Epigenetic silencing of LDHB promotes hepatocellular carcinoma by remodeling the tumor microenvironment.

Lactate dehydrogenase B (LDHB) reversibly catalyzes the conversion of pyruvate to lactate or lactate to pyruvate and expressed in various malignancies. However, the role of LDHB in modulating immune responses against hepatocellular carcinoma (HCC) remains largely unknown. Here, we found that down-regulation of lactate dehydrogenase B (LDHB) was coupled with the promoter hypermethylation and knocking down the DNA methyltransferase 3A (DNMT 3A) restored LDHB expression levels in HCC cell lines. Bioinformatics analysis of the HCC cohort from The Cancer Genome Atlas revealed a significant positive correlation between LDHB expression and immune regulatory signaling pathways and immune cell infiltrations. Moreover, immune checkpoint inhibitors (ICIs) have shown considerable promise for HCC treatment and patients with higher LDHB expression responded better to ICIs. Finally, we found that overexpression of LDHB suppressed HCC growth in immunocompetent but not in immunodeficient mice, suggesting that the host immune system was involved in the LDHB-medicated tumor suppression. Our findings indicate that DNMT3A-mediated epigenetic silencing of LDHB may contribute to HCC progression through remodeling the tumor immune microenvironment, and LDHB may become a potential prognostic biomarker and therapeutic target for HCC immunotherapy.

Integrated analysis of single cell-RNA sequencing and Mendelian randomization identifies lactate dehydrogenase B as a target of melatonin in ischemic stroke.

AIMS: Despite the success of single-cell RNA sequencing in identifying cellular heterogeneity in ischemic stroke, clarifying the mechanisms underlying these associations of differently expressed genes remains challenging. Several studies that integrate gene expression and gene expression quantitative trait loci (eQTLs) with genome wide-association study (GWAS) data to determine their causal role have been proposed. METHODS: Here, we combined Mendelian randomization (MR) framework and single cell (sc) RNA sequencing to study how differently expressed genes (DEGs) mediating the effect of gene expression on ischemic stroke. The hub gene was further validated in the in vitro model. RESULTS: We identified 2339 DEGs in 10 cell clusters. Among these DEGs, 58 genes were associated with the risk of ischemic stroke. After external validation with eQTL dataset, lactate dehydrogenase B (LDHB) is identified to be positively associated with ischemic stroke. The expression of LDHB has also been validated in sc RNA-seq with dominant expression in microglia and astrocytes, and melatonin is able to reduce the LDHB expression and activity in vitro ischemic models. CONCLUSION: Our study identifies LDHB as a novel biomarker for ischemic stroke via combining the sc RNA-seq and MR analysis.

Expression of neuronal NO synthase α- and ß-isoforms in skeletal muscle of mice.

Knowledge of the primary structure of neuronal NO synthase (nNOS) in skeletal muscle is still conflicting and needs further clarification. To elucidate the expression patterns of nNOS isoforms at both mRNA and protein level, systematic reverse transcription (RT)-PCR and epitope mapping by qualitative immunoblot analysis on skeletal muscle of C57/BL6 mice were performed. The ability of the nNOS isoforms to form aggregates was characterized by native low-temperature polyacrylamide electrophoresis (LT-PAGE). The molecular analysis was focused on the rectus femoris (RF) muscle, a skeletal muscle with a nearly balanced ratio of nNOS α- and ß-isoforms. RT-PCR amplificates from RF muscles showed exclusive exon-1d mRNA expression, either with or without exon-µ. Epitope mapping demonstrated the simultaneous expression of the nNOS splice variants α/µ, α/non-µ, ß/µ and ß/non-µ. Furthermore, immunoblotting suggests that the transition between nNOS α- and ß-isoforms lies within exon-3. In LT-PAGE, three protein nNOS associated aggregates were detected in homogenates of RF muscle and tibialis anterior muscle: a 320 kDa band containing nNOS α-isoforms, while 250 and 300 kDa bands consist of nNOS ß-isoforms that form homodimers or heterodimers with non-nNOS proteins.

The role of genetic polymorphisms in the sulfation of pregnenolone by human cytosolic sulfotransferase SULT2B1a.

