Comparative Studies on Retroviral Proteases: Substrate Specificity

Exogenous retroviruses are subclassified into seven genera and include viruses that cause diseases in humans. The viral Gag and Gag-Pro-Pol polyproteins are processed by the retroviral protease in the last stage of replication and inhibitors of the HIV-1 protease are widely used in AIDS therapy. Resistant mutations occur in response to the drug therapy introducing residues that are frequently found in the equivalent position of other retroviral proteases. Therefore, besides helping to understand the general and specific features of these enzymes, comparative studies of retroviral proteases may help to understand the mutational capacity of the HIV-1 protease

Bias in food intake reporting in children and adolescents with type 1 diabetes: the role of body size, age and gender

An assessment of total daily energy intake is helpful in planning the overall treatment of children with type 1 diabetes (T1D). However, energy intake misreporting may hinder nutritional intervention.Aims: To assess the plausibility of energy intake reporting and the potential role of gender, body mass index (BMI) z-score (z-BMI), disease duration and insulin requirement in energy intake misreporting in a sample of children and adolescents with T1D.Methods: The study included 58 children and adolescents aged 8–16 yr with T1D. Anthropometry, blood pressure and glycated hemoglobin (HbA1c) were measured. Subjects were instructed to wear a SenseWear Pro Armband (SWA) for 3 consecutive days, including a weekend day and to fill out with their parents a weighed dietary record for the same days. Predicted energy expenditure (pEE) was calculated by age and gender specific equations, including gender, age, weight, height and physical activity level (assessed by SWA). The percent reported energy intake (rEI)/pEE ratio was used as an estimate of the plausibility of dietary reporting.Results: Misreporting of food intake, especially under-reporting, was common in children and adolescents with T1D: more than one-third of participants were classified as under- reporters and 10% as over-reporters. Age, z-BMI and male gender were associated with the risk of under-reporting (model R2 = 0.5). Waist circumference was negatively associated with the risk of over-reporting (model R2 = 0.25).Conclusions: Children and adolescents with T1D frequently under-report their food intake. Age, gender and z-BMI contribute to identify potential under-reporters

Design of HIV-1-PR inhibitors which do not create resistance: blocking the folding of single monomers

One of the main problems of drug design is that of optimizing the drug--target interaction. In the case in which the target is a viral protein displaying a high mutation rate, a second problem arises, namely the eventual development of resistance. We wish to suggest a scheme for the design of non--conventional drugs which do not face any of these problems and apply it to the case of HIV--1 protease. It is based on the knowledge that the folding of single--domain proteins, like e.g. each of the monomers forming the HIV--1--PR homodimer, is controlled by local elementary structures (LES), stabilized by local contacts among hydrophobic, strongly interacting and highly conserved amino acids which play a central role in the folding process. Because LES have evolved over myriads of generations to recognize and strongly interact with each other so as to make the protein fold fast as well as to avoid aggregation with other proteins, highly specific (and thus little toxic) as well as effective folding--inhibitor drugs suggest themselves: short peptides (or eventually their mimetic molecules), displaying the same amino acid sequence of that of LES (p--LES). Aside from being specific and efficient, these inhibitors are expected not to induce resistance: in fact, mutations which successfully avoid their action imply the destabilization of one or more LES and thus should lead to protein denaturation. Making use of Monte Carlo simulations within the framework of a simple although not oversimplified model, which is able to reproduce the main thermodynamic as well as dynamic properties of monoglobular proteins, we first identify the LES of the HIV--1--PR and then show that the corresponding p--LES peptides act as effective inhibitors of the folding of the protease which do not create resistance

Changes in γ-secretase activity and specificity caused by the introduction of consensus aspartyl protease active motif in Presenilin 1

