Biochemical characterization of a low molecular weight aspartic protease inhibitor from thermo-tolerant Bacillus licheniformis: Kinetic interactions with Pepsin

The present article reports a low molecular weight aspartic protease inhibitor, API, from a newly isolated thermo-tolerant Bacillus licheniformis. The inhibitor was purified to homogeneity as shown by rp-HPLC and SDS-PAGE. API is found to be stable over a broad pH range of 2–11 and at temperature 90 °C for 2 1/2 h. It has a Mr (relative molecular mass) of 1363 Da as shown by MALDI-TOF spectra and 1358 Da as analyzed by SDS-PAGE .The amino acid analysis of the peptide shows the presence of 12 amino acid residues having Mr of 1425 Da. The secondary structure of API as analyzed by the CD spectra showed 7% α-helix, 49% β-sheet and 44% aperiodic structure. The Kinetic studies of Pepsin–API interactions reveal that API is a slow-tight binding competitive inhibitor with the IC₅₀ and K_i values 4.0 nM and (3.83 nM–5.31 nM) respectively. The overall inhibition constant K_i? value is 0.107 ± 0.015 nM. The progress curves are time-dependent and consistent with slow-tight binding inhibition: E + I ? (k₄, k₅) EI ? (k₆, k₇) EI?. Rate constant k₆ = 2.73 ± 0.32 s^− 1 reveals a fast isomerization of enzyme–inhibitor complex and very slow dissociation as proved by k₇ = 0.068 ± 0.009 s^− 1. The Rate constants from the intrinsic tryptophanyl fluorescence data is in agreement with those obtained from the kinetic analysis; therefore, the induced conformational changes were correlated to the isomerization of EI to EI?.     

The congenital bicuspid aortic valve can experience high-frequency unsteady shear stresses on its leaflet surface

The bicuspid aortic valve (BAV) is a common congenital malformation of the aortic valve (AV) affecting 1% to 2% of the population. The BAV is predisposed to early degenerative calcification of valve leaflets, and BAV patients constitute 50% of AV stenosis patients. Although evidence shows that genetic defects can play a role in calcification of the BAV leaflets, we hypothesize that drastic changes in the mechanical environment of the BAV elicit pathological responses from the valve and might be concurrently responsible for early calcification. An in vitro model of the BAV was constructed by surgically manipulating a native trileaflet porcine AV. The BAV valve model and a trileaflet AV (TAV) model were tested in an in vitro pulsatile flow loop mimicking physiological hemodynamics. Laser Doppler velocimetry was used to make measurements of fluid shear stresses on the leaflet of the valve models using previously established methodologies. Furthermore, particle image velocimetry was used to visualize the flow fields downstream of the valves and in the sinuses. In the BAV model, flow near the leaflets and fluid shear stresses on the leaflets were much more unsteady than for the TAV model, most likely due to the moderate stenosis in the BAV and the skewed forward flow jet that collided with the aorta wall. This additional unsteadiness occurred during mid- to late-systole and was composed of cycle-to-cycle magnitude variability as well as high-frequency fluctuations about the mean shear stress. It has been demonstrated that the BAV geometry can lead to unsteady shear stresses under physiological flow and pressure conditions. Such altered shear stresses could play a role in accelerated calcification in BAVs.     

Metabolism of Vertebrate Amino Sugars with N-Glycolyl Groups: INTRACELLULAR β-O-LINKED N-GLYCOLYLGLUCOSAMINE (GlcNGc), UDP-GlcNGc, AND THE BIOCHEMICAL AND STRUCTURAL RATIONALE FOR THE SUBSTRATE TOLERANCE OF β-O-LINKED β-N-ACETYLGLUCOSAMINIDASE

The O-GlcNAc modification involves the attachment of single β-O-linked N-acetylglucosamine residues to serine and threonine residues of nucleocytoplasmic proteins. Interestingly, previous biochemical and structural studies have shown that O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc from proteins, has an active site pocket that tolerates various N-acyl groups in addition to the N-acetyl group of GlcNAc. The remarkable sequence and structural conservation of residues comprising this pocket suggest functional importance. We hypothesized this pocket enables processing of metabolic variants of O-GlcNAc that could be formed due to inaccuracy within the metabolic machinery of the hexosamine biosynthetic pathway. In the accompanying paper (Bergfeld, A. K., Pearce, O. M., Diaz, S. L., Pham, T., and Varki, A. (2012) J. Biol. Chem. 287, 28865-28881), N-glycolylglucosamine (GlcNGc) was shown to be a catabolite of NeuNGc. Here, we show that the hexosamine salvage pathway can convert GlcNGc to UDP-GlcNGc, which is then used to modify proteins with O-GlcNGc. The kinetics of incorporation and removal of O-GlcNGc in cells occur in a dynamic manner on a time frame similar to that of O-GlcNAc. Enzymatic activity of O-GlcNAcase (OGA) toward a GlcNGc glycoside reveals OGA can process glycolyl-containing substrates fairly efficiently. A bacterial homolog (BtGH84) of OGA, from a human gut symbiont, also processes O-GlcNGc substrates, and the structure of this enzyme bound to a GlcNGc-derived species reveals the molecular basis for tolerance and binding of GlcNGc. Together, these results demonstrate that analogs of GlcNAc, such as GlcNGc, are metabolically viable species and that the conserved active site pocket of OGA likely evolved to enable processing of mis-incorporated analogs of O-GlcNAc and thereby prevent their accumulation. Such plasticity in carbohydrate processing enzymes may be a general feature arising from inaccuracy in hexosamine metabolic pathways.     

