Catalytic Water Co-Existing with a Product Peptide in the Active Site of HIV-1 Protease Revealed by X-Ray Structure Analysis

BACKGROUND: It is known that HIV-1 protease is an important target for design of antiviral compounds in the treatment of Acquired Immuno Deficiency Syndrome (AIDS). In this context, understanding the catalytic mechanism of the enzyme is of crucial importance as transition state structure directs inhibitor design. Most mechanistic proposals invoke nucleophilic attack on the scissile peptide bond by a water molecule. But such a water molecule coexisting with any ligand in the active site has not been found so far in the crystal structures. PRINCIPAL FINDINGS: We report here the first observation of the coexistence in the active site, of a water molecule WAT1, along with the carboxyl terminal product (Q product) peptide. The product peptide has been generated in situ through cleavage of the full-length substrate. The N-terminal product (P product) has diffused out and is replaced by a set of water molecules while the Q product is still held in the active site through hydrogen bonds. The position of WAT1, which hydrogen bonds to both the catalytic aspartates, is different from when there is no substrate bound in the active site. We propose WAT1 to be the position from where catalytic water attacks the scissile peptide bond. Comparison of structures of HIV-1 protease complexed with the same oligopeptide substrate, but at pH 2.0 and at pH 7.0 shows interesting changes in the conformation and hydrogen bonding interactions from the catalytic aspartates. CONCLUSIONS/SIGNIFICANCE: The structure is suggestive of the repositioning, during substrate binding, of the catalytic water for activation and subsequent nucleophilic attack. The structure could be a snap shot of the enzyme active site primed for the next round of catalysis. This structure further suggests that to achieve the goal of designing inhibitors mimicking the transition-state, the hydrogen-bonding pattern between WAT1 and the enzyme should be replicated

Lacticacidaemia due to pyruvate dehydrogenase deficiency, with evidence of protein polymorphism in the α-subunit of the enzyme

In three infants with neonatal lacticacidaemia, a deficiency in the E 1 (pyruvate dehydrogenase) component of the pyruvate dehydrogenase complex was demonstrated in skin fibroblast cultures. Residual activites of the pyruvate dehydrogenase complex in the activated state were 1.6%, 3.9% and 18.8% of control values, respectively. Immunoprecipitation of extracts of cultures skin fibroblasts grown on 35 S-methionine with anti-pyruvate dehydrogenase complex antibody revealed an abnormality in the E 1 α-component of these three patients when visualised after sodium dodecyl sulphate/polyacrylamide gel electrophoresis. This component appeared to have a slightly lower molecular weight than did this protein from control cell strains. Cell strains from other patients with a deficiency of the pyruvate dehydrogenase complex did not exhibit this defect. Three patients also showed dysmorphism and developmental abnormalities of the central nervous system.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47532/1/431_2004_Article_BF00441736.pd

Chronic Alcohol Exposure Alters Behavioral and Synaptic Plasticity of the Rodent Prefrontal Cortex

In the present study, we used a mouse model of chronic intermittent ethanol (CIE) exposure to examine how CIE alters the plasticity of the medial prefrontal cortex (mPFC). In acute slices obtained either immediately or 1-week after the last episode of alcohol exposure, voltage-clamp recording of excitatory post-synaptic currents (EPSCs) in mPFC layer V pyramidal neurons revealed that CIE exposure resulted in an increase in the NMDA/AMPA current ratio. This increase appeared to result from a selective increase in the NMDA component of the EPSC. Consistent with this, Western blot analysis of the postsynaptic density fraction showed that while there was no change in expression of the AMPA GluR1 subunit, NMDA NR1 and NRB subunits were significantly increased in CIE exposed mice when examined immediately after the last episode of alcohol exposure. Unexpectedly, this increase in NR1 and NR2B was no longer observed after 1-week of withdrawal in spite of a persistent increase in synaptic NMDA currents. Analysis of spines on the basal dendrites of layer V neurons revealed that while the total density of spines was not altered, there was a selective increase in the density of mushroom-type spines following CIE exposure. Examination of NMDA-receptor mediated spike-timing-dependent plasticity (STDP) showed that CIE exposure was associated with altered expression of long-term potentiation (LTP). Lastly, behavioral studies using an attentional set-shifting task that depends upon the mPFC for optimal performance revealed deficits in cognitive flexibility in CIE exposed mice when tested up to 1-week after the last episode of alcohol exposure. Taken together, these observations are consistent with those in human alcoholics showing protracted deficits in executive function, and suggest these deficits may be associated with alterations in synaptic plasticity in the mPFC

