Insights into the regulation of malate dehydrogenase: inhibitors, activators, and allosteric modulation by small molecules.

Cellular metabolism comprises a complex network of biochemical anabolic and catabolic processes that fuel the growth and survival of living organisms. The enzyme malate dehydrogenase (MDH) is most known for its role in oxidizing malate to oxaloacetate (OAA) in the last step of the tricarboxylic acid (TCA) cycle, but it also participates in the malate-aspartate shuttle in the mitochondria as well as the glyoxylate cycle in plants. These pathways and the specific reactions within them are dynamic and must be carefully calibrated to ensure a balance between nutrient/energy supply and demand. MDH structural and functional complexity requires a variety of regulatory mechanisms, including allosteric regulation, feedback, and competitive inhibition, which are often dependent on whether the enzyme is catalyzing its forward or reverse reaction. Given the role of MDH in central metabolism and its potential as a target for therapeutics in both cancer and infectious diseases, there is a need to better understand its regulation. The involvement of MDH in multiple pathways makes it challenging to identify which effectors are critical to its activity. Many of the in vitro experiments examining MDH regulation were done decades ago, and though allosteric sites have been proposed, none to date have been specifically mapped. This review aims to provide an overview of the current knowledge surrounding MDH regulation by its substrate, products, and other intermediates of the TCA cycle while highlighting all the gaps in our understanding of its regulatory mechanisms.

Genetic and functional evidence links a missense variant in B4GALT1 to lower LDL and fibrinogen.

Increased blood levels of low-density lipoprotein cholesterol (LDL-C) and fibrinogen are independent risk factors for cardiovascular disease. We identified associations between an Amish-enriched missense variant (p.Asn352Ser) in a functional domain of beta-1,4-galactosyltransferase 1 (B4GALT1) and 13.9 milligrams per deciliter lower LDL-C (P = 4.1 × 10­19) and 29 milligrams per deciliter lower plasma fibrinogen (P = 1.3 × 10­5). B4GALT1 gene­based analysis in 544,955 subjects showed an association with decreased coronary artery disease (odds ratio = 0.64, P = 0.006). The mutant protein had 50% lower galactosyltransferase activity compared with the wild-type protein. N-linked glycan profiling of human serum found serine 352 allele to be associated with decreased galactosylation and sialylation of apolipoprotein B100, fibrinogen, immunoglobulin G, and transferrin. B4galt1 353Ser knock-in mice showed decreases in LDL-C and fibrinogen. Our findings suggest that targeted modulation of protein galactosylation may represent a therapeutic approach to decreasing cardiovascular disease.

Allele-specific enhancers mediate associations between LCAT and ABCA1 polymorphisms and HDL metabolism.

For most complex traits, the majority of SNPs identified through genome-wide association studies (GWAS) reside within noncoding regions that have no known function. However, these regions are enriched for the regulatory enhancers specific to the cells relevant to the specific trait. Indeed, many of the GWAS loci that have been functionally characterized lie within enhancers that regulate expression levels of key genes. In order to identify polymorphisms with potential allele-specific regulatory effects, we developed a bioinformatics pipeline that harnesses epigenetic signatures as well as transcription factor (TF) binding motifs to identify putative enhancers containing a SNP with potential allele-specific TF binding in linkage disequilibrium (LD) with a GWAS-identified SNP. We applied the approach to GWAS findings for blood lipids, revealing 7 putative enhancers harboring associated SNPs, 3 of which lie within the introns of LCAT and ABCA1, genes that play crucial roles in cholesterol biogenesis and lipoprotein metabolism. All 3 enhancers demonstrated allele-specific in vitro regulatory activity in liver-derived cell lines. We demonstrated that these putative enhancers are in close physical proximity to the promoters of their respective genes, in situ, likely through chromatin looping. In addition, the associated alleles altered the likelihood of transcription activator STAT3 binding. Our results demonstrate that through our approach, the LD blocks that contain GWAS signals, often hundreds of kilobases in size with multiple SNPs serving as statistical proxies to the true functional site, can provide an experimentally testable hypothesis for the underlying regulatory mechanism linking genetic variants to complex traits.

Modulation of Jaw Muscle Motor Response and Wake-Time Parafunctional Tooth Clenching With Music.