Pregnenolone is a key intermediate in the biosynthesis of many steroid hormones and neuroprotective steroids. Sulfotransferase family cytosolic 2B member 1 (SULT2B1a) has been reported to be highly selective to sulfate pregnenolone. This study aimed to clarify the effect of missense single nucleotide polymorphisms (SNPs) of the human SULT2B1 gene on the sulfating activity of coded SULT2B1a allozymes toward Pregnenolone. To investigate the effects of single nucleotide polymorphisms of the SULT2B1 gene on the sulfation of pregnenolone by SULT2B1a allozymes, 13 recombinant SULT2B1a allozymes were generated, expressed, and purified using established procedures. Human SULT2B1a SNPs were identified by a comprehensive database search. 13 SULT2B1a nonsynonymous missense coding SNPs (cSNPs) were selected, and site-directed mutagenesis was used to generate the corresponding cDNAs, packaged in pGEX-2TK expression vector, encoding these 13 SULT2B1a allozymes, which were bacterially expressed in BL21 E. coli cells and purified by glutathione-Sepharose affinity chromatography. Purified SULT2B1a allozymes were analyzed for sulfating activities towards pregnenolone. In comparison with the wild-type SULT2B1a, of the 13 allozymes, 11 showed reduced activity toward pregnenolone at 0.1 µM. Specifically, P134L and R259Q allozymes, reported to be involved in autosomal-recessive congenital ichthyosis, displayed low activity (1-10%) toward pregnenolone. The findings of this study may demonstrate the impact of genetic polymorphism on the sulfation of pregnenolone in individuals with different SULT2B1 genotypes.

LDHB contributes to the regulation of lactate levels and basal insulin secretion in human pancreatic ß cells.

Using 13C6 glucose labeling coupled to gas chromatography-mass spectrometry and 2D 1H-13C heteronuclear single quantum coherence NMR spectroscopy, we have obtained a comparative high-resolution map of glucose fate underpinning ß cell function. In both mouse and human islets, the contribution of glucose to the tricarboxylic acid (TCA) cycle is similar. Pyruvate fueling of the TCA cycle is primarily mediated by the activity of pyruvate dehydrogenase, with lower flux through pyruvate carboxylase. While the conversion of pyruvate to lactate by lactate dehydrogenase (LDH) can be detected in islets of both species, lactate accumulation is 6-fold higher in human islets. Human islets express LDH, with low-moderate LDHA expression and ß cell-specific LDHB expression. LDHB inhibition amplifies LDHA-dependent lactate generation in mouse and human ß cells and increases basal insulin release. Lastly, cis-instrument Mendelian randomization shows that low LDHB expression levels correlate with elevated fasting insulin in humans. Thus, LDHB limits lactate generation in ß cells to maintain appropriate insulin release.

The Expression and Secretion Profile of TRAP5 Isoforms in Gaucher Disease.

BACKGROUND: Gaucher disease (GD) is caused by glucocerebrosidase (GCase) enzyme deficiency, leading to glycosylceramide (Gb-1) and glucosylsphingosine (Lyso-Gb-1) accumulation. The pathological hallmark for GD is an accumulation of large macrophages called Gaucher cells (GCs) in the liver, spleen, and bone marrow, which are associated with chronic organ enlargement, bone manifestations, and inflammation. Tartrate-resistant acid phosphatase type 5 (TRAP5 protein, ACP5 gene) has long been a nonspecific biomarker of macrophage/GCs activation; however, the discovery of two isoforms of TRAP5 has expanded its significance. The discovery of TRAP5's two isoforms revealed that it is more than just a biomarker of macrophage activity. While TRAP5a is highly expressed in macrophages, TRAP5b is secreted by osteoclasts. Recently, we have shown that the elevation of TRAP5b in plasma is associated with osteoporosis in GD. However, the role of TRAP isoforms in GD and how the accumulation of Gb-1 and Lyso-Gb-1 affects TRAP expression is unknown. METHODS: 39 patients with GD were categorized into cohorts based on bone mineral density (BMD). TRAP5a and TRAP5b plasma levels were quantified by ELISA. ACP5 mRNA was estimated using RT-PCR. RESULTS: An increase in TRAP5b was associated with reduced BMD and correlated with Lyso-Gb-1 and immune activator chemokine ligand 18 (CCL18). In contrast, the elevation of TRAP5a correlated with chitotriosidase activity in GD. Lyso-Gb-1 and plasma seemed to influence the expression of ACP5 in macrophages. CONCLUSIONS: As an early indicator of BMD alteration, measurement of circulating TRAP5b is a valuable tool for assessing osteopenia-osteoporosis in GD, while TRAP5a serves as a biomarker of macrophage activation in GD. Understanding the distinct expression pattern of TRAP5 isoforms offers valuable insight into both bone disease and the broader implications for immune system activation in GD.

