Phosphorylation of mammalian cytosolic and mitochondrial malate dehydrogenase: insights into regulation.

Malate dehydrogenase (MDH) is a key enzyme in mammalian metabolic pathways in cytosolic and mitochondrial compartments. Regulation of MDH through phosphorylation remains an underexplored area. In this review we consolidate evidence supporting the potential role of phosphorylation in modulating the function of mammalian MDH. Parallels are drawn with the phosphorylation of lactate dehydrogenase, a homologous enzyme, to reveal its regulatory significance and to suggest a similar regulatory strategy for MDH. Comprehensive mining of phosphorylation databases, provides substantial experimental (primarily mass spectrometry) evidence of MDH phosphorylation in mammalian cells. Experimentally identified phosphorylation sites are overlaid with MDH's functional domains, offering perspective on how these modifications could influence enzyme activity. Preliminary results are presented from phosphomimetic mutations (serine/threonine residues changed to aspartate) generated in recombinant MDH proteins serving as a proof of concept for the regulatory impact of phosphorylation. We also examine and highlight several approaches to probe the structural and cellular impact of phosphorylation. This review highlights the need to explore the dynamic nature of MDH phosphorylation and calls for identifying the responsible kinases and the physiological conditions underpinning this modification. The synthesis of current evidence and experimental data aims to provide insights for future research on understanding MDH regulation, offering new avenues for therapeutic interventions in metabolic disorders and cancer.

Roles of the oncometabolite enantiomers of 2-hydroxyglutarate and their metabolism by diverse dehydrogenases.

2-Hydroxyglutarate (2HG) is an oncometabolite that can contribute to tumor progression. Two enantiomer forms, L-2HG and D-2HG, arise from independent pathways starting from the precursor α-ketoglutarate (αKG). L-2HG production occurs through the promiscuous activities of malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) under acidic and/or hypoxic conditions. D-2HG frequently accumulates by gain-of-function mutations in the genes encoding two isoforms of isocitrate dehydrogenase (IDH1 and IDH2). Cognate metabolite repair enzymes, L- and D-2-hydroxyglutarate dehydrogenases, oxidize the enantiomers and cause abnormally high 2HG accumulation and disease when mutated. Elevated levels of either oncometabolite affect redox homeostasis, metabolism, and immune system functioning. Moreover, the oncometabolites inhibit several α-ketoglutarate-dependent dioxygenases resulting in epigenetic changes such as DNA and histone hypermethylation as well as deficiencies in DNA repair. L-2HG, and D-2HG in some cases, inhibit degradation of hypoxia-inducible factor (HIF1α), a transcription factor that alters gene expression to adapt to hypoxic conditions, favoring tumorigenesis. Patients with the rare disease 2-hydroxyglutaric aciduria (2HGA) have exceedingly high levels of 2HG, which is neurotoxic, causing developmental delays and brain abnormalities. D-2HG also has specific effects on collagen production and NADPH pools. Recently, D-2HG has been targeted in new chemotherapies aimed at disrupting the gain-of-function IDH1 and IDH2 mutants, resulting in successful clinical trials for several cancers.

External Collaboration Results in Student Learning Gains and Positive STEM Attitudes in CUREs.

The implementation of course-based undergraduate research experiences (CUREs) has made it possible to expose large undergraduate populations to research experiences. For these research experiences to be authentic, they should reflect the increasingly collaborative nature of research. While some CUREs have expanded, involving multiple schools across the nation, it is still unclear how a structured extramural collaboration between students and faculty from an outside institution affects student outcomes. In this study, we established three cohorts of students: 1) no-CURE, 2) single-institution CURE (CURE), and 3) external collaborative CURE (ec-CURE), and assessed academic and attitudinal outcomes. The ec-CURE differs from a regular CURE in that students work with faculty member from an external institution to refine their hypotheses and discuss their data. The sharing of ideas, data, and materials with an external faculty member allowed students to experience a level of collaboration not typically found in an undergraduate setting. Students in the ec-CURE had the greatest gains in experimental design; self-reported course benefits; scientific skills; and science, technology, engineering, and mathematics (STEM) importance. Importantly this study occurred in a diverse community of STEM disciplinary faculty from 2- and 4-year institutions, illustrating that exposing students to structured external collaboration is both feasible and beneficial to student learning.

Crowdsourcing biocuration: The Community Assessment of Community Annotation with Ontologies (CACAO).

