Branched-chain amino acids (BCAA) are strongly associated with dysregulated glucose and lipid metabolism, but the underlying mechanisms are poorly understood. We report that inhibition of the kinase ...(BDK) or overexpression of the phosphatase (PPM1K) that regulates branched-chain ketoacid dehydrogenase (BCKDH), the committed step of BCAA catabolism, lowers circulating BCAA, reduces hepatic steatosis, and improves glucose tolerance in the absence of weight loss in Zucker fatty rats. Phosphoproteomics analysis identified ATP-citrate lyase (ACL) as an alternate substrate of BDK and PPM1K. Hepatic overexpression of BDK increased ACL phosphorylation and activated de novo lipogenesis. BDK and PPM1K transcript levels were increased and repressed, respectively, in response to fructose feeding or expression of the ChREBP-β transcription factor. These studies identify BDK and PPM1K as a ChREBP-regulated node that integrates BCAA and lipid metabolism. Moreover, manipulation of the BDK:PPM1K ratio relieves key metabolic disease phenotypes in a genetic model of severe obesity.
Display omitted
•Lowering the BDK:PPM1K ratio improves glucose and lipid homeostasis in obese rats•ATP-citrate lyase (ACL) is an alternate BDK/PPM1K substrate•Overexpression of BDK in liver increases ACL phosphorylation and de novo lipogenesis•The ChREBP-β transcription factor increases BDK and decreases PPM1K expression
Branched-chain amino acids (BCAA) are strongly associated with metabolic diseases. White et al. demonstrate that the kinase (BDK) and phosphatase (PPM1K) that regulate a rate-limiting BCAA metabolic enzyme, BCKDH, also regulate ATP-citrate lyase, a key lipogenic enzyme, thus identifying a new regulatory node that integrates BCAA and lipid metabolism.
Isothermal titration calorimetry (ITC) has long been established as an excellent means to determine the thermodynamic parameters of biomolecular interactions. More recently, efforts have focused on ...exploiting the power/time trace (the “thermogram”) resulting from ITC experiments to glean kinetic association and dissociation rates for these interactions. The success of such analyses rests on the ability of algorithms to simulate with high accuracy the output of the calorimeter. Thus, several critical factors must be taken into account: the injection protocol, the kinetics of the interaction, accurate discovery of the instrumental response to heat signals, and the addition of unrelated signals. All of these aspects of extracting kinetic constants from thermograms have been considered and addressed in the current work. To validate the resultant methods, we performed several ITC experiments, titrating small-molecule inhibitors into solutions of bovine carbonic anhydrase II or titrating lysozyme into solutions of anti-lysozyme nanobodies. We found that our methods could arrive at kinetic constants that were close to the known values for these interactions taken from other methods. Finally, the effort to improve ITC kinetic characterizations uncovered a set of best practices for both the calorimetric experiment and the subsequent analyses (termed “kinetically optimized ITC” or “KO-ITC”) that is detailed in this work.
The mechanisms of energy generation and carbon-source utilization in the syphilis spirochete Treponema pallidum have remained enigmatic despite complete genomic sequence information. Whereas the ...bacterium harbors enzymes for glycolysis, the apparatus for more efficient use of glucose catabolites, namely the citric-acid cycle, is apparently not present. Yet, the organism's energy needs likely exceed the modest output from glycolysis alone. Recently, building on our structure-function studies of T. pallidum lipoproteins, we proposed a "flavin-centric" metabolic lifestyle for the organism that partially resolves this conundrum. As a part of the hypothesis, we have proposed that T. pallidum contains an acetogenic energy-conservation pathway that catabolizes D-lactate, yielding acetate, reducing equivalents for the generation and maintenance of chemiosmotic potential, and ATP. We already have confirmed the D-lactate dehydrogenase activity in T. pallidum necessary for this pathway to operate. In the current study, we focused on another enzyme ostensibly involved in treponemal acetogenesis, phosphotransacetylase (Pta). This enzyme is putatively identified as TP0094 and, in this study, we determined a high-resolution (1.95 Å) X-ray crystal structure of the protein, finding that its fold comports with other known Pta enzymes. Further studies on its solution behavior and enzyme activity confirmed that it has the properties of a Pta. These results are consistent with the proposed acetogenesis pathway in T. pallidum, and we propose that the protein be referred to henceforth as TpPta.
Biophysical and biochemical studies on the lipoproteins and other periplasmic proteins from the spirochetal species Treponema pallidum have yielded numerous insights into the functioning of the ...organism's peculiar membrane organization, its nutritional requirements, and intermediary metabolism. However, not all T. pallidum proteins have proven to be amenable to biophysical studies. One such recalcitrant protein is Tp0309, a putative polar‐amino‐acid‐binding protein of an ABC transporter system. To gain further information on its possible function, a homolog of the protein from the related species T. vincentii was used as a surrogate. This protein, Tv2483, was crystallized, resulting in the determination of its crystal structure at a resolution of 1.75 Å. The protein has a typical fold for a ligand‐binding protein, and a single molecule of l‐arginine was bound between its two lobes. Differential scanning fluorimetry and isothermal titration calorimetry experiments confirmed that l‐arginine bound to the protein with unusually high selectivity. However, further comparison to Tp0309 showed differences in key amino‐acid‐binding residues may impart an alternate specificity for the T. pallidum protein.