Identification of Bromophenols' glucuronidation and its induction on UDP- glucuronosyltransferases isoforms.

Bromophenols (BPs) are prominent environmental pollutants extensively utilized in aquaculture, pharmaceuticals, and chemical manufacturing. This study aims to identify UDP- glucuronosyltransferases (UGTs) isoforms involved in the metabolic elimination of BPs. Mono-glucuronides of BPs were detected in human liver microsomes (HLMs) incubated with the co-factor uridine-diphosphate glucuronic acid (UDPGA). The glucuronidation metabolism reactions catalyzed by HLMs followed Michaelis-Menten or substrate inhibition kinetics. Recombinant enzymes and inhibition experiments with chemical reagents were employed to phenotype the principal UGT isoforms participating in BP glucuronidation. UGT1A6 emerged as the major enzyme in the glucuronidation of 4-Bromophenol (4-BP), while UGT1A1, UGT1A6, and UGT1A8 were identified as the most essential isoforms for metabolizing 2,4-dibromophenol (2,4-DBP). UGT1A1, UGT1A8, and UGT2B4 were deemed the most critical isoforms in the catalysis of 2,4,6-tribromophenol (2,4,6-TBP) glucuronidation. Species differences were investigated using the liver microsomes of pig (PLM), rat (RLM), monkey (MyLM), and dog (DLM). Additionally, 2,4,6-TBP effects on the expression of UGT1A1 and UGT2B7 in HepG2 cells were evaluated. The results demonstrated potential induction of UGT1A1 and UGT2B7 upon exposure to 2,4,6-TBP at a concentration of 50 µM. Collectively, these findings contribute to elucidating the metabolic elimination and toxicity of BPs.

Comparison of the Backbone Dynamics of Dehaloperoxidase-Hemoglobin Isoenzymes.

Dehaloperoxidase (DHP) is a multifunctional hemeprotein with a functional switch generally regulated by the chemical class of the substrate. Its two isoforms, DHP-A and DHP-B, differ by only five amino acids and have an almost identical protein fold. However, the catalytic efficiency of DHP-B for oxidation by a peroxidase mechanism ranges from 2- to 6-fold greater than that of DHP-A depending on the conditions. X-ray crystallography has shown that many substrates and ligands have nearly identical binding in the two isoenzymes, suggesting that the difference in catalytic efficiency could be due to differences in the conformational dynamics. We compared the backbone dynamics of the DHP isoenzymes at pH 7 through heteronuclear relaxation dynamics at 11.75, 16.45, and 19.97 T in combination with four 300 ns MD simulations. While the overall dynamics of the isoenzymes are similar, there are specific local differences in functional regions of each protein. In DHP-A, Phe35 undergoes a slow chemical exchange between two conformational states likely coupled to a swinging motion of Tyr34. Moreover, Asn37 undergoes fast chemical exchange in DHP-A. Given that Phe35 and Asn37 are adjacent to Tyr34 and Tyr38, it is possible that their dynamics modulate the formation and migration of the active tyrosyl radicals in DHP-A at pH 7. Another significant difference is that both distal and proximal histidines have a 15-18% smaller S2 value in DHP-B, thus their greater flexibility could account for the higher catalytic activity. The distal histidine grants substrate access to the distal pocket. The greater flexibility of the proximal histidine could also accelerate H2O2 activation at the heme Fe by increased coupling of an amino acid charge relay to stabilize the ferryl Fe(IV) oxidation state in a Poulos-Kraut "push-pull"-type peroxidase mechanism.

The significance of the two cytosolic glutamine synthetase enzymes, GLN1;3 and GLN1;5, in the context of seed development and germination in Arabidopsis thaliana.