Presenilin (PS1 or PS2) is an essential component of the active γ-secretase complex that liberates the Aβ peptides from amyloid precursor protein (APP). PS1 is regarded as an atypical aspartyl protease harboring two essential aspartic acids in the context of the sequence D257LV and D385FI, respectively, rather than the typical DTG...DTG catalytic motif of classical aspartyl proteases. In the present studies, we introduced the sequence DTG in PS1 at and around the catalytic D257 and D385 residues to generate three PS1 mutants: D257TG, D385TG, and the double-mutant D257TG/D385TG. The effects of these changes on the γ-secretase activity in the presence or absence of γ-secretase inhibitors and modulators were investigated. The results showed that PS1 mutants having D385TG robustly enhanced Aβ42 production compared to the wild type (wt), and were more sensitive than wt to inhibition by a classical aspartyl protease transition state mimic, and fenchylamine, a sulfonamide derivative. Unlike wt PS1 and some of its clinical mutants, all three PS1 artificial mutants decreased cleavage of Notch S3-site, suggesting that these artificial mutations may trigger conformational changes at the substrate docking and catalytic site that cause alteration of substrate specificity and inhibition pattern. Consistent with this notion, we have found that NSAID enzymatic inhibitors of COX, known modulators of the γ-secretase activity, cause PS1 mutants containing D385TG to produce higher levels of both Aβ38 and Aβ42, but to reduce levels of Aβ39, showing a pattern of Aβ formation different from that observed with wild type PS1 and its clinical mutants. This study provides an important structural clue for the rational design of drugs to inhibit processing of APP at the γ-site without interfering with Notch processing

Multiple Routes and Milestones in the Folding of HIV–1 Protease Monomer

Proteins fold on a time scale incompatible with a mechanism of random search in conformational space thus indicating that somehow they are guided to the native state through a funneled energetic landscape. At the same time the heterogeneous kinetics suggests the existence of several different folding routes. Here we propose a scenario for the folding mechanism of the monomer of HIV–1 protease in which multiple pathways and milestone events coexist. A variety of computational approaches supports this picture. These include very long all-atom molecular dynamics simulations in explicit solvent, an analysis of the network of clusters found in multiple high-temperature unfolding simulations and a complete characterization of free-energy surfaces carried out using a structure-based potential at atomistic resolution and a combination of metadynamics and parallel tempering. Our results confirm that the monomer in solution is stable toward unfolding and show that at least two unfolding pathways exist. In our scenario, the formation of a hydrophobic core is a milestone in the folding process which must occur along all the routes that lead this protein towards its native state. Furthermore, the ensemble of folding pathways proposed here substantiates a rational drug design strategy based on inhibiting the folding of HIV–1 protease

Adenylate Kinase and AMP Signaling Networks: Metabolic Monitoring, Signal Communication and Body Energy Sensing

Adenylate kinase and downstream AMP signaling is an integrated metabolic monitoring system which reads the cellular energy state in order to tune and report signals to metabolic sensors. A network of adenylate kinase isoforms (AK1-AK7) are distributed throughout intracellular compartments, interstitial space and body fluids to regulate energetic and metabolic signaling circuits, securing efficient cell energy economy, signal communication and stress response. The dynamics of adenylate kinase-catalyzed phosphotransfer regulates multiple intracellular and extracellular energy-dependent and nucleotide signaling processes, including excitation-contraction coupling, hormone secretion, cell and ciliary motility, nuclear transport, energetics of cell cycle, DNA synthesis and repair, and developmental programming. Metabolomic analyses indicate that cellular, interstitial and blood AMP levels are potential metabolic signals associated with vital functions including body energy sensing, sleep, hibernation and food intake. Either low or excess AMP signaling has been linked to human disease such as diabetes, obesity and hypertrophic cardiomyopathy. Recent studies indicate that derangements in adenylate kinase-mediated energetic signaling due to mutations in AK1, AK2 or AK7 isoforms are associated with hemolytic anemia, reticular dysgenesis and ciliary dyskinesia. Moreover, hormonal, food and antidiabetic drug actions are frequently coupled to alterations of cellular AMP levels and associated signaling. Thus, by monitoring energy state and generating and distributing AMP metabolic signals adenylate kinase represents a unique hub within the cellular homeostatic network