Leishmania donovani exploits host deubiquitinating enzyme A20, a negative regulator of TLR signaling, to subvert host immune response

TLRs, which form an interface between mammalian host and microbe, play a key role in pathogen recognition and initiation of proinflammatory response thus stimulating antimicrobial activity and host survival. However, certain intracellular pathogens such as Leishmania can successfully manipulate the TLR signaling, thus hijacking the defensive strategies of the host. Despite the presence of lipophosphoglycan, a TLR2 ligand capable of eliciting host-defensive cytokine response, on the surface of Leishmania, the strategies adopted by the parasite to silence the TLR2-mediated proinflammatory response is not understood. In this study, we showed that Leishmania donovani modulates the TLR2-mediated pathway in macrophages through inhibition of the IKK-NF-κB cascade and suppression of IL-12 and TNF-α production. This may be due to impairment of the association of TRAF6 with the TAK-TAB complex, thus inhibiting the recruitment of TRAF6 in TLR2 signaling. L. donovani infection drastically reduced Lys 63-linked ubiquitination of TRAF6, and the deubiquitinating enzyme A20 was found to be significantly upregulated in infected macrophages. Small interfering RNA-mediated silencing of A20 restored the Lys 63-linked ubiquitination of TRAF6 as well as IL-12 and TNF-α levels with a concomitant decrease in IL-10 and TGF-β synthesis in infected macrophages. Knockdown of A20 led to lower parasite survival within macrophages. Moreover, in vivo silencing of A20 by short hairpin RNA in BALB/c mice led to increased NF-κB DNA binding and host-protective proinflammatory cytokine response resulting in effective parasite clearance. These results suggest that L. donovani might exploit host A20 to inhibit the TLR2-mediated proinflammatory gene expression, thus escaping the immune responses of the host.     

AMP-activated protein kinase: an emerging drug target to regulate imbalances in lipid and carbohydrate metabolism to treat cardio-metabolic diseases: Thematic Review Series: New Lipid and Lipoprotein Targets for the Treatment of Cardiometabolic Diseases

The adenosine monophosphate-activated protein kinase (AMPK) is a metabolic sensor of energy metabolism at the cellular as well as whole-body level. It is activated by low energy status that triggers a switch from ATP-consuming anabolic pathways to ATP-producing catabolic pathways. AMPK is involved in a wide range of biological activities that normalizes lipid, glucose, and energy imbalances. These pathways are dysregulated in patients with metabolic syndrome (MetS), which represents a clustering of major cardiovascular risk factors including diabetes, lipid abnormalities, and energy imbalances. Clearly, there is an unmet medical need to find a molecule to treat alarming number of patients with MetS. AMPK, with multifaceted activities in various tissues, has emerged as an attractive drug target to manage lipid and glucose abnormalities and maintain energy homeostasis. A number of AMPK activators have been tested in preclinical models, but many of them have yet to reach to the clinic. This review focuses on the structure-function and role of AMPK in lipid, carbohydrate, and energy metabolism. The mode of action of AMPK activators, mechanism of anti-inflammatory activities, and preclinical and clinical findings as well as future prospects of AMPK as a drug target in treating cardio-metabolic disease are discussed.     

Pd-mediated functionalization of polysubstituted pyrroles: Their evaluation as potential inhibitors of PDE4

Novel polysubstituted pyrroles have been designed and accessed via a one-pot multicomponent reaction followed by Pd-mediated C-C bond forming reactions. All the compounds synthesized were tested for their PDE4B inhibitory properties in vitro and two of them obtained via Heck reaction showed significant inhibition. The docking results suggested that these alkenyl derivatives containing ester moiety interact well with the PDE4B protein in silico where the ester carbonyl oxygen played a key role. The pyrrole framework presented here could be a new template for the identification of small molecule based novel inhibitors of PDE4. The single crystal X-ray data of a representative compound is presented.     

Modeling and experimental analyses reveals signaling plasticity in a bi-modular assembly of CD40 receptor activated kinases

Depending on the strength of signal dose, CD40 receptor (CD40) controls ERK-1/2 and p38MAPK activation. At low signal dose, ERK-1/2 is maximally phosphorylated but p38MAPK is minimally phosphorylated; as the signal dose increases, ERK-1/2 phosphorylation is reduced whereas p38MAPK phosphorylation is reciprocally enhanced. The mechanism of reciprocal activation of these two MAPKs remains un-elucidated. Here, our computational model, coupled to experimental perturbations, shows that the observed reciprocity is a system-level behavior of an assembly of kinases arranged in two modules. Experimental perturbations with kinase inhibitors suggest that a minimum of two trans-modular negative feedback loops are required to reproduce the experimentally observed reciprocity. The bi-modular architecture of the signaling pathways endows the system with an inherent plasticity which is further expressed in the skewing of the CD40-induced productions of IL-10 and IL-12, the respective anti-inflammatory and pro-inflammatory cytokines. Targeting the plasticity of CD40 signaling significantly reduces Leishmania major infection in a susceptible mouse strain. Thus, for the first time, using CD40 signaling as a model, we show how a bi-modular assembly of kinases imposes reciprocity to a receptor signaling. The findings unravel that the signalling plasticity is inherent to a reciprocal system and that the principle can be used for designing a therapy.