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In Escherichia coli MinE induces MinC/MinD to oscillate between the ends of the cell, contributing to the precise placement of the Z ring at midcell. To do this, MinE undergoes a remarkable conformational change from a latent 6β-stranded form that diffuses in the cytoplasm to an active 4β-stranded form bound to the membrane and MinD. How this conformational switch occurs is not known. Here, using hydrogen–deuterium exchange coupled to mass spectrometry (HDX-MS) we rule out a model in which the two forms are in rapid equilibrium. Furthermore, HDX-MS revealed that a MinE mutant (D45A/V49A), previously shown to produce an aberrant oscillation and unable to assemble a MinE ring, is more rigid than WT MinE. This mutant has a defect in interaction with MinD, suggesting it has difficulty in switching to the active form. Analysis of intragenic suppressors of this mutant suggests it has difficulty in releasing the N-terminal membrane targeting sequences (MTS). These results indicate that the dynamic association of the MTS with the β-sheet is fine-tuned to balance MinE’s need to sense MinD on the membrane with its need to diffuse in the cytoplasm, both of which are necessary for the oscillation. The results lead to models for MinE activation and MinE ring formation.

In tissue engineering, an ideal scaffold attracts and supports cells thus providing them with the necessary mechanical support and architecture as they reconstruct new tissue in vitro and in vivo. This manuscript details a novel matrix derived from decellularized Wharton's jelly (WJ) obtained from human umbilical cord for use as a scaffold for tissue engineering application. This decellularized Wharton's jelly matrix (DWJM) contained 0.66 ± 0.12 μg/mg sulfated glycosaminoglycans (GAGs), and was abundant in hyaluronic acid, and completely devoid of cells. Mass spectroscopy revealed the presence of collagen types II, VI and XII, fibronectin-I, and lumican I. When seeded onto DWJM, WJ mesenchymal stem cells (WJMSCs), successfully attached to, and penetrated the porous matrix resulting in a slower rate of cell proliferation. Gene expression analysis of WJ and bone marrow (BM) MSCs cultured on DWJM demonstrated decreased expression of proliferation genes with no clear pattern of differentiation. When this matrix was implanted into a murine calvarial defect model with, green fluorescent protein (GFP) labeled osteocytes, the osteocytes were observed to migrate into the matrix as early as 24 hours. They were also identified in the matrix up to 14 days after transplantation. Together with these findings, we conclude that DWJM can be used as a 3D porous, bioactive and biocompatible scaffold for tissue engineering and regenerative medicine applications.

Metabolic dysfunction‐associated liver disease (MASLD) is a leading cause of liver disease in children. There is a paucity of data on potential biomarkers and therapeutic targets, especially in pediatric MASLD. We used mass spectrometry (MS)‐mediated proteomics followed by enzyme‐linked immunosorbent assay (ELISA) to identify potential biomarkers and therapeutic targets in pediatric MASLD. Serum samples were collected from pediatric subjects without (n = 56) and with MASLD (n = 72). Initial screen using MS‐based proteomics identified 6 upregulated (adenosine deaminase 2, sex hormone‐binding globulin (SHBG), inter‐alpha‐trypsin inhibitor heavy chain H1 (ITIH1), fructose‐bisphosphate aldolase A, type II cytoskeletal 2 epidermal keratine, N‐acetylmuramoyl‐L‐alanine amidase) and 3 downregulated (alcohol dehydrogenase 4 (ADH4), fructose‐bisphosphate aldolase B (ALDOB), serum albumin) proteins in the MASLD group. Confirmatory studies using ELISA were performed for the 2 strongest upregulated proteins (SHBG and ITIH1) and two top downregulated proteins (ADH4 and ALDOB). Correlation of ELISA results with clinical data revealed that SHBG had strong associations with BMI, ALT, and HgbA1c (p < 0.05). ADH4 had strong associations with BMI and HgbA1c (p < 0.05). ITIH1 and ALDOB had no strong correlations with common clinical parameters of MASLD. Area under ROC Curve revealed statistically significant ability of SHBG (494 nmol/L, sensitivity = 98%, specificity 80%) and ADH4 (2.14 ng/mL, sensitivity = 65%, specificity = 66%) to diagnosis MASLD (p < 0.05). MS with confirmation ELISA identified SHBG and ADH4 as potential biomarkers of pediatric MASLD.