Glutamine synthetase (GS), an initial enzyme in nitrogen (N) plant metabolism, exists as a group of isoenzymes found in both cytosolic (GS1) and plastids (GS2) and has gathered significant attention for enhancing N use efficiency and crop yield. This work focuses on the A. thaliana GLN1;3 and GLN1;5 genes, the two predicted most expressed genes in seeds, among the five isogenes encoding GS1 in this species. The expression patterns were studied using transgenic marker line plants and qPCR during seed development and germination. The observed patterns highlight distinct functions for the two genes and confirm GLN1;5 as the most highly expressed GS1 gene in seeds. The GLN1;5, expression, oriented towards hypocotyl and cotyledons, suggests a role in protein turnover during germination, while the radicle-oriented expression of GLN1;3 supports a function in early external N uptake. While the single mutants exhibited a normal phenotype, except for a decrease in seed parameters, the double gln1;3/gln1;5 mutant displayed a germination delay, substantial impairment in growth, nitrogen metabolism, and number and quality of the seeds, as well as a diminishing in flowering. Although seed and pollen-specific, GLN1;5 expression is upregulated in the meristems of the gln1;3 mutants, filling the lack of GLN1;3 and ensuring the normal functioning of the gln1;3 mutants. These findings validate earlier in silico data on the expression patterns of GLN1;3 and GL1;5 genes in seeds, explore their different functions, and underscore their essential role in plant growth, seed production, germination, and early stages of plant development.

Substrate Specificities of Variants of Barley (1,3)- and (1,3;1,4)-ß-d-Glucanases Resulting from Mutagenesis and Segment Hybridization.

Barley (1,3;1,4)-ß-d-glucanase is believed to have evolved from an ancestral monocotyledon (1,3)-ß-d-glucanase, enabling the hydrolysis of (1,3;1,4)-ß-d-glucans in the cell walls of leaves and germinating grains. In the present study, we investigated the substrate specificities of variants of the barley enzymes (1,3;1,4)-ß-d-glucan endohydrolase [(1,3;1,4)-ß-d-glucanase] isoenzyme EII (HvEII) and (1,3)-ß-d-glucan endohydrolase [(1,3)-ß-d-glucanase] isoenzyme GII (HvGII) obtained by protein segment hybridization and site-directed mutagenesis. Using protein segment hybridization, we obtained three variants of HvEII in which the substrate specificity was that of a (1,3)-ß-d-glucanase and one variant that hydrolyzed both (1,3)-ß-d-glucans and (1,3;1,4)-ß-d-glucans; the wild-type enzyme hydrolyzed only (1,3;1,4)-ß-d-glucans. Using substitutions of specific amino acid residues, we obtained one variant of HvEII that hydrolyzed both substrates. However, neither protein segment hybridization nor substitutions of specific amino acid residues gave variants of HvGII that could hydrolyze (1,3;1,4)-ß-d-glucans; the wild-type enzyme hydrolyzed only (1,3)-ß-d-glucans. Other HvEII and HvGII variants showed changes in specific activity and their ability to degrade the (1,3;1,4)-ß-d-glucans or (1,3)-ß-d-glucans to larger oligosaccharides. We also used molecular dynamics simulations to identify amino-acid residues or structural regions of wild-type HvEII and HvGII that interact with (1,3;1,4)-ß-d-glucans and (1,3)-ß-d-glucans, respectively, and may be responsible for the substrate specificities of the two enzymes.

KAT7 enhances the proliferation and metastasis of head and neck squamous carcinoma by promoting the acetylation level of LDHA.

Lysine acetyltransferase 7 (KAT7), a histone acetyltransferase, has recently been identified as an oncoprotein and has been implicated in the development of various malignancies. However, its specific role in head and neck squamous carcinoma (HNSCC) has not been fully elucidated. Our study revealed that high expression of KAT7 in HNSCC patients is associated with poor survival prognosis and silencing KAT7 inhibits the Warburg effect, leading to reduced proliferation, invasion, and metastatic potential of HNSCC. Further investigation uncovered a link between the high expression of KAT7 in HNSCC and tumor-specific glycolytic metabolism. Notably, KAT7 positively regulates Lactate dehydrogenase A (LDHA), a key enzyme in metabolism, to promote lactate production and create a conducive environment for tumor proliferation and metastasis. Additionally, KAT7 enhances LDHA activity and upregulates LDHA protein expression by acetylating the lysine 118 site of LDHA. Treatment with WM3835, a KAT7 inhibitor, effectively suppressed the growth of subcutaneously implanted HNSCC cells in mice. In conclusion, our findings suggest that KAT7 exerts pro-cancer effects in HNSCC by acetylating LDHA and may serve as a potential therapeutic target. Inhibiting KAT7 or LDHA expression holds promise as a therapeutic strategy to suppress the growth and progression of HNSCC.