AIMS: To evaluate the effects of Guided Music Listening (GML) on masticatory muscles and on the amplitude of wake-time tooth clenching in individuals with higher vs lower frequency of clenching episodes. METHODS: The electromyographic (EMG) activity of the right masseter was recorded during three 20-minute music (relaxing, stress/tension, and favorite) tasks and a control no-music task in 10 (mean age ± standard deviation [SD] = 21.4 ± 3.0 years) and 11 (22.6 ± 2.9 years) healthy volunteers with higher (HP) vs lower (LP) frequency of tooth-clenching episodes, respectively. EMG episodes greater than 10% of the maximum voluntary contraction (EMG activity of the masseter during tooth clenching) and below 10% (EMG activity during rest) were analyzed. Nonparametric tests were used to assess between-group and within-group (between-task) differences in primary outcome measures. RESULTS: In both groups, EMG activity during rest was the greatest during the stress/tension task, and it was the lowest during the favorite task in the LP group and the relaxing task in the HP group (all P < .001). In the HP group, the amplitude of clenching episodes was significantly lower during the favorite and stress/tension tasks than during the relaxing task (all P < .05), while in the LP group, it was significantly lower during the stress/tension task than during the control task (P = .001). The experiment did not affect the frequency or duration of clenching episodes. CONCLUSION: GML modulates masticatory muscle activity. The response to GML depends on the frequency of clenching and the type of music.

An APOO Pseudogene on Chromosome 5q Is Associated With Low-Density Lipoprotein Cholesterol Levels.

BACKGROUND: Elevated levels of low-density lipoprotein cholesterol (LDL-C) are a major risk factor for cardiovascular disease via its contribution to the development and progression of atherosclerotic lesions. Although the genetic basis of LDL-C has been studied extensively, currently known genetic variants account for only ≈20% of the variation in LDL-C levels. METHODS: Through an array-based association analysis in 1102 Amish subjects, we identified a variant strongly associated with LDL-C levels. Using a combination of genetic analyses, zebrafish models, and in vitro experiments, we sought to identify the causal gene driving this association. RESULTS: We identified a founder haplotype associated with a 15 mg/dL increase in LDL-C on chromosome 5. After recombination mapping, the associated region contained 8 candidate genes. Using a zebrafish model to evaluate the relevance of these genes to cholesterol metabolism, we found that expression of the transcribed pseudogene, APOOP1, increased LDL-C and vascular plaque formation. CONCLUSIONS: Based on these data, we propose that APOOP1 regulates levels of LDL-C in humans, thus identifying a novel mechanism of lipid homeostasis.

Characterization, mutagenesis and mechanistic analysis of an ancient algal sterol C24-methyltransferase: Implications for understanding sterol evolution in the green lineage.

Sterol C24-methyltransferases (SMTs) constitute a group of sequence-related proteins that catalyze the pattern of sterol diversity across eukaryotic kingdoms. The only gene for sterol alkylation in green algae was identified and the corresponding catalyst from Chlamydomonas reinhardtii (Cr) was characterized kinetically and for product distributions. The properties of CrSMT were similar to those predicted for an ancient SMT expected to possess broad C3-anchoring requirements for substrate binding and formation of 24ß-methyl/ethyl Δ(25(27))-olefin products typical of primitive organisms. Unnatural Δ(24(25))-sterol substrates, missing a C4ß-angular methyl group involved with binding orientation, convert to product ratios in favor of Δ(24(28))-products. Remodeling the active site to alter the electronics of Try110 (to Leu) results in delayed timing of the hydride migration from methyl attack of the Δ(24)-bond, that thereby produces metabolic switching of product ratios in favor of Δ(25(27))-olefins or impairs the second C1-transfer activity. Incubation of [27-(13)C]lanosterol or [methyl-(2)H3]SAM as co-substrates established the CrSMT catalyzes a sterol methylation pathway by the "algal" Δ(25(27))-olefin route, where methylation proceeds by a conserved SN2 reaction and de-protonation proceeds from the pro-Z methyl group on lanosterol corresponding to C27. This previously unrecognized catalytic competence for an enzyme of sterol biosynthesis, together with phylogenomic analyses, suggest that mutational divergence of a promiscuous SMT produced substrate- and phyla-specific SMT1 (catalyzes first biomethylation) and SMT2 (catalyzes second biomethylation) isoforms in red and green algae, respectively, and in the case of SMT2 selection afforded modification in reaction channeling necessary for the switch in ergosterol (24ß-methyl) biosynthesis to stigmasterol (24α-ethyl) biosynthesis during the course of land plant evolution.

Sterol C24-methyltransferase: Physio- and stereo-chemical features of the sterol C3 group required for catalytic competence.