Experimental data about gene functions curated from the primary literature have enormous value for research scientists in understanding biology. Using the Gene Ontology (GO), manual curation by experts has provided an important resource for studying gene function, especially within model organisms. Unprecedented expansion of the scientific literature and validation of the predicted proteins have increased both data value and the challenges of keeping pace. Capturing literature-based functional annotations is limited by the ability of biocurators to handle the massive and rapidly growing scientific literature. Within the community-oriented wiki framework for GO annotation called the Gene Ontology Normal Usage Tracking System (GONUTS), we describe an approach to expand biocuration through crowdsourcing with undergraduates. This multiplies the number of high-quality annotations in international databases, enriches our coverage of the literature on normal gene function, and pushes the field in new directions. From an intercollegiate competition judged by experienced biocurators, Community Assessment of Community Annotation with Ontologies (CACAO), we have contributed nearly 5,000 literature-based annotations. Many of those annotations are to organisms not currently well-represented within GO. Over a 10-year history, our community contributors have spurred changes to the ontology not traditionally covered by professional biocurators. The CACAO principle of relying on community members to participate in and shape the future of biocuration in GO is a powerful and scalable model used to promote the scientific enterprise. It also provides undergraduate students with a unique and enriching introduction to critical reading of primary literature and acquisition of marketable skills.

An idea to explore: Use of augmented reality for teaching three-dimensional biomolecular structures.

Biology and biochemistry students must learn to visualize and comprehend the complex three-dimensional (3D) structures of macromolecules such as proteins or DNA. However, most tools available for teaching biomolecular structures typically operate in two dimensions. Here, we present protocols and pedagogical approaches for using immersive augmented reality (AR) visors, specifically the Microsoft HoloLens, to reinforce learning with large scale 3D holographic structures. We developed a novel workflow to render vividly colored custom biomolecules in AR visors. In addition, we developed AR exercises to review concepts relevant to protein or DNA structure and then implemented the exercises in four different biology and biochemistry courses. Surveys showed that students reported greater interest in biomolecular structures after the exercise. We also highlight some of the advantages and disadvantages of the software and hardware of this upcoming technology.

Superior Dispersal Ability Can Lead to Persistent Ecological Dominance throughout Succession.

A large number of descriptive surveys have shown that microbial communities experience successional changes over time and that ecological dominance is common in the microbial world. However, direct evidence for the ecological processes mediating succession or causing ecological dominance remains rare. Different dispersal abilities among species may be a key mechanism. We surveyed fungal diversity within a metacommunity of pitchers of the model carnivorous plant Sarracenia purpurea and discovered that the yeast Candida pseudoglaebosa was ecologically dominant. Its frequency in the metacommunity increased during the growing season, and it was not replaced by other taxa. We next measured its competitive ability in a manipulative laboratory experiment and tracked its dispersal over time in nature. Despite its dominance, C. pseudoglaebosa is not a superior competitor. Instead, it is a superior disperser: it arrives in pitchers earlier, and disperses into more pitchers, than other fungi. Differential dispersal across the spatially structured metacommunity of individual pitchers emerges as a key driver of the continuous dominance of C. pseudoglaebosa during succession.IMPORTANCE Microbial communities are ubiquitous and occupy nearly every imaginable habitat and resource, including human-influenced habitats (e.g., fermenting food and hospital surfaces) and habitats with little human influence (e.g., aquatic communities living in carnivorous plant pitchers). We studied yeast communities living in pitchers of the carnivorous purple pitcher plant to understand how and why microbial communities change over time. We found that dispersal ability is not only important for fungal communities early in their existence, it can also determine which species is dominant (here, the yeast Candida pseudoglaebosa) long after the species and its competitors have arrived. These results contrast with observations from many human-influenced habitats, in which a good competitor eventually outcompetes good dispersers, since humans often design these habitats to favor a specific competitor. This study will help microbiologists understand the qualities of microbial species that enable takeover of new habitats in both natural and human-influenced environments.

Stressing Escherichia coli to educate students about research: A CURE to investigate multiple levels of gene regulation.