PDB Code(s): 6DET
Microtubules are heavily regulated dynamic polymers of αβ-tubulin that are required for proper chromosome segregation and organization of the cytoplasm. Polymerases in the XMAP215 family use arrayed ...TOG domains to promote faster microtubule elongation. Regulatory factors in the cytoplasmic linker associated protein (CLASP) family that reduce catastrophe and/or increase rescue also contain arrayed TOGs, but how CLASP TOGs contribute to activity is poorly understood. Here, using Saccharomyces cerevisiae Stu1 as a model CLASP, we report structural, biochemical, and reconstitution studies that clarify functional properties of CLASP TOGs. The two TOGs in Stu1 have very different tubulin-binding properties: TOG2 binds to both unpolymerized and polymerized tubulin, and TOG1 binds very weakly to either. The structure of Stu1-TOG2 reveals a CLASP-specific residue that likely confers distinctive tubulin-binding properties. The isolated TOG2 domain strongly suppresses microtubule catastrophe and increases microtubule rescue in vitro, contradicting the expectation that regulatory activity requires an array of TOGs. Single point mutations on the tubulin-binding surface of TOG2 ablate its anti-catastrophe and rescue activity in vitro, and Stu1 function in cells. Revealing that an isolated CLASP TOG can regulate polymerization dynamics without being part of an array provides insight into the mechanism of CLASPs and diversifies the understanding of TOG function.
Pyruvate dehydrogenase kinase isoforms (PDKs 1–4) negatively regulate activity of the mitochondrial pyruvate dehydrogenase complex by reversible phosphorylation. PDK isoforms are up-regulated in ...obesity, diabetes, heart failure, and cancer and are potential therapeutic targets for these important human diseases. Here, we employed a structure-guided design to convert a known Hsp90 inhibitor to a series of highly specific PDK inhibitors, based on structural conservation in the ATP-binding pocket. The key step involved the substitution of a carbonyl group in the parent compound with a sulfonyl in the PDK inhibitors. The final compound of this series, 2-(2,4-dihydroxyphenyl)sulfonylisoindoline-4,6-diol, designated PS10, inhibits all four PDK isoforms with IC50 = 0.8 μm for PDK2. The administration of PS10 (70 mg/kg) to diet-induced obese mice significantly augments pyruvate dehydrogenase complex activity with reduced phosphorylation in different tissues. Prolonged PS10 treatments result in improved glucose tolerance and notably lessened hepatic steatosis in the mouse model. The results support the pharmacological approach of targeting PDK to control both glucose and fat levels in obesity and type 2 diabetes.
Up-regulated pyruvate dehydrogenase kinase isoforms (PDKs) are associated with impaired glucose homeostasis in diabetes.
Novel PDK inhibitors were developed using structure-based design, which improves glucose tolerance with reduced hepatic steatosis in diet-induced obese mice.
Obesity phenotypes are effectively treated by chemical intervention with PDK inhibitors.
PDKs are potential drug targets for obesity and type 2 diabetes.
Detergents are widely used in modern in vitro biochemistry and biophysics, in particular to aid the characterization of integral membrane proteins. An important characteristic of these chemicals in ...aqueous solutions is the concentration above which their molecular monomers self-associate to form micelles, termed the critical micellar concentration (CMC). Micelles are supramolecular assemblies arranged with the hydrophobic portions oriented inward and the hydrophilic head groups positioned outward to interact with the aqueous solvent. Knowledge of the CMC is not only of practical relevance but also of theoretical interest because it provides thermodynamic insights. Isothermal titration calorimetry (ITC) is a powerful method to determine CMCs, as it furnishes additional information on the enthalpy and entropy of micellization. Here we describe our extension of previous methods to determine CMCs and other thermodynamic parameters from ITC demicellization curves. The new algorithm, incorporated into the stand-alone software package D/STAIN, analyzes ITC demicellization curves by taking advantage of state-of-the-art thermogram-integration techniques and automatically providing rigorous confidence intervals on the refined parameters. As a demonstration of the software’s capabilities, we undertook ITC experiments to determine the respective CMCs of n-octyl β-d-glucopyranoside (OG), n-dodecyl β-d-maltopyranoside (DDM), and lauryldimethylamine N-oxide (LDAO). Motivated by the fact that in vitro membrane protein studies often require additives such as precipitants (e.g., polyethylene glycol (PEG)), we also carried out ITC demicellization studies in the presence of PEG3350, finding in all cases that PEG had significant effects on the thermodynamics of detergent micellization.
