Type 2 diabetes is associated with increased fracture risk and delayed facture healing; the underlying mechanism, however, remains poorly understood. We systematically investigated skeletal pathology in leptin receptor–deficient diabetic mice on a C57BLKS background (db). Compared with wild type (wt), db mice displayed reduced peak bone mass and age-related trabecular and cortical bone loss. Poor skeletal outcome in db mice contributed high-glucose– and nonesterified fatty acid–induced osteoblast apoptosis that was associated with peroxisome proliferator–activated receptor γ coactivator 1-α (PGC-1α) downregulation and upregulation of skeletal muscle atrogenes in osteoblasts. Osteoblast depletion of the atrogene muscle ring finger protein-1 (MuRF1) protected against gluco- and lipotoxicity-induced apoptosis. Osteoblast-specific PGC-1α upregulation by 6-C-β-d-glucopyranosyl-(2S,3S)-(+)-5,7,3′,4′-tetrahydroxydihydroflavonol (GTDF), an adiponectin receptor 1 (AdipoR1) agonist, as well as metformin in db mice that lacked AdipoR1 expression in muscle but not bone restored osteopenia to wt levels without improving diabetes. Both GTDF and metformin protected against gluco- and lipotoxicity-induced osteoblast apoptosis, and depletion of PGC-1α abolished this protection. Although AdipoR1 but not AdipoR2 depletion abolished protection by GTDF, metformin action was not blocked by AdipoR depletion. We conclude that PGC-1α upregulation in osteoblasts could reverse type 2 diabetes–associated deterioration in skeletal health.
M. tuberculosis harbors an essential phosphoserine phosphatase (MtSerB2, Rv3042c) that contains two small- molecule binding ACT-domains (Pfam 01842) at the N-terminus followed by the phosphoserine phosphatase (PSP) domain. We found that exogenously added MtSerB2 elicits microtubule rearrangements in THP-1 cells. Mutational analysis demonstrates that phosphatase activity is co-related to the elicited rearrangements, while addition of the ACT-domains alone elicits no rearrangements. The enzyme is dimeric, exhibits divalent metal- ion dependency, and is more specific for l- phosphoserine unlike other classical PSPases. Binding of a variety of amino acids to the ACT-domains influences MtSerB2 activity by either acting as activators/inhibitors/have no effects. Additionally, reduced activity of the PSP domain can be enhanced by equimolar addition of the ACT domains. Further, we identified that G18 and G108 of the respective ACT-domains are necessary for ligand-binding and their mutations to G18A and G108A abolish the binding of ligands like l- serine. A specific transition to higher order oligomers is observed upon the addition of l- serine at ∼0.8 molar ratio as supported by Isothermal calorimetry and Size exclusion chromatography experiments. Mutational analysis shows that the transition is dependent on binding of l- serine to the ACT-domains. Furthermore, the higher-order oligomeric form of MtSerB2 is inactive, suggesting that its formation is a mechanism for feedback control of enzyme activity. Inhibition studies involving over eight inhibitors, MtSerB2, and the PSP domain respectively, suggests that targeting the ACT-domains can be an effective strategy for the development of inhibitors.