Course-based undergraduate research experiences (CUREs) have been shown to increase student retention and learning in the biological sciences. Most CURES cover only one aspect of gene regulation, such as transcriptional control. Here we present a new inquiry-based lab that engages understanding of gene expression from multiple perspectives. Students carry out a forward genetic screen to identify regulators of the stationary phase master regulator RpoS in the model organism Escherichia coli and then use a series of reporter fusions to determine if the regulation is at the level of transcription or the post-transcription level. This easy-to-implement course has been run both as a 9-week long project and a condensed 5-6 week version in three different schools and types of courses. A majority of the genes found in the screen are novel, thus giving students the opportunity to contribute to original findings to the field. Assessments of this CURE show student gains in learning in many knowledge areas. In addition, attitudinal surveys suggest the students are enthusiastic about the screen and their learning about gene regulation. In summary, this lab would be an appropriate addition to an intermediate or advanced level Molecular Biology, Genetics, or Microbiology curriculum. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(5):449-458, 2017.

Mechanical slowing-down of cytoplasmic diffusion allows in vivo counting of proteins in individual cells.

Many key regulatory proteins in bacteria are present in too low numbers to be detected with conventional methods, which poses a particular challenge for single-cell analyses because such proteins can contribute greatly to phenotypic heterogeneity. Here we develop a microfluidics-based platform that enables single-molecule counting of low-abundance proteins by mechanically slowing-down their diffusion within the cytoplasm of live Escherichia coli (E. coli) cells. Our technique also allows for automated microscopy at high throughput with minimal perturbation to native physiology, as well as viable enrichment/retrieval. We illustrate the method by analysing the control of the master regulator of the E. coli stress response, RpoS, by its adapter protein, SprE (RssB). Quantification of SprE numbers shows that though SprE is necessary for RpoS degradation, it is expressed at levels as low as 3-4 molecules per average cell cycle, and fluctuations in SprE are approximately Poisson distributed during exponential phase with no sign of bursting.

RpoS proteolysis is controlled directly by ATP levels in Escherichia coli.

The master regulator of stationary phase in Escherichia coli, RpoS, responds to carbon availability through changes in stability, but the individual steps in the pathway are unknown. Here we systematically block key steps of glycolysis and the citric acid cycle and monitor the effect on RpoS degradation in vivo. Nutrient upshifts trigger RpoS degradation independently of protein synthesis by activating metabolic pathways that generate small energy molecules. Using metabolic mutants and inhibitors, we show that ATP, but not GTP or NADH, is necessary for RpoS degradation. In vitro reconstitution assays directly demonstrate that ClpXP fails to degrade RpoS, but not other proteins, at low ATP hydrolysis rates. These data suggest that cellular ATP levels directly control RpoS stability.

Bacterial diversity across individual lichens.

Symbioses are unique habitats for bacteria. We surveyed the spatial diversity of bacterial communities across multiple individuals of closely related lichens using terminal restriction fragment length polymorphism (T-RFLP) and pyrosequencing. Centers of lichens house richer, more consistent assemblages than species-poor and compositionally disparate lichen edges, suggesting that ecological succession plays a role in structuring these communities.

A keystone predator controls bacterial diversity in the pitcher-plant (Sarracenia purpurea) microecosystem.

The community of organisms inhabiting the water-filled leaves of the carnivorous pitcher-plant Sarracenia purpurea includes arthropods, protozoa and bacteria, and serves as a model system for studies of food web dynamics. Despite the wealth of data collected by ecologists and zoologists on this food web, very little is known about the bacterial assemblage in this microecosystem. We used terminal restriction fragment length polymorphism (T-RFLP) analysis to quantify bacterial diversity within the pitchers as a function of pitcher size, pH of the pitcher fluid and the presence of the keystone predator in this food web, larvae of the pitcher-plant mosquito Wyeomyia smithii. Results were analysed at two spatial scales: within a single bog and across three isolated bogs. Pitchers were sterile before they opened and composition of the bacterial assemblage was more variable between different bogs than within bogs. Measures of bacterial richness and diversity were greater in the presence of W. smithii and increased with increasing pitcher size. Our results suggest that fundamental ecological concepts derived from macroscopic food webs can also be used to predict the bacterial assemblages in pitcher plants.

Decline in ribosomal fidelity contributes to the accumulation and stabilization of the master stress response regulator sigmaS upon carbon starvation.

