Biochemical Journal最新文献

Bacteria evolve mechanisms to compete for limited resources and survive in new niches. Here we study the mechanism of isethionate import from the sulfate-reducing bacterium Oleidesulfovibrio alaskensis. The catabolism of isethionate by Desulfovibrio species has been implicated in human disease, due to hydrogen sulfide production, and has potential for industrial applications. O. alaskensis employs a tripartite ATP-independent periplasmic (TRAP) transporter (OaIsePQM) to import isethionate, which relies on the substrate-binding protein (OaIseP) to scavenge isethionate and deliver it to the membrane transporter component (OaIseQM) for import into the cell. We determined the binding affinity of isethionate to OaIseP by isothermal titration calorimetry, KD = 0.95 µM (68% CI = 0.6-1.4 µM), which is weaker compared with other TRAP substrate-binding proteins. The X-ray crystal structures of OaIseP in the ligand-free and isethionate-bound forms were obtained and showed that in the presence of isethionate, OaIseP adopts a closed conformation whereby two domains of the protein fold over the substrate. We serendipitously discovered two crystal forms with sulfonate-containing buffers (HEPES and MES) bound in the isethionate-binding site. However, these do not evoke domain closure, presumably because of the larger ligand size. Together, our data elucidate the molecular details of how a TRAP substrate-binding protein binds a sulfonate-containing substrate, rather than a typical carboxylate-containing substrate. These results may inform future antibiotic development to target TRAP transporters and provide insights into protein engineering of TRAP transporter substrate-binding proteins.

Regulation of protein longevity via the ubiquitin (Ub) - proteasome pathway is fundamental to eukaryotic biology. Ubiquitin E3 ligases (E3s) interact with substrate proteins and provide specificity to the pathway. A small subset of E3s bind to specific exposed N-termini (N-degrons) and promote the ubiquitination of the bound protein. Collectively these E3s, and other N-degron binding proteins, are known as N-recognins. There is considerable functional divergence between fungi, animal, and plant N-recognins. In plants, at least three proteins (PRT1, PRT6, and BIG) participate in the Arg/N-degron pathway. PRT1 has demonstrated E3 ligase activity, whereas PRT6 and BIG are candidate E3s. The Arg/N-degron pathway plays a central role in plant development, germination, and submersion tolerance. The pathway has been manipulated both to improve crop performance and for conditional protein degradation. A more detailed structural and biochemical understanding of the Arg/N-recognins and their substrates is required to fully realise the biotechnological potential of the pathway. This perspective focuses on the structural and molecular details of substrate recognition and ubiquitination in the plant Arg/N-degron pathway. While PRT1 appears to be plant specific, the PRT6 and BIG proteins are similar to UBR1 and UBR4, respectively. Analysis of the cryo-EM structures of Saccharomyces UBR1 suggests that the mode of ubiquitin conjugating enzyme (E2) and substrate recruitment is conserved in PRT6, but regulation of the two N-recognins may be significantly different. The structurally characterised domains from human UBR4 are also likely to be conserved in BIG, however, there are sizeable gaps in our understanding of both proteins.

The Phosphatases of Regenerating Liver (PRLs) are members of the protein tyrosine phosphatase (PTP) superfamily that play pro-oncogenic roles in cell proliferation, migration, and survival. We previously demonstrated that PRLs can post-translationally downregulate PTEN, a tumor suppressor frequently inactivated in human cancers, by dephosphorylating PTEN at Tyr336, which promotes the NEDD4-mediated PTEN ubiquitination and proteasomal degradation. Here we report that PRLs can also reduce PTEN expression by upregulating MicroRNA-21 (miR-21), which is one of the most frequently overexpressed miRNAs in solid tumors. We observe a broad correlation between PRL and miR-21 levels in multiple human cancers. Mechanistically, PRL2, the most abundant and ubiquitously expressed PRL family member, promotes the JAK2/STAT3 pathway-mediated miR-21 expression by directly dephosphorylating JAK2 at Tyr570. Finally, we confirm that the PRL2-mediated miR-21 expression contributes to its oncogenic potential in breast cancer cells. Our study defines a new functional role of PRL2 in PTEN regulation through a miR-21-dependent post-transcriptional mechanism, in addition to our previously reported NEDD4-dependent post-translational PTEN regulation. Together, these studies further establish the PRLs as negative regulators of PTEN.

Mild uncoupling of oxidative phosphorylation is an intrinsic property of all mitochondria, allowing for adjustments in cellular energy metabolism to maintain metabolic homeostasis. Small molecule uncouplers have been extensively studied for their potential to increase metabolic rate, and recent research has focused on developing safe and effective mitochondrial uncoupling agents for the treatment of obesity and cardiometabolic syndrome (CMS). Here, we provide a brief overview of CMS and cover the recent mechanisms by which chemical uncouplers regulate CMS-associated risk-factors and comorbidities, including dyslipidemia, insulin resistance, steatotic liver disease, type 2 diabetes, and atherosclerosis. Additionally, we review the current landscape of uncoupling agents, focusing on repurposed FDA-approved drugs and compounds in advanced preclinical or early-stage clinical development. Lastly, we discuss recent molecular insights by which chemical uncouplers enhance cellular energy expenditure, highlighting their potential as a new addition to the current CMS drug landscape, and outline several limitations that need to be addressed before these agents can successfully be introduced into clinical practice.

