Monospecific inhibitors show that both mannan-binding lectin-associated serine protease (MASP)-1 and -2 are essential for lectin pathway activation and reveal structural plasticity of MASP-2.

The lectin pathway is an antibody-independent activation route of the complement system. It provides immediate defense against pathogens and altered self-cells, but it also causes severe tissue damage after stroke, heart attack and other ischemia reperfusion injuries. The pathway is triggered by target-binding of pattern recognition molecules leading to the activation of zymogen mannan-binding lectin-associated serine proteases (MASPs). MASP-2 is considered as the autonomous pathway- activator while MASP-1 as an auxiliary component. We evolved a pair of monospecific MASP inhibitors. In accordance with the key role of MASP-2, the MASP-2 inhibitor completely blocks the lectin pathway activation. Importantly, the MASP-1 inhibitor does the same demonstrating that MASP-1 is not an auxiliary but an essential pathway component. We report the first Michaelis- like complex structures of MASP-1 and MASP-2 formed with substrate-like inhibitors. The 1.28 A resolution MASP-2 structure reveals significant plasticity of the protease suggesting that either an induced fit or a conformational selection mechanism should contribute to the extreme specificity of the enzyme

Glutamate 270 plays an essential role in K activation and domain closure of Thermus thermophilus isopropylmalate dehydrogenase

The mutant E270A of Thermus thermophilus 3-isopropylmalate dehydrogenase exhibits largely reduced (∼1%) catalytic activity and negligible activation by K+ compared to the wild-type enzyme. A 3–4 kcal/mol increase in the activation energy of the catalysed reaction upon this mutation could also be predicted by QM/MM calculations. In the X-ray structure of the E270A mutant a water molecule was observed to take the place of K+. SAXS and FRET experiments revealed the essential role of E270 in stabilisation of the active domain-closed conformation of the enzyme. In addition, E270 seems to position K+ into close proximity of the nicotinamide ring of NAD+ and the electron-withdrawing effect of K+ may help to polarise the aromatic ring in order to aid the hydride-transfer

PICALM modulates autophagy activity and tau accumulation.

Genome-wide association studies have identified several loci associated with Alzheimer's disease (AD), including proteins involved in endocytic trafficking such as PICALM/CALM (phosphatidylinositol binding clathrin assembly protein). It is unclear how these loci may contribute to AD pathology. Here we show that CALM modulates autophagy and alters clearance of tau, a protein which is a known autophagy substrate and which is causatively linked to AD, both in vitro and in vivo. Furthermore, altered CALM expression exacerbates tau-mediated toxicity in zebrafish transgenic models. CALM influences autophagy by regulating the endocytosis of SNAREs, such as VAMP2, VAMP3 and VAMP8, which have diverse effects on different stages of the autophagy pathway, from autophagosome formation to autophagosome degradation. This study suggests that the AD genetic risk factor CALM modulates autophagy, and this may affect disease in a number of ways including modulation of tau turnover.We are grateful for funding from a Wellcome Trust Principal Research Fellowship (D.C.R.), a Wellcome Trust/MRC Strategic Grant on Neurodegeneration (D.C.R., C.J.O’.K.), a Wellcome Trust Strategic Award to Cambridge Institute for Medical Research, Wellcome Trust Studentship (E.Z.), the Alzheimer’s disease Biomedical Research Unit and Addenbrooke’s Hospital, the Tau Consortium, a fellowship from University of Granada (A.L.R.), a V Foundation/Applebee’s Research Grant (D.S.W.) and NCI R01 CA 109281 (D.S.W.).This is the final published version. It is also available from Nature Publishing at http://www.nature.com/ncomms/2014/140922/ncomms5998/full/ncomms5998.html

BioDMET: a physiologically based pharmacokinetic simulation tool for assessing proposed solutions to complex biological problems

We developed a detailed, whole-body physiologically based pharmacokinetic (PBPK) modeling tool for calculating the distribution of pharmaceutical agents in the various tissues and organs of a human or animal as a function of time. Ordinary differential equations (ODEs) represent the circulation of body fluids through organs and tissues at the macroscopic level, and the biological transport mechanisms and biotransformations within cells and their organelles at the molecular scale. Each major organ in the body is modeled as composed of one or more tissues. Tissues are made up of cells and fluid spaces. The model accounts for the circulation of arterial and venous blood as well as lymph. Since its development was fueled by the need to accurately predict the pharmacokinetic properties of imaging agents, BioDMET is more complex than most PBPK models. The anatomical details of the model are important for the imaging simulation endpoints. Model complexity has also been crucial for quickly adapting the tool to different problems without the need to generate a new model for every problem. When simpler models are preferred, the non-critical compartments can be dynamically collapsed to reduce unnecessary complexity. BioDMET has been used for imaging feasibility calculations in oncology, neurology, cardiology, and diabetes. For this purpose, the time concentration data generated by the model is inputted into a physics-based image simulator to establish imageability criteria. These are then used to define agent and physiology property ranges required for successful imaging. BioDMET has lately been adapted to aid the development of antimicrobial therapeutics. Given a range of built-in features and its inherent flexibility to customization, the model can be used to study a variety of pharmacokinetic and pharmacodynamic problems such as the effects of inter-individual differences and disease-states on drug pharmacokinetics and pharmacodynamics, dosing optimization, and inter-species scaling. While developing a tool to aid imaging agent and drug development, we aimed at accelerating the acceptance and broad use of PBPK modeling by providing a free mechanistic PBPK software that is user friendly, easy to adapt to a wide range of problems even by non-programmers, provided with ready-to-use parameterized models and benchmarking data collected from the peer-reviewed literature

Atypical parkinsonism-associated retromer mutant alters endosomal sorting of specific cargo proteins