Sterol C24-methyltransferases (24-SMTs) catalyze the electrophilic alkylation of Δ(24)-sterols to a variety of sterol side chain constructions, and the C3- moiety is the primary determinant for substrate binding by these enzymes. To determine what specific structural features of the C3-polar group ensure sterol catalysis, a series of structurally related C3-analogs of lanosterol that differed in stereochemistry, bulk and electronic properties were examined against the fungal 24-SMT from Paracoccidioides brasiliensis (Pb) which recognize lanosterol as the natural substrate. Analysis of the magnitude of sterol C24-methylation activity (based on the kinetic constants of V(max)/K(m) and product distributions determined by GC-MS) resulting from changes at the C3-position in which the 3ß-OH was replaced by 3α-OH, 3ß-acetyl, 3-oxo, 3-OMe, 3ß-F, 3ß-NH(2) (protonated species) or 3H group revealed that lanosterol and five substrate analogs were catalyzed and yielded identical side chain products whereas neither the 3H- or 3α-OH lanosterol derivatives were productively bound. Taken together, our results demonstrate a chemical complementarity involving hydrogen bonding formation of specific active site contacts to the nucleophilic C3-group of sterol is required for proper orientation of the substrate C-methyl intermediate in the activated complex.

A comparison of stage of presentation for pancreatic and colorectal cancer in Pennsylvania 2000-2005.

BACKGROUND: The goal of this study was to examine how rurality, socioeconomic status (SES) and access to medical care are related to the stage at presentation of patients with colorectal (CRC) and pancreatic cancer (PC) in Pennsylvania. MATERIALS AND METHODS: Incident CRC and PC cases were identified from the Pennsylvania Department of Health. Demographic, SES, and access variables were collected at the county level. RESULTS: Increased urbanization, younger age, and male gender were shown to be significantly related to later stage at diagnosis for PC. Age and education level were significant predictors of the rate of PC, while age, education level, insurance status, rurality, and the ratio of oncologists to primary care physicians were significant predictors of the rate of CRC. CONCLUSION: Based on county-level data, urban residence, younger age, and male gender were shown to be predictors of later stage at diagnosis for PC. These findings should help guide further research into factors that may be important predictors of later stage of diagnosis.

The VARL gene family and the evolutionary origins of the master cell-type regulatory gene, regA, in Volvox carteri.

Chlamydomonas reinhardtii, Volvox carteri, and their relatives in the family Volvocaceae provide an excellent opportunity for studying how multicellular organisms with differentiated cell types evolved from unicellular ancestors. While C. reinhardtii is unicellular, V. carteri is multicellular with two cell types, one of which resembles C. reinhardtii cytologically but is terminally differentiated. Maintenance of this "somatic cell" fate is controlled by RegA, a putative transcription factor. We recently showed that RegA shares a conserved region with several predicted V. carteri and C. reinhardtii proteins and that this region, the VARL domain, is likely to include a DNA-binding SAND domain. As the next step toward understanding the evolutionary origins of the regA gene, we analyzed the genome sequences of C. reinhardtii and V. carteri to identify additional genes with the potential to encode VARL domain proteins. Here we report that the VARL gene family, which consists of 12 members in C. reinhardtii and 14 in V. carteri, has experienced a complex evolutionary history in which members of the family have been both gained and lost over time, although several pairs of potentially orthologous genes can still be identified. We find that regA is part of a tandem array of four VARL genes in V. carteri but that a similar array is absent in C. reinhardtii. Most importantly, our phylogenetic analysis suggests that a proto-regA gene was present in a common unicellular ancestor of V. carteri and C. reinhardtii and that this gene was lost in the latter lineage.

Orthologs and paralogs of regA, a master cell-type regulatory gene in Volvox carteri.

The multicellular green alga Volvox carteri forma nagariensis has only two cell types: terminally differentiated somatic cells and reproductive cells. The regA gene maintains the terminally differentiated state of the somatic cells, apparently by repressing transcription of genes required for chloroplast biogenesis and thereby preventing cell growth. Because the RegA protein sequence bore no obvious motifs, we are attempting to identify regions of functional importance by searching for strongly conserved domains in RegA orthologs. Here we report the cloning and characterization of regA from the most closely related known taxon, V. carteri f. kawasakiensis. Given the closeness of the relationship between these two formas, their regA genes are surprisingly different: they differ in the number of introns and by several lengthy indels, and they encode proteins that are only 80% identical. We also serendipitously discovered a paralogous gene immediately upstream of each regA locus. The two regA genes, both upstream paralogs and several genes in Chlamydomonas (the closest unicellular relative of Volvox) encode a conserved region (the VARL domain) that contains what appears to be a DNA-binding SAND domain. This discovery has opened up a new avenue for exploring how regA and the terminally differentiated state that it controls evolved.