dPiscidin-1 possesses significant antimicrobial and cytotoxic activities. To recognize the primary amino acid sequence(s) in piscidin-1 that could be important for its biological activity, a long heptad repeat sequence located in the region from amino acids 2 to 19 was identified. To comprehend the possible role of this motif, six analogs of piscidin-1 were designed by selectively replacing a single isoleucine residue at a d (5th) position or at an a (9th or 16th) position with either an alanine or a valine residue. Two more analogs, namely, I5F,F6A-piscidin-1 and V12I-piscidin-1, were designed for investigating the effect of interchanging an alanine residue at a d position with an adjacent phenylalanine residue and replacing a valine residue with an isoleucine residue at another d position of the heptad repeat of piscidin-1, respectively. Single alanine-substituted analogs exhibited significantly reduced cytotoxicity against mammalian cells compared with that of piscidin-1 but appreciably retained the antibacterial and antiendotoxin activities of piscidin-1. All the single valine-substituted piscidin-1 analogs and I5F,F6A-piscidin-1 showed cytotoxicity greater than that of the corresponding alanine-substituted analogs, antibacterial activity marginally greater than or similar to that of the corresponding alanine-substituted analogs, and also antiendotoxin activity superior to that of the corresponding alanine-substituted analogs. Interestingly, among these peptides, V12I-piscidin-1 showed the highest cytotoxicity and antibacterial and antiendotoxin activities. Lipopolysaccharide (12 mg/kg of body weight)-treated mice, further treated with I16A-piscidin-1, the piscidin-1 analog with the highest therapeutic index, at a single dose of 1 or 2 mg/kg of body weight, showed 80 and 100% survival, respectively. Structural and functional characterization of these peptides revealed the basis of their biological activity and demonstrated that nontoxic piscidin-1 analogs with significant antimicrobial and antiendotoxin activities can be designed by incorporating single alanine substitutions in the piscidin-1 heptad repeat. Fish antimicrobial peptide (AMP) piscidin-1, which was discovered in 2001, possesses versatile biological activities. Piscidin-1 shows significant activity against bacteria, fungi, parasites, and cancer cells (1-7). It can also neutralize lipopolysaccharide (LPS)-induced proinflammatory responses in macrophage cells (5). Along with these desired biological activities, piscidin-1 also exhibits very significant lytic activity against normal mammalian cells, which is an obstacle for employing it as a lead molecule for the development of a new antimicrobial agent. Therefore, deciphering of the basis of cytotoxicity in piscidin-1 and the design of nontoxic analogs of piscidin-1 with desired biological activity were the objectives of the present investigation. Toward this end, we intended to identify the important primary sequence in piscidin-1 that could have a strong impact on its structural, functional, and ...
Feast/famine regulatory proteins (FFRPs), 5 also known as Lrp/AsnC family proteins, bind to a variety of effectors like amino acids that modulate the respective regulatory functions. They are involved in the formation of globular nucleoprotein structures, chromosome structure organization, and other regulatory functions (1). Directly or indirectly, they globally regulate a variety of metabolic processes in response to amino acids and nitrogen bases present in the environment. The general understanding is that FFRPs adopt a variety of quaternary structures upon binding/release of effectors. This presumably allows for binding to target promoter regions or for disrupting the nucleoprotein structures formed by them (2). Fine tuning/ selection of the target promoter-FFRP interactions is also generally thought to occur due to binding of the effector molecules and in some cases can elicit subtle structural changes as opposed to changes to the oligomeric association itself (3, 4). Escherichia coli Lrp, a better studied member of the family, is known to be important for changes that occur from a "feasting" to a "famine" state, and it controls ϳ10% of gene expression (5, 6). The latter protein binds to a variety of amino acids like Leu, Ala, Pro, and Val, and that in turn elicits either positive or negative regulation of the target genes (7).Crystal structures of several FFRPs/Lrp/AsnC-type proteins have been reported from bacterial and archaeal sources. These include structures of the E. coli Lrp, E. coli AsnC, Mycobacterium tuberculosis FFRP (also called LrpA), Lrp proteins from Pyrococcus furiosus, and Pyrococcus sp. OT3 FL11. Complexes of some of them with several amino acids, DNA, etc. have also been reported (8 -10). The studies reveal that the basic functional unit of FFRPs is a dimer where each chain folds into two domains. The N-terminal domain normally faces outside and contains the DNA-binding helix-turn-helix motif and a C-terminal domain that is involved in effector binding and in oligomerization. Both of the domains are connected by a rather long linker region. Some FFRPs like the M. tuberculosis FFRP and E. coli Lrp have been shown to adopt the rare "open" quaternary structure that seems to be an operating principle in these proteins. This presumably allows them to bind to non-symmetrical target sites (10 -12). In general, instances of deviations from oligomeric symmetry in proteins are rare, and wherever they are observed, it is attributed to strong functional reasons (13). Helical cylindrical arrangements are the other kind of assembly observed in P. furiosus sp. OT3 Lrp. FFRPs may have one or more types of effector binding sites (e.g. E. coli AsnC has one binding site, whereas those like M. tuberculosis FFRP have at least two types of binding sites, each with different hypothesized roles) (8). The type I binding site is a common site in the FFRP family that occurs at the interdimer interface at the C-terminal oligomerization domain, whereas the type II site was identified in M. tuberculosis FFRP at the i...
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