Protein arginine methyl transferase 1 (PRMT1) is the main enzyme for cellular arginine methylation. It regulates many aspects of liver biology including inflammation, lipid metabolism, and proliferation. Previously we identified that PRMT1 is necessary for protection from alcohol‐induced liver injury. However, many PRMT1 targets in the liver after alcohol exposure are not yet identified. We studied the changes in the PRMT1‐dependent arginine methylated proteome after alcohol feeding in mouse liver using mass spectrometry. We found that arginine methylation of the RNA‐binding protein (heterogeneous nuclear ribonucleoprotein [hnRNP]) H1 is mediated by PRMT1 and is altered in alcohol‐fed mice. PRMT1‐dependent methylation suppressed hnRNP H1 binding to several messenger RNAs of complement pathway including complement component C3. We found that PRMT1‐dependent hnRNP H methylation suppressed complement component expression in vitro, and phosphorylation is required for this function of PRMT1. In agreement with that finding, hepatocyte‐specific PRMT1 knockout mice had an increase in complement component expression in the liver. Excessive complement expression in alcohol‐fed PRMT1 knockout mice resulted in further complement activation and an increase in serum C3a and C5a levels, which correlated with inflammation in multiple organs including lung and adipose tissue. Using specific inhibitors to block C3aR and C5aR receptors, we were able to prevent lung and adipose tissue inflammation without affecting inflammation in the liver or liver injury. Conclusion: Taken together, these data suggest that PRMT1‐dependent suppression of complement production in the liver is necessary for prevention of systemic inflammation in alcohol‐fed mice. C3a and C5a play a role in this liver–lung and liver–adipose interaction in alcohol‐fed mice deficient in liver arginine methylation.

The adenosine A2B receptor (A2BAR) is a member of a family of G-protein coupled receptors (GPCRs), which has a low affinity for adenosine and is now implicated in several pathophysiological conditions. We have demonstrated the beneficial effects of A2BAR activation in enhancing recovery after acute liver injury induced by an acetaminophen (APAP) overdose. While receptor trafficking within the cell is recognized to play a role in GPCR signaling, its role in the mediation of A2BAR effects in the context of APAP-induced liver injury is not well understood. This was investigated here, where C57BL/6J mice were subjected to an APAP overdose (300 mg/kg), and the temporal course of A2BAR intracellular localization was examined. The impact of A2BAR activation or inhibition on trafficking was examined by utilizing the A2BAR agonist BAY 60-6583 or antagonist PSB 603. The modulation of A2BAR trafficking via APAP-induced cell signaling was explored by using 4-methylpyrazole (4MP), an inhibitor of Cyp2E1 and JNK activation. Our results indicate that APAP overdose induced the translocation of A2BAR to mitochondria, which was prevented via 4MP treatment. Furthermore, we demonstrated that A2BAR is localized on the mitochondrial outer membrane and interacts with progesterone receptor membrane component 1 (PGRMC1). While the activation of A2BAR enhanced mitochondrial localization, its inhibition decreased PGRMC1 mitochondria levels and blunted mitochondrial Cyp2E1 activity. Thus, our data reveal a hitherto unrecognized consequence of A2BAR trafficking to mitochondria and its interaction with PGRMC1, which regulates mitochondrial Cyp2E1 activity and modulates APAP-induced liver injury.

is the predominant bacterial pathogen affecting the Chilean salmonid industry. This bacterium is the etiological agent of piscirickettsiosis, a significant fish disease. Membrane vesicles (MVs) released by deliver several virulence factors to host cells. To improve on existing knowledge for the pathogenicity-associated functions of MVs, we studied the proteome of purified MVs from the LF-89 type strain using multidimensional protein identification technology. Initially, the cytotoxicity of different MV concentration purified from LF-89 was confirmed in an adult zebrafish infection model. The cumulative mortality of zebrafish injected with MVs showed a dose-dependent pattern. Analyses identified 452 proteins of different subcellular origins; most of them were associated with the cytoplasmic compartment and were mainly related to key functions for pathogen survival. Interestingly, previously unidentified putative virulence-related proteins were identified in MVs, such as outer membrane porin F and hemolysin. Additionally, five amino acid sequences corresponding to the toxin subunit 1 and two amino acid sequences corresponding to the heat-labile enterotoxin alpha chain of were located in the MV proteome. Curiously, these putative toxins were located in a plasmid region of LF-89. Based on the identified proteins, we propose that the protein composition of LF-89 MVs could reflect total protein characteristics of this type strain.

Development of drugs that allosterically regulate enzyme functions to treat disease is a costly venture. Amino acid susbstitutions that mimic allosteric effectors in vitro will identify therapeutic regulatory targets enhancing the likelihood of developing a disease treatment at a reasonable cost. We demonstrate the potential of this approach utilizing human liver pyruvate kinase (hLPYK) as a model. Inhibition of hLPYK was the first desired outcome of this study. We identified individual point mutations that: 1) mimicked allosteric inhibition by alanine, 2) mimicked inhibition by protein phosphorylation, and 3) prevented binding of fructose-1,6-bisphosphate (Fru-1,6-BP). Our second desired outcome was activation of hLPYK. We identified individual point mutations that: 1) prevented hLPYK from binding alanine, the allosteric inhibitor, 2) prevented inhibitory protein phosphorylation, or 3) mimicked allosteric activation by Fru-1,6-BP. Combining the three activating point mutations produced a constitutively activated enzyme that was unresponsive to regulators. Expression of a mutant hLPYK transgene containing these three mutations in a mouse model was not lethal. Thus, mutational mimics of allosteric effectors will be useful to confirm whether allosteric activation of hLPYK will control glycolytic flux in the diabetic liver to reduce hepatic glucose production and, in turn, reduce or prevent hyperglycemia.

