👤 Kevin Bain

As many as 700,000 unique sequences in the human genome are predicted to fold into G-quadruplexes (G4s), non-canonical structures formed by Hoogsteen guanine-guanine pairing within G-rich nucleic acids. G4s play both physiological and pathological roles in many vital cellular processes including DNA replication, DNA repair and RNA transcription. Several reagents have been developed to visualize G4s in vitro and in cells. Recently, Zhen et al. synthesized a small protein G4P based on the G4 recognition motif from RHAU (DHX36) helicase (RHAU specific motif, RSM). G4P was reported to bind the G4 structures in cells and in vitro, and to display better selectivity toward G4s than the previously published BG4 antibody. To get insight into G4P- G4 interaction kinetics and selectivity, we purified G4P and its expanded variants, and analyzed their G4 binding using single-molecule total internal reflection fluorescence microscopy and mass photometry. We found that G4P binds to various G4s with affinities defined mostly by the association rate. Doubling the number of the RSM units in the G4P increases the protein's affinity for telomeric G4s and its ability to interact with sequences folding into multiple G4s. Show less

As many as 700,000 unique sequences in the human genome are predicted to fold into G-quadruplexes (G4s), non-canonical structures formed by Hoogsteen guanine-guanine pairing within G-rich nucleic acids. G4s play both physiological and pathological roles in many vital cellular processes including DNA replication, DNA repair and RNA transcription. Several reagents have been developed to visualize G4s Show less

A strong association of obesity and insulin resistance with increased circulating levels of branched-chain and aromatic amino acids and decreased glycine levels has been recognized in human subjects for decades. More recently, human metabolomics and genetic studies have confirmed and expanded upon these observations, accompanied by a surge in preclinical studies that have identified mechanisms involved in the perturbation of amino acid homeostasis- how these events are connected to dysregulated glucose and lipid metabolism, and how elevations in branched-chain amino acids (BCAA) may participate in the development of insulin resistance, type 2 diabetes (T2D), and other cardiometabolic diseases and conditions. In human cohorts, BCAA and related metabolites are now well established as among the strongest biomarkers of obesity, insulin resistance, T2D, and cardiovascular diseases. Lowering of BCAA and branched-chain ketoacid (BCKA) levels by feeding BCAA-restricted diet or by the activation of the rate-limiting enzyme in BCAA catabolism, branched-chain ketoacid dehydrogenase (BCKDH), in rodent models of obesity have clear salutary effects on glucose and lipid homeostasis, but BCAA restriction has more modest effects in short-term studies in human T2D subjects. Feeding of rats with diets enriched in sucrose or fructose result in the induction of the ChREBP transcription factor in the liver to increase expression of the BCKDH kinase (BDK) and suppress the expression of its phosphatase (PPM1K) resulting in the inactivation of BCKDH and activation of the key lipogenic enzyme ATP-citrate lyase (ACLY). These and other emergent links between BCAA, glucose, and lipid metabolism motivate ongoing studies of possible causal actions of BCAA and related metabolites in the development of cardiometabolic diseases. Show less

In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators. Show less