Sulfonyl thiourea derivatives from 2-aminodiarylpyrimidines: In vitro and in silico evaluation as potential carbonic anhydrase I, II, IX, and XII inhibitors.

A series of synthesized sulfonyl thiourea derivatives (7a-o) of substituted 2-amino-4,6-diarylpyrimidines (4a-o) exhibited the remarkable inhibitory activity against some the human carbonic anhydrases (hCAs), including hCA I, II, IX, and XII isoforms. The inhibitory efficacy of synthesized sulfonyl thiourea derivatives were experimentally validated by in vitro enzymatic assays. 7a (KI = 46.14 nM), 7j (KI = 48.92 nM), and 7m (KI = 62.59 nM) (for isoform hCA I); 7f (KI = 42.72 nM), 7i (KI = 40.98 nM), and 7j (KI = 33.40 nM) (for isoform hCA II); 7j (KI = 228.5 nM), 7m (KI = 195.4 nM), and 7n (KI = 210.1 nM) (for isoform hCA IX); 7l (KI = 116.9 nM), 7m (KI = 118.8 nM), and 7n (KI = 147.2 nM) (for isoform hCA XII) in comparison with KI values of 452.1, 327.3, 437.2, and 338.9 nM, respectively, of the standard drug AAZ. These compounds also had significantly more potent inhibitory action against cytosolic isoform hCA I and tumor-associated isoforms hCA IX and hCA XII. Furthermore, the potential inhibitory compounds were subjected to in silico screening for molecular docking and molecular dynamics simulations. The results of in vitro and in silico studies revealed that compounds 7a, 7j, and 7m were the most promising derivatives in this series due to their significant effects on studied hCA I, II, IX, and XII isoforms, respectively. The results showed that the sulfonyl thiourea moiety was accommodated deeply in the active site and interacted with the zinc ion in the receptors.

Chemical Versatility in Catalysis and Inhibition of the Class IIb Histone Deacetylases.

ConspectusThe zinc-dependent histone deacetylases (HDACs 1-11) belong to the arginase-deacetylase superfamily of proteins, members of which share a common α/ß fold and catalytic metal binding site. While several HDACs play a role in epigenetic regulation by catalyzing acetyllysine hydrolysis in histone proteins, the biological activities of HDACs extend far beyond histones. HDACs also deacetylate nonhistone proteins in the nucleus as well as the cytosol to regulate myriad cellular processes. The substrate pool is even more diverse in that certain HDACs can hydrolyze other covalent modifications. For example, HDAC6 is also a lysine decrotonylase, and HDAC11 is a lysine-fatty acid deacylase. Surprisingly, HDAC10 is not a lysine deacetylase but instead is a polyamine deacetylase. Thus, the HDACs are biologically and chemically versatile catalysts as they regulate the function of diverse protein and nonprotein substrates throughout the cell.Owing to their critical regulatory functions, HDACs serve as prominent targets for drug design. At present, four HDAC inhibitors are FDA-approved for cancer chemotherapy. However, these inhibitors are active against multiple HDAC isozymes, and a lack of selectivity is thought to contribute to undesirable side effects. Current medicinal chemistry campaigns focus on the development of isozyme-selective inhibitors, and many such studies largely focus on HDAC6 and HDAC10. HDAC6 is a target for therapeutic intervention due to its cellular role as a tubulin deacetylase and tau deacetylase, and selective inhibitors are being studied in cancer chemotherapy and the treatment of peripheral neuropathy. Crystal structures of enzyme-inhibitor complexes reveal how various features of inhibitor design, such as zinc-coordinating groups, bifurcated capping groups, and aromatic fluorination patterns, contribute to affinity and isozyme selectivity. The polyamine deacetylase HDAC10 is also an emerging target for cancer chemotherapy. Crystal structures of intact substrates trapped in the HDAC10 active site reveal the molecular basis of strikingly narrow substrate specificity for N8-acetylspermidine hydrolysis. Active site features responsible for substrate specificity have been successfully exploited in the design of potent and selective inhibitors.In this Account, I review the structural chemistry and inhibition of HDACs, highlighting recent X-ray crystallographic and functional studies of HDAC6 and HDAC10 in my laboratory. These studies have yielded fascinating snapshots of catalysis as well as novel chemical transformations involving bound inhibitors. The zinc-bound water molecule in the HDAC active site is the catalytic nucleophile in the deacetylation reaction, but this activated water molecule can also react with inhibitor C═O or C═N groups to yield unanticipated reaction products that bind exceptionally tightly. Versatile active site chemistry unleashes the full inhibitory potential of such compounds, and X-ray crystallography allows us to view this chemistry in action.