Cysteine S-conjugate β-lyases are pyridoxal 5′-phosphate-containing enzymes that catalyze β-elimination reactions with cysteine S-conjugates that possess a good leaving group in the β-position. The end products are aminoacrylate and a sulfur-containing fragment. The aminoacrylate tautomerizes and hydrolyzes to pyruvate and ammonia. The mammalian cysteine S-conjugate β-lyases thus far identified are enzymes involved in amino acid metabolism that catalyze β-lyase reactions as non-physiological side reactions. Most are aminotransferases. In some cases the lyase is inactivated by reaction products. The cysteine S-conjugate β-lyases are of much interest to toxicologists because they play an important key role in the bioactivation (toxication) of halogenated alkenes, some of which are produced on an industrial scale and are environmental contaminants. The cysteine S-conjugate β-lyases have been reviewed in this journal previously (Cooper and Pinto in Amino Acids 30:1-15, 2006). Here, we focus on more recent findings regarding: (1) the identification of enzymes associated with high-M r cysteine S-conjugate β-lyases in the cytosolic and mitochondrial fractions of rat liver and kidney; (2) the mechanism of syncatalytic inactivation of rat liver mitochondrial aspartate aminotransferase by the nephrotoxic β-lyase substrate S-(1,1,2,2-tetrafluoroethyl)-l-cysteine (the cysteine S-conjugate of tetrafluoroethylene); (3) toxicant channeling of reactive fragments from the active site of mitochondrial aspartate aminotransferase to susceptible proteins in the mitochondria; (4) the involvement of cysteine S-conjugate β-lyases in the metabolism/bioactivation of drugs and natural products; and (5) the role of cysteine S-conjugate β-lyases in the metabolism of selenocysteine Se-conjugates. This review emphasizes the fact that the cysteine S-conjugate β-lyases are biologically more important than hitherto appreciated.

Protein arginine methyl transferase 1 (PRMT1) is the main enzyme for cellular arginine methylation. It regulates many aspects of liver biology including inflammation, lipid metabolism, and proliferation. Previously we identified that PRMT1 is necessary for protection from alcohol‐induced liver injury. However, many PRMT1 targets in the liver after alcohol exposure are not yet identified. We studied the changes in the PRMT1‐dependent arginine methylated proteome after alcohol feeding in mouse liver using mass spectrometry. We found that arginine methylation of the RNA‐binding protein (heterogeneous nuclear ribonucleoprotein [hnRNP]) H1 is mediated by PRMT1 and is altered in alcohol‐fed mice. PRMT1‐dependent methylation suppressed hnRNP H1 binding to several messenger RNAs of complement pathway including complement component C3. We found that PRMT1‐dependent hnRNP H methylation suppressed complement component expression in vitro, and phosphorylation is required for this function of PRMT1. In agreement with that finding, hepatocyte‐specific PRMT1 knockout mice had an increase in complement component expression in the liver. Excessive complement expression in alcohol‐fed PRMT1 knockout mice resulted in further complement activation and an increase in serum C3a and C5a levels, which correlated with inflammation in multiple organs including lung and adipose tissue. Using specific inhibitors to block C3aR and C5aR receptors, we were able to prevent lung and adipose tissue inflammation without affecting inflammation in the liver or liver injury. Conclusion: Taken together, these data suggest that PRMT1‐dependent suppression of complement production in the liver is necessary for prevention of systemic inflammation in alcohol‐fed mice. C3a and C5a play a role in this liver–lung and liver–adipose interaction in alcohol‐fed mice deficient in liver arginine methylation.

Protein arginine methyl transferase 1 (PRMT1) is the main enzyme for cellular arginine methylation. It regulates many aspects of liver biology including inflammation, lipid metabolism, and proliferation. Previously we identified that PRMT1 is necessary for protection from alcohol-induced liver injury. However, many PRMT1 targets in the liver after alcohol exposure are not yet identified. We studied the changes in the PRMT1-dependent arginine methylated proteome after alcohol feeding in mouse liver using mass spectrometry. We found that arginine methylation of the RNA-binding protein (heterogeneous nuclear ribonucleoprotein [hnRNP]) H1 is mediated by PRMT1 and is altered in alcohol-fed mice. PRMT1-dependent methylation suppressed hnRNP H1 binding to several messenger RNAs of complement pathway including complement component C3. We found that PRMT1-dependent hnRNP H methylation suppressed complement component expression , and phosphorylation is required for this function of PRMT1. In agreement with that finding, hepatocyte-specific PRMT1 knockout mice had an increase in complement component expression in the liver. Excessive complement expression in alcohol-fed PRMT1 knockout mice resulted in further complement activation and an increase in serum C3a and C5a levels, which correlated with inflammation in multiple organs including lung and adipose tissue. Using specific inhibitors to block C3aR and C5aR receptors, we were able to prevent lung and adipose tissue inflammation without affecting inflammation in the liver or liver injury. Taken together, these data suggest that PRMT1-dependent suppression of complement production in the liver is necessary for prevention of systemic inflammation in alcohol-fed mice. C3a and C5a play a role in this liver-lung and liver-adipose interaction in alcohol-fed mice deficient in liver arginine methylation.