Drug Off-Target Effects Predicted Using Structural Analysis in the Context of a Metabolic Network Model

Recent advances in structural bioinformatics have enabled the prediction of protein-drug off-targets based on their ligand binding sites. Concurrent developments in systems biology allow for prediction of the functional effects of system perturbations using large-scale network models. Integration of these two capabilities provides a framework for evaluating metabolic drug response phenotypes in silico. This combined approach was applied to investigate the hypertensive side effect of the cholesteryl ester transfer protein inhibitor torcetrapib in the context of human renal function. A metabolic kidney model was generated in which to simulate drug treatment. Causal drug off-targets were predicted that have previously been observed to impact renal function in gene-deficient patients and may play a role in the adverse side effects observed in clinical trials. Genetic risk factors for drug treatment were also predicted that correspond to both characterized and unknown renal metabolic disorders as well as cryptic genetic deficiencies that are not expected to exhibit a renal disorder phenotype except under drug treatment. This study represents a novel integration of structural and systems biology and a first step towards computational systems medicine. The methodology introduced herein has important implications for drug development and personalized medicine

Rationale and design of the United Kingdom Heart Failure with Preserved Ejection Fraction Registry

\ua9 Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY. Published by BMJ.Objective: Heart failure with preserved ejection fraction (HFpEF) is a common heterogeneous syndrome that remains imprecisely defined and consequently has limited treatment options and poor outcomes. Methods: The UK Heart Failure with Preserved Ejection Fraction Registry (UK HFpEF) is a prospective data-enabled cohort and platform study. The study will develop a large, highly characterised cohort of patients with HFpEF. A biobank will be established. Deep clinical phenotyping, imaging, multiomics and centrally held national electronic health record data will be integrated at scale, in order to reclassify HFpEF into distinct subgroups, improve understanding of disease mechanisms and identify new biological pathways and molecular targets. Together, these will form the basis for developing diagnostics and targeted therapeutics specific to subgroups. It will be a platform for more effective and efficient trials, focusing on subgroups in whom targeted interventions are expected to be effective, with consent in place to facilitate rapid recruitment, and linkage for follow-up. Patients with a diagnosis of HFpEF made by a heart failure specialist, who have had natriuretic peptide levels measured and a left ventricular ejection fraction >40% are eligible. Patients with an ejection fraction between 40% and 49% will be limited to no more than 25% of the cohort. Conclusions: UK HFpEF will develop a rich, multimodal data resource to enable the identification of disease endotypes and develop more effective diagnostic strategies, precise risk stratification and targeted therapeutics. Trial registration number: NCT05441839

Gut microbiota and sirtuins in obesity-related inflammation and bowel dysfunction

Obesity is a chronic disease characterized by persistent low-grade inflammation with alterations in gut motility. Motor abnormalities suggest that obesity has effects on the enteric nervous system (ENS), which controls virtually all gut functions. Recent studies have revealed that the gut microbiota can affect obesity and increase inflammatory tone by modulating mucosal barrier function. Furthermore, the observation that inflammatory conditions influence the excitability of enteric neurons may add to the gut dysfunction in obesity. In this article, we discuss recent advances in understanding the role of gut microbiota and inflammation in the pathogenesis of obesity and obesity-related gastrointestinal dysfunction. The potential contribution of sirtuins in protecting or regulating the circuitry of the ENS under inflamed states is also considered

Persistent left superior vena cava: Review of the literature, clinical implications, and relevance of alterations in thoracic central venous anatomy as pertaining to the general principles of central venous access device placement and venography in cancer patients

Persistent left superior vena cava (PLSVC) represents the most common congenital venous anomaly of the thoracic systemic venous return, occurring in 0.3% to 0.5% of individuals in the general population, and in up to 12% of individuals with other documented congential heart abnormalities. In this regard, there is very little in the literature that specifically addresses the potential importance of the incidental finding of PLSVC to surgeons, interventional radiologists, and other physicians actively involved in central venous access device placement in cancer patients. In the current review, we have attempted to comprehensively evaluate the available literature regarding PLSVC. Additionally, we have discussed the clinical implications and relevance of such congenital aberrancies, as well as of treatment-induced or disease-induced alterations in the anatomy of the thoracic central venous system, as they pertain to the general principles of successful placement of central venous access devices in cancer patients. Specifically regarding PLSVC, it is critical to recognize its presence during attempted central venous access device placement and to fully characterize the pattern of cardiac venous return (i.e., to the right atrium or to the left atrium) in any patient suspected of PLSVC prior to initiation of use of their central venous access device