Benzothiadiazinone-1,1-Dioxide Carbonic Anhydrase Inhibitors Suppress the Growth of Drug-Resistant Mycobacterium tuberculosis Strains.

2H-Benzo[e][1,2,4]thiadiazin-3(4H)-one 1,1-dioxide (BTD) based carbonic anhydrase (CA) inhibitors are here explored as new anti-mycobacterial agents. The chemical features of BTD derivatives meet the criteria for a potent inhibition of ß-class CA isozymes. BTD derivatives show chemical features meeting the criteria for a potent inhibition of ß-class CA isozymes. Specifically, three ß-CAs (MtCA1, MtCA2, and MtCA3) were identified in Mycobacterium tuberculosis and their inhibition was shown to exert an antitubercular action. BTDs derivatives 2a-q effectively inhibited the mycobacterial CAs, especially MtCA2 and MtCA3, with Ki values up to a low nanomolar range (MtCA3, Ki = 15.1-2250 nM; MtCA2, Ki = 38.1-4480 nM) and with a significant selectivity ratio over the off-target human CAs I and II. A computational study was conducted to elucidate the compound structure-activity relationship. Importantly, the most potent MtCA inhibitors demonstrated efficacy in inhibiting the growth of M. tuberculosis strains resistant to both rifampicin and isoniazid-standard reference drugs for Tuberculosis treatment.

The dopamine D2 receptors antagonist Veralipride inhibits carbonic anhydrases: solution and crystallographic insights on human isoforms.

The inhibitory effects of veralipride, a benzamide-class antipsychotic acting as dopamine D2 receptors antagonist incorporates a primary sulfonamide moiety and was investigated for its interactions with carbonic anhydrase (CA) isoforms. In vitro profiling using the stopped-flow technique revealed that veralipride exhibited potent inhibitory activity across all tested hCA isoforms, with exception of hCA III. Comparative analysis with standard inhibitors, acetazolamide (AAZ), and sulpiride, provided insights for understanding the relative efficacy of veralipride as CA inhibitor. The study reports the X-ray crystal structure analysis of the veralipride adduct with three human (h) isoforms, hCA I, II, and CA XII mimic, allowing the understanding of the molecular interactions rationalizing its inhibitory effects against each isoform. These findings contribute to our understanding of veralipride pharmacological properties and for the design of structural analogs endowed with polypharmacological properties.

Physiological function and regulation of ascorbate peroxidase isoforms.

Ascorbate peroxidase (APX) reduces H2O2 to H2O by utilizing ascorbate as a specific electron donor and constitutes the ascorbate-glutathione cycle in organelles of plants including chloroplasts, cytosol, mitochondria, and peroxisomes. It has been almost 40 years since APX was discovered as an important plant-specific H2O2-scavenging enzyme, during which time many research groups have conducted molecular physiological analyses. It is now clear that APX isoforms function not only just as antioxidant enzymes but also as important factors in intracellular redox regulation through the metabolism of reactive oxygen species. The function of APX isoforms is regulated at multiple steps, from the transcriptional level to post-translational modifications of enzymes, thereby allowing them to respond flexibly to ever-changing environmental factors and physiological phenomena such as cell growth and signal transduction. In this review, we summarize the physiological functions and regulation mechanisms of expression of each APX isoform.

Protein kinase C signaling "in" and "to" the nucleus: Master kinases in transcriptional regulation.

