetylated lysine residues in human α2-antiplasmin

BackgroundThe post-translational protein modification via lysine residues can significantly alter its function. α2-antiplasmin, a key inhibitor of fibrinolysis, contains 19 lysine residues.AimWe sought to identify sites of glycation and acetylation in human α2-antiplasmin and test whether the competition might occur on the lysine residues of α2-antiplasmin.MethodsWe analyzed human α2-antiplasmin (1) untreated; (2) incubated with increasing concentrations of β-d-glucose (0, 5, 10, 50 mM); (3) incubated with 1.6 mM acetylsalicylic acid (ASA) and (4) incubated with 1.6 mM ASA and 50 mM β-d-glucose, using the ultraperformance liquid chromatography system coupled to mass spectrometer.ResultsEleven glycation sites and 10 acetylation sites were found in α2-antiplasmin. Incubation with β-d-glucose was associated with glycation of 4 (K-418, K-427, K-434, K-441) out of 6 lysine residues, known to be important for mediating the interaction with plasmin. Glycation and acetylation overlapped at 9 sites in samples incubated with β-d-glucose or ASA. Incubation with concomitant ASA and β-d-glucose was associated with the decreased acetylation at all sites overlapping with glycation sites. At K-182 and K-448, decreased acetylation was associated with increased glycation when compared with α2-antiplasmin incubated with 50 mM β-d-glucose alone. Although K-24 located in the proximity of the α2-antiplasmin cleavage site, was found to be only acetylated, incubation with ASA and 50 mM β-d-glucose was associated the absence of acetylation at that site.ConclusionHuman α2-antiplasmin is glycated and acetylated at several sites, with the possible competition between acetylation and glycation at K-182 and K-448. Our finding suggests possibly relevant alterations to α2-antiplasmin function at high glycemia and during aspirin use.     

Sorting through the extensive and confusing roles of sortilin in metabolic disease

Sortilin is a post-Golgi trafficking receptor homologous to the yeast vacuolar protein sorting receptor 10 (VPS10). The VPS10 motif on sortilin is a 10-bladed β-propeller structure capable of binding more than 50 proteins, covering a wide range of biological functions including lipid and lipoprotein metabolism, neuronal growth and death, inflammation, and lysosomal degradation. Sortilin has a complex cellular trafficking itinerary, where it functions as a receptor in the trans-Golgi network, endosomes, secretory vesicles, multivesicular bodies, and at the cell surface. In addition, sortilin is associated with hypercholesterolemia, Alzheimer’s disease, prion diseases, Parkinson’s disease, and inflammation syndromes. The 1p13.3 locus containing SORT1, the gene encoding sortilin, carries the strongest association with LDL-C of all loci in human genome-wide association studies. However, the mechanism by which sortilin influences LDL-C is unclear. Here, we review the role sortilin plays in cardiovascular and metabolic diseases and describe in detail the large and often contradictory literature on the role of sortilin in the regulation of LDL-C levels.