The retromer complex acts as a scaffold for endosomal protein complexes that sort integral membrane proteins to various cellular destinations. The retromer complex is a heterotrimer of VPS29, VPS35, and VPS26. Two of these paralogues, VPS26A and VPS26B, are expressed in humans. Retromer dysfunction is associated with neurodegenerative disease, and recently, three VPS26A mutations (p.K93E, p.M112V, and p.K297X) were discovered to be associated with atypical parkinsonism. Here, we apply quantitative proteomics to provide a detailed description of the retromer interactome. By establishing a comparative proteomic methodology, we identify how this interactome is perturbed in atypical parkinsonism-associated VPS26A mutants. In particular, we describe a selective defect in the association of VPS26A (p.K297X) with the SNX27 cargo adaptor. By showing how a retromer mutant leads to altered endosomal sorting of specific PDZ ligand–containing cargo proteins, we reveal a new mechanism for perturbed endosomal cargo sorting in atypical parkinsonism

A Rigidifying Salt-Bridge Favors the Activity of Thermophilic Enzyme at High Temperatures at the Expense of Low-Temperature Activity

Although enzymes from thermophiles thriving in hot habitats are more stable than their mesophilic homologs, they are often less active at low temperatures. One theory suggests that extra stabilizing interactions found in thermophilic enzymes may increase their rigidity and decrease enzymatic activity at lower temperatures. We used acylphosphatase as a model to study how flexibility affects enzymatic activity. This enzyme has a unique structural feature in that an invariant arginine residue, which takes part in catalysis, is restrained by a salt-bridge in the thermophilic homologs but not in its mesophilic homologs. Here, we demonstrate the trade-offs between flexibility and enzymatic activity by disrupting the salt-bridge in a thermophilic acylphosphatase and introducing it in the mesophilic human homolog. Our results suggest that the salt-bridge is a structural adaptation for thermophilic acylphosphatases as it entropically favors enzymatic activity at high temperatures by restricting the flexibility of the active-site residue. However, at low temperatures the salt-bridge reduces the enzymatic activity because of a steeper temperature-dependency of activity

Membrane protein dynamics: limited lipid control

Correlation of lipid disorder with membrane protein dynamics has been studied with infrared spectroscopy, by combining data characterizing lipid phase, protein structure and, via hydrogen-deuterium (H/D) exchange, protein dynamics. The key element was a new measuring scheme, by which the combined effects of time and temperature on the H/D exchange could be separated. Cyanobacterial and plant thylakoid membranes, mammalian mitochondria membranes, and for comparison, lysozyme were investigated. In dissolved lysozyme, as a function of temperature, H/D exchange involved only reversible movements (the secondary structure did not change considerably); heat-denaturing was a separate event at much higher temperature. Around the low-temperature functioning limit of the biomembranes, lipids affected protein dynamics since changes in fatty acyl chain disorders and H/D exchange exhibited certain correlation. H/D exchange remained low in all membranes over physiological temperatures. Around the high-temperature functioning limit of the membranes, the exchange rates became higher. When temperature was further increased, H/D exchange rates went over a maximum and afterwards decreased (due to full H/D exchange and/or protein denaturing). Maximal H/D exchange rate temperatures correlated neither with the disorder nor with the unsaturation of lipids. In membrane proteins, in contrast to lysozyme, the onsets of sizable H/D exchange rates were the onsets of irreversible denaturing as well. Seemingly, at temperatures where protein self-dynamics allows large-scale H/D exchange, lipid-protein coupling is so weak that proteins prefer aggregating to limit the exposure of their hydrophobic surface regions to water. In all membranes studied, dynamics seemed to be governed by lipids around the low-temperature limit, and by proteins around the high-temperature limit of membrane functionality

Retromer and Its Role in Regulating Signaling at Endosomes.

The retromer complex is a key element of the endosomal protein sorting machinery being involved in trafficking of proteins from endosomes to the Golgi and also endosomes to the cell surface. There is now accumulating evidence that retromer also has a prominent role in regulating the activity of many diverse signaling proteins that traffic through endosomes and this activity has profound implications for the functioning of many different cell and tissue types from neuronal cells to cells of the immune system to specialized polarized epithelial cells of the retina. In this review, the protein composition of the retromer complex will be described along with many of the accessory factors that facilitate retromer-mediated endosomal protein sorting to detail how retromer activity contributes to the regulation of several distinct signaling pathways

Role of Active Site Rigidity in Activity: MD Simulation and Fluorescence Study on a Lipase Mutant

Relationship between stability and activity of enzymes is maintained by underlying conformational flexibility. In thermophilic enzymes, a decrease in flexibility causes low enzyme activity while in less stable proteins such as mesophiles and psychrophiles, an increase in flexibility is associated with enhanced enzyme activity. Recently, we identified a mutant of a lipase whose stability and activity were enhanced simultaneously. In this work, we probed the conformational dynamics of the mutant and the wild type lipase, particularly flexibility of their active site using molecular dynamic simulations and time-resolved fluorescence techniques. In contrast to the earlier observations, our data show that active site of the mutant is more rigid than wild type enzyme. Further investigation suggests that this lipase needs minimal reorganization/flexibility of active site residues during its catalytic cycle. Molecular dynamic simulations suggest that catalytically competent active site geometry of the mutant is relatively more preserved than wild type lipase, which might have led to its higher enzyme activity. Our study implies that widely accepted positive correlation between conformation flexibility and enzyme activity need not be stringent and draws attention to the possibility that high enzyme activity can still be accomplished in a rigid active site and stable protein structures. This finding has a significant implication towards better understanding of involvement of dynamic motions in enzyme catalysis and enzyme engineering through mutations in active site