PKC is a multifunctional family of Ser-Thr kinases widely implicated in the regulation of fundamental cellular functions, including proliferation, polarity, motility, and differentiation. Notwithstanding their primary cytoplasmic localization and stringent activation by cell surface receptors, PKC isozymes impel prominent nuclear signaling ultimately impacting gene expression. While transcriptional regulation may be wielded by nuclear PKCs, it most often relies on cytoplasmic phosphorylation events that result in nuclear shuttling of PKC downstream effectors, including transcription factors. As expected from the unique coupling of PKC isozymes to signaling effector pathways, glaring disparities in gene activation/repression are observed upon targeting individual PKC family members. Notably, specific PKCs control the expression and activation of transcription factors implicated in cell cycle/mitogenesis, epithelial-to-mesenchymal transition and immune function. Additionally, PKCs isozymes tightly regulate transcription factors involved in stepwise differentiation of pluripotent stem cells toward specific epithelial, mesenchymal, and hematopoietic cell lineages. Aberrant PKC expression and/or activation in pathological conditions, such as in cancer, leads to profound alterations in gene expression, leading to an extensive rewiring of transcriptional networks associated with mitogenesis, invasiveness, stemness, and tumor microenvironment dysregulation. In this review, we outline the current understanding of PKC signaling "in" and "to" the nucleus, with significant focus on established paradigms of PKC-mediated transcriptional control. Dissecting these complexities would allow the identification of relevant molecular targets implicated in a wide spectrum of diseases.

Isoenzyme characterization of Leishmania infantum toward checking the antioxidant activity of superoxide dismutase and glutathione peroxidase.

BACKGROUND: Leishmania infantum is the major causative agent of visceral leishmaniasis in Mediterranean regions. Isoenzyme electrophoresis (IE), as a biochemical technique, is applied in the characterization of Leishmania species. The current study attempted to investigate the isoenzyme patterns of logarithmic and stationary promastigotes and axenic amastigotes (amastigote-like) of L. infantum using IE. The antioxidant activity of superoxide dismutase (SOD) and glutathione peroxidase (GPX) was also checked in the aforementioned forms. METHOD: After L. infantum cultivation and obtaining logarithmic and stationary promastigotes, axenic amastigotes were achieved by incubation of stationary promastigotes at 37 °C for 48 h. The lysate samples were prepared and examined for six enzymatic systems including glucose-6-phosphate dehydrogenase (G6PD), nucleoside hydrolase 1 (NH1), malate dehydrogenase (MDH), glucose-phosphate isomerase (GPI), malic enzyme (ME), and phosphoglucomutase (PGM). Additionally, the antioxidant activity of SOD and GPX was measured. RESULTS: GPI, MDH, NH1, and G6PD enzymatic systems represented different patterns in logarithmic and stationary promastigotes and axenic amastigotes of L. infantum. PGM and ME showed similar patterns in the aforementioned forms of parasite. The highest level of SOD activity was determined in the axenic amastigote form and GPX activity was not detected in different forms of L. infantum. CONCLUSION: The characterization of leishmanial-isoenzyme patterns and the measurement of antioxidant activity of crucial antioxidant enzymes, including SOD and GPX, might reveal more information in the biology, pathogenicity, and metabolic pathways of Leishmania parasites and consequently drive to designing novel therapeutic strategies in leishmaniasis treatment.

Inhibitory effects of the main metabolites of Apatinib on CYP450 isozymes in human and rat liver microsomes.