The active site Cys residue in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is sensitive to oxidation by hydrogen peroxide (H2O2), with this resulting in enzyme inactivation. This re-routes the carbon flux from glycolysis to the pentose phosphate pathway favoring the formation of NADPH and synthetic intermediates required for antioxidant defense and repair systems. Consequently, GAPDH inactivation serves as a redox switch for metabolic adaptation under conditions of oxidative stress. However, there is a major knowledge gap as to how GAPDH is efficiently oxidized and inactivated, when the increase in intracellular H2O2 is modest, and there is a high concentration of alternative (non-signaling) thiols and efficient peroxide removing systems. We have therefore explored whether GAPDH inactivation is enhanced by two factors of in vivo relevance: macromolecular crowding, an inherent property of biological environments, and the presence of bicarbonate, an abundant biological buffer. Bicarbonate is already known to modulate H2O2 metabolism via formation of peroxymonocarbonate. GAPDH activity was assessed in experiments with low doses of H2O2 under both dilute and crowded conditions (induced by inert high molecular mass polymers and small molecules), in both the absence and presence of 25 mM sodium bicarbonate. H2O2-induced inactivation of GAPDH was observed to be significantly enhanced under macromolecular crowding conditions, with bicarbonate having an additional effect. These data strongly suggest that these two factors are of major importance in redox switch mechanisms involving GAPDH (and possibly other thiol-dependent systems) within the cellular environment.

PI3Kα, consisting of the p110α isoform of the catalytic subunit of PI 3-kinase (encoded by PIK3CA) and the p85α regulatory subunit (encoded by PI3KR1) is activated by growth factor receptors. The identification of common oncogenic mutations in PIK3CA has driven the development of many inhibitors that bind to the ATP-binding site in the p110α subunit. Upon activation, PI3Kα undergoes conformational changes that promote its membrane interaction and catalytic activity, yet the effects of ATP-site directed inhibitors on the PI3Kα membrane interaction are unknown. Using FRET and biolayer interferometry assays, we show that a class of ATP-site directed inhibitors represented by GSK2126458 block the growth factor activated PI3KαWT membrane interaction, an activity dependent on the ligand forming specific ATP-site interactions. The membrane interaction for hot spot oncogenic mutations that bypass normal p85α regulatory mechanisms was insensitive to GSK2126458, while GSK2126458 could regulate mutations found outside of these hot spot regions. Our data show that the effect of GSK126458 on the membrane interaction requires the enzyme to revert from its growth factor activated state to a basal state. We find that an ATP substrate analogue can increase the wild type PI3Kα membrane interaction, uncovering a substrate based regulatory event that can be mimicked by different inhibitor chemotypes. Our findings, together with the discovery of small molecule allosteric activators of PI3Kα illustrate that PI3Kα membrane interactions can be modulated by factors related to ligand binding both within the ATP site and at allosteric sites.

Per- and polyfluorinated chemicals (PFAS) are of rising concern due to environmental persistence and emerging evidence of health risks to humans. Environmental persistence is largely attributed to a failure of microbes to degrade PFAS. PFAS recalcitrance has been proposed to result from chemistry, specifically C-F bond strength, or biology, largely negative selection from fluoride toxicity. Given natural evolution has many hurdles, this review advocates for a strategy of laboratory engineering and evolution. Enzymes identified to participate in defluorination reactions have been discovered in all Enzyme Commission classes, providing a palette for metabolic engineering. In vivo PFAS biodegradation will require multiple types of reactions and powerful fluoride mitigation mechanisms to act in concert. The necessary steps are to: (1) engineer bacteria that survive very high, unnatural levels of fluoride, (2) design, evolve, and screen for enzymes that cleave C-F bonds in a broader array of substrates, and (3) create overall physiological conditions that make for positive selective pressure with PFAS substrates.

Sequence variation among homologous proteins can shed light on their function and ancestry. In this study, we analyze variation at catalytic residues among MCR (mobile colistin resistance) proteins, which confer resistance to the last resort antibiotic, colistin, in gram-negative bacteria. We show that not all naturally occurring variants at a lipid A-binding residue, Ser284, are tolerated in MCR-1. In particular, the substitution of Ser284 with Asp, found naturally in MCR-5, resulted in diminished colistin resistance. Using phylogenetic analyses and structure predictions we trace back variation at this site among MCRs to their ancestors, i.e. EptA phosphoethanolamine transferases that are encoded by diverse bacterial genomes. Mutational studies and AlphaFold-based structural modeling revealed that the functional importance of position 284 varies between phylogenetically distant MCRs, i.e. MCR-1 and MCR-5. Despite a high degree of similarity among their catalytic domains, inter-domain interactions were not conserved between MCR-1 and MCR-5 due to their different ancestries, providing a mechanistic basis behind the different phenotypes of similar mutations at position 284. Our study thus uncovers subtle differences in the organization of domains among MCR proteins that can lead to substantial differences in their catalytic properties and mutational tolerances.

Erythrokeratodermia variabilis et progressiva (EKVP) is a rare hereditary skin disorder characterized by hyperkeratotic plaques and erythematous patches that progressively worsen with age. This disorder has been associated with variants in three connexin encoding genes (GJA1, GJB3, GJB4) and four unrelated genes (KRT83, KDSR, TRPM4, PERP). Most cases of connexin-linked EKVP exhibit an autosomal dominant mode of inheritance, with rare autosomal recessive cases. Collectively, evidence suggests that connexin variants associated with EKVP elicit a plethora of molecular defects including impaired gap junction (GJ) formation, dysregulated hemichannel and/or GJ channel function, cytotoxicity, dominant disruption of co-expressed connexins, and/or altered turnover kinetics. Here, we review the progress made in understanding the genetic and molecular basis of EKVP associated with connexin gene variants. We also discuss the landscape of treatment options used for this disorder and the future directions for research into this rare condition.