The emergence of microscale thermophoresis (MST) as a technique for determining the dissociation constants for bimolecular interactions has enabled these quantities to be measured in systems that ...were previously difficult or impracticable. However, most models for analyses of these data featured the assumption of a simple 1:1 binding interaction. The only model widely used for multiple binding sites was the Hill equation. Here, we describe two new MST analytic models that assume a 1:2 binding scheme: the first features two microscopic binding constants (KD(1) and KD(2)), while the other assumes symmetry in the bivalent molecule, culminating in a model with a single macroscopic dissociation constant (KD,M) and a single factor (α) that accounts for apparent cooperativity in the binding. We also discuss the general applicability of the Hill equation for MST data. The performances of the algorithms on both real and simulated data are assessed, and implementation of the algorithms in the MST analysis program PALMIST is discussed.
Mitochondrial pyruvate dehydrogenase kinases 1–4 (PDKs1–4) negatively regulate activity of the pyruvate dehydrogenase complex (PDC) by reversible phosphorylation. PDKs play a pivotal role in ...maintaining energy homeostasis and contribute to metabolic flexibility by attenuating PDC activity in various mammalian tissues. Cumulative evidence has shown that the up-regulation of PDK4 expression is tightly associated with obesity and diabetes. In this investigation, we test the central hypothesis that PDKs1-4 are a pharmacological target for lowering glucose levels and restoring insulin sensitivity in obesity and type 2 diabetes (T2D).
Diet-induced obese (DIO) mice were treated with a liver-specific pan-PDK inhibitor 2-(2,4-dihydroxyphenyl) sulfonylisoindoline-4,6-diol (PS10) for four weeks, and results compared with PDK2/PDK4 double knockout (DKO) mice on the same high fat diet (HFD).
Both PS10-treated DIO mice and HFD-fed DKO mice showed significantly improved glucose, insulin and pyruvate tolerance, compared to DIO controls, with lower plasma insulin levels and increased insulin signaling in liver. In response to lower glucose levels, phosphorylated AMPK in PS10-treated DIO and HFD-fed DKO mice is upregulated, accompanied by decreased nuclear carbohydrate-responsive element binding protein (ChREBP). The reduced ChREBP signaling correlates with down-regulation of hepatic lipogenic enzymes (ACC1, FAS, and SCD1), leading to markedly diminished hepatic steatosis in both study groups, with lower circulating cholesterol and triacylglyceride levels as well as reduced fat mass. PS10-treated DIO as well as DKO mice showed predominant fatty acid over glucose oxidation. However, unlike systemic DKO mice, increased hepatic PDC activity alone in PS10-treated DIO mice does not raise the plasma total ketone body level.
Our findings establish that specific targeting of hepatic PDKs with the PDK inhibitor PS10 is an effective therapeutic approach to maintaining glucose and lipid homeostasis in obesity and T2D, without the harmful ketoacidosis associated with systemic inhibition of PDKs.
•Diet-induced obese (DIO) mice were treated with a novel liver-specific pyruvate dehydrogenase kinase (PDK) inhibitor PS10.•PS10-treated DIO mice and PDK2/PDK4-DKO mice fed indentical HFD showed improved glucose, insulin and pyruvate tolerance.•PS10-treated DIO and untreated DKO mice manifest reduced hepatic steatosis and attenuation of ChREBP-mediated lipogenesis.•Unlike DKO mice, targeting hepatic PDK alone in DIO mice with PS10 does not increase total plasma ketone-body levels.
Phosphorylation of proteins on tyrosine (Tyr) residues evolved in metazoan organisms as a mechanism of coordinating tissue growth
. Multicellular eukaryotes typically have more than 50 distinct ...protein Tyr kinases that catalyse the phosphorylation of thousands of Tyr residues throughout the proteome
. How a given Tyr kinase can phosphorylate a specific subset of proteins at unique Tyr sites is only partially understood
. Here we used combinatorial peptide arrays to profile the substrate sequence specificity of all human Tyr kinases. Globally, the Tyr kinases demonstrate considerable diversity in optimal patterns of residues surrounding the site of phosphorylation, revealing the functional organization of the human Tyr kinome by substrate motif preference. Using this information, Tyr kinases that are most compatible with phosphorylating any Tyr site can be identified. Analysis of mass spectrometry phosphoproteomic datasets using this compendium of kinase specificities accurately identifies specific Tyr kinases that are dysregulated in cells after stimulation with growth factors, treatment with anti-cancer drugs or expression of oncogenic variants. Furthermore, the topology of known Tyr signalling networks naturally emerged from a comparison of the sequence specificities of the Tyr kinases and the SH2 phosphotyrosine (pTyr)-binding domains. Finally we show that the intrinsic substrate specificity of Tyr kinases has remained fundamentally unchanged from worms to humans, suggesting that the fidelity between Tyr kinases and their protein substrate sequences has been maintained across hundreds of millions of years of evolution.
PDF