PURPOSE: The inhibitory effect of Apatinib on cytochrome P450 (CYP450) enzymes has been studied. However, it is unknown whether the inhibition is related to the major metabolites, M1-1, M1-2 and M1-6. METHODS: A 5-in-1 cocktail system composed of CYP2B6/Cyp2b1, CYP2C9/Cyp2c11, CYP2E1/Cyp2e1, CYP2D6/Cyp2d1 and CYP3A/Cyp3a2 was used in this study. Firstly, the effects of APA and its main metabolites on the activities of HLMs, RLMs and recombinant isoforms were examined. The reaction mixture included HLMs, RLMs or recombinant isoforms (CYP3A4.1, CYP2D6.1, CYP2D6.10 or CYP2C9.1), analyte (APA, M1-1, M1-2 or M1-6), probe substrates. The reactions were pre-incubated for 5 min at 37 °C, followed by the addition of NAPDH to initiate the reactions, which continued for 40 min. Secondly, IC50 experiments were conducted to determine if the inhibitions were reversible. The reaction mixture of the "+ NADPH Group" included HLMs or RLMs, 0 to 100 of µM M1-1 or M1-2, probe substrates. The reactions were pre-incubated for 5 min at 37 °C, and then NAPDH was added to initiate reactions, which proceeded for 40 min. The reaction mixture of the "- NADPH Group" included HLMs or RLMs, probe substrates, NAPDH. The reactions were pre-incubated for 30 min at 37 °C, and then 0 to 100 µM of M1-1 or M1-2 was added to initiate the reactions, which proceeded for 40 min. Finally, the reversible inhibition of M1-1 and M1-2 on isozymes was determined. The reaction mixture included HLMs or RLMs, 0 to 10 µM of M1-1 or M1-2, probe substrates with concentrations ranging from 0.25Km to 2Km. RESULTS: Under the influence of M1-6, the activity of CYP2B6, 2C9, 2E1 and 3A4/5 was increased to 193.92%, 210.82%, 235.67% and 380.12% respectively; the activity of CYP2D6 was reduced to 92.61%. The inhibitory effects of M1-1 on CYP3A4/5 in HLMs and on Cyp2d1 in RLMs, as well as the effect of M1-2 on CYP3A in HLMs, were determined to be noncompetitive inhibition, with the Ki values equal to 1.340 µM, 1.151 µM and 1.829 µM, respectively. The inhibitory effect of M1-1 on CYP2B6 and CYP2D6 in HLMs, as well as the effect of M1-2 on CYP2C9 and CYP2D6 in HLMs, were determined to be competitive inhibition, with the Ki values equal to 12.280 µM, 2.046 µM, 0.560 µM and 4.377 µM, respectively. The inhibitory effects of M1-1 on CYP2C9 in HLMs and M1-2 on Cyp2d1 in RLMs were determined to be mixed-type, with the Ki values equal to 0.998 µM and 0.884 µM. The parameters could not be obtained due to the atypical kinetics of CYP2E1 in HLMs under the impact of M1-2. CONCLUSIONS: M1-1 and M1-2 exhibited inhibition for several CYP450 isozymes, especially CYP2B6, 2C9, 2D6 and 3A4/5. This observation may uncover potential drug-drug interactions and provide valuable insights for the clinical application of APA.

Effect of the N-terminal extension in myosin essential light chain A1 on the mechanism of actomyosin ATP hydrolysis.

Myosin essential light chains A1 and A2 are identical isoforms except for an extension of ∼40 amino acids at the N terminus of A1 that binds F-actin. The extension has no bearing on the burst hydrolysis rate (M-ATP → M-ADP-Pi) as determined by chemical quench flow (100 µM isoenzyme). Whereas actomyosin-S1A2 steady state MgATPase (low ionic strength, 20 °C) is hyperbolically dependent on concentration: Vmax 7.6 s-1, Kapp 6.4 µM (F-actin) and Vmax 10.1 s-1, Kapp 5.5 µM (native thin filaments, pCa 4), the relationship for myosin-S1A1 is bimodal; an initial rise at low concentration followed by a decline to one-third the Vmax of S1A2, indicative of more than one rate-limiting step and A1-enforced flux through the slower actomyosin-limited hydrolysis pathway. In double-mixing stopped-flow with an indicator, Ca(II)-mediated activation of Pi dissociation (regulatedAM-ADP-Pi → regulatedAM-ADP + Pi) is attenuated by A1 attachment to thin filaments (pCa 4). The maximum accelerated rates of Pi dissociation are: 81 s-1 (S1A1, Kapp 8.9 µM) versus 129 s-1 (S1A2, Kapp 58 µM). To investigate apomyosin-S1-mediated activation, thin filaments (EGTA) are premixed with a given isomyosin-S1 and double-mixing is repeated with myosin-S1A1 in the first mix. Similar maximum rates of Pi dissociation are observed, 44.5 s-1 (S1A1) and 47.1 s-1 (S1A2), which are lower than for Ca(II) activation. Overall, these results biochemically demonstrate how the longer light chain A1 can contribute to slower contraction and higher force and the shorter version A2 to faster contraction and lower force, consistent with their distribution in different types of striated muscle.