The sigma(S) subunit of RNA polymerase is a master regulator of Escherichia coli that retards cellular senescence and bestows cells with general stress protective functions during growth arrest. We show that mutations and drugs triggering translational errors elevate sigma(S) levels and stability. Furthermore, mutations enhancing translational fidelity attenuate induction of the rpoS regulon and prevent stabilization of sigma(S) upon carbon starvation. Destabilization of sigma(S) by increased proofreading requires the presence of the sigma(S) recognition factor SprE (RssB) and the ClpXP protease. The data further suggest that sigma(S) becomes stabilized upon starvation as a result of ClpP sequestration and this sequestration is enhanced by oxidative modifications of aberrant proteins produced by erroneous translation. ClpP overproduction counteracted starvation-induced stabilization of sigma(S), whereas overproduction of a ClpXP substrate (ssrA-tagged GFP) stabilized sigma(S) in exponentially growing cells. We present a model for the sequence of events leading to the accumulation and activation of sigma(S) upon carbon starvation, which are linked to alterations in both ribosomal fidelity and efficiency.

LrhA regulates rpoS translation in response to the Rcs phosphorelay system in Escherichia coli.

Regulation of the Escherichia coli stationary-phase sigma factor RpoS is complex and occurs at multiple levels in response to different environmental stresses. One protein that reduces RpoS levels is the transcription factor LrhA, a global regulator of flagellar synthesis. Here we clarify the mechanism of this repression and provide insight into the signaling pathways that feed into this regulation. We show that LrhA represses RpoS at the level of translation in a manner that is dependent on the small RNA (sRNA) chaperone Hfq. Although LrhA also represses the transcription of the sRNA RprA, its regulation of RpoS mainly occurs independently of RprA. To better understand the physiological signals affecting this pathway, a transposon mutagenesis screen was carried out to find factors affecting LrhA activity levels. The RcsCDB phosphorelay system, a cell envelope stress-sensing pathway, was found to repress lrhA synthesis. In addition, mutations in the gene encoding the DNA motor protein FtsK induce lrhA synthesis, which may explain why such strains fail to accumulate RpoS in stationary phase.

RpoS proteolysis is regulated by a mechanism that does not require the SprE (RssB) response regulator phosphorylation site.

In Escherichia coli the response regulator SprE (RssB) facilitates degradation of the sigma factor RpoS by delivering it to the ClpXP protease. This process is regulated: RpoS is degraded in logarithmic phase but becomes stable upon carbon starvation, resulting in its accumulation. Because SprE contains a CheY domain with a conserved phosphorylation site (D58), the prevailing model posits that this control is mediated by phosphorylation. To test this model, we mutated the conserved response regulator phosphorylation site (D58A) of the chromosomal allele of sprE and monitored RpoS levels in response to carbon starvation. Though phosphorylation contributed to the SprE basal activity, we found that RpoS proteolysis was still regulated upon carbon starvation. Furthermore, our results indicate that phosphorylation of wild-type SprE occurs by a mechanism that is independent of acetyl phosphate.

Interactions of enantiomers of 2',3'-didehydro-2',3'-dideoxy-fluorocytidine with wild type and M184V mutant HIV-1 reverse transcriptase.

Both the beta-D-(+) and beta-L-(-)-enantiomers of 2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine (D4FC) are clinically relevant compounds because of their potent anti-HIV and anti-HBV activities. Cross-resistance to L-D4FC with HBV containing a mutation in the conserved polymerase YMDD region has been observed. In order to better understand the effects of stereochemistry on planar 5-fluorinated cytidine analogs and to gain insight into resistance caused by YMDD mutations in HIV-1 reverse transcriptase (RT), a combination of transient kinetic studies and computer modeling were employed. In contrast to studies with the (+) and (-) isomers of 3TC-TP and FTC-TP, it was found that wild type RT had a high enantiomeric selectivity between the D-(+) and L-(-) isomers of D4FC-TP. While no resistance was conferred by the methionine 184 to valine mutation to D-D4FC-TP, L-D4FC-TP was incorporated 50- to 70-fold less efficiently. The kinetic parameters of incorporation in the presence of L-D4FC-TP by RT(WT) and the mechanism of resistance by RT(M184V) were found to be distinct from those seen with the corresponding L-isomers containing an oxathiolane ring: (-)-3TC-TP and (-)-FTC-TP. Molecular modeling suggests that L- and D-D4FC-TP are positioned in the active site favorably for incorporation by RT(WT) and that L-D4FC-TP, but not D-D4FC-TP, is sterically hindered by the addition of a beta branched amino acid at position 184 of RT(M184V).