Structural Role of S100 Proteins in Membrane Repair

Membrane repair can be modulated by the association of an S100A10 dimer with the calcium- and phospholipid-binding protein annexin A2. This heterotetrameric complex has the ability to form larger multiprotein assemblies such as those with the enlargeosome protein AHNAK and members of the transmembrane ferlin family. The main goals of this thesis were to design, synthesize and characterize a molecule that would facilitate assembly of larger S100 multiprotein complexes, investigate the arrangement of the proteins, stoichiometry and affinity of AHNAK for the S100A10-annexin A2 complex and identify structural details of the ternary complex formed between S100A10, annexin A2 and AHNAK. Successful expression, purification and characterization of two S100-target peptide hybrid proteins comprised of S100A10 and S100B linked in tandem to annexin A2 (residues 1-15) (referred to as A10A2) and CapZ (TRTK12) (referred to as BT12), respectively, was achieved. Different protease cleavage sites were incorporated into the linkers of the hybrid proteins. Since in situ proteolytic cleavage showed the linker did not perturb the structures of the S100A10-annexin A2 or S100B-TRTK12 complexes, this approach was used as a scaffold for larger S100 complexes. Peptide array experiments identified the regions of interaction within the C-terminus of AHNAK. Of the eight consensus regions observed, one of the sequences of AHNAK (AHNAK5) which showed the strongest interaction was used as a synthetic peptide to identify the binding region(s), stoichiometry and affinity with the A10A2 complex. Using NMR, fluorescence and non-denaturing electrospray mass spectrometries, a novel asymmetric arrangement between a single AHNAK5 peptide and the A10A2 dimer having an affinity near 3 nM was identified. The binding region of AHNAK5 on A10A2 was determined by NMR spectroscopy and X-ray crystallography. X-ray crystallography of the A10A2-AHNAK5 complex showed that the backbone structure of A10A2-AHNAK5 is similar to that of S100A10 bound to the N-terminal peptide of annexin A2 (Rety et al. 1999). From the structure, it was clear that only one AHNAK5 was bound to a dimeric A10A2 through helix IV in S100A10 and the C-terminal portion from the annexin A2 peptide. These results provide a novel mode of interaction for AHNAK5 in an S100-target complex

The effects of oligomerization on Saccharomyces cerevisiae Mcm4/6/7 function

<p>Abstract</p> <p>Background</p> <p>Minichromosome maintenance proteins (Mcm) 2, 3, 4, 5, 6 and 7 are related by sequence and form a variety of complexes that unwind DNA, including Mcm4/6/7. A Mcm4/6/7 trimer forms one half of the Mcm2-7 hexameric ring and can be thought of as the catalytic core of Mcm2-7, the replicative helicase in eukaryotic cells. Oligomeric analysis of Mcm4/6/7 suggests that it forms a hexamer containing two Mcm4/6/7 trimers, however, under certain conditions trimeric Mcm4/6/7 has also been observed. The functional significance of the different Mcm4/6/7 oligomeric states has not been assessed. The results of such an assessment would have implications for studies of both Mcm4/6/7 and Mcm2-7.</p> <p>Results</p> <p>Here, we show that <it>Saccharomyces cerevisiae </it>Mcm4/6/7 reconstituted from individual subunits exists in an equilibrium of oligomeric forms in which smaller oligomers predominate in the absence of ATP. In addition, we found that ATP, which is required for Mcm4/6/7 activity, shifts the equilibrium towards larger oligomers, likely hexamers of Mcm4/6/7. ATPγS and to a lesser extent ADP also shift the equilibrium towards hexamers. Study of Mcm4/6/7 complexes containing mutations that interfere with the formation of inter-subunit ATP sites (arginine finger mutants) indicates that full activity of Mcm4/6/7 requires all of its ATP sites, which are formed in a hexamer and not a trimer. In keeping with this observation, Mcm4/6/7 binds DNA as a hexamer.</p> <p>Conclusions</p> <p>The minimal functional unit of Mcm4/6/7 is a hexamer. One of the roles of ATP binding by Mcm4/6/7 may be to stabilize formation of hexamers.</p

Structure of an Asymmetric Ternary Protein Complex Provides Insight for Membrane Interaction

SummaryPlasma membrane repair involves the coordinated effort of proteins and the inner phospholipid surface to mend the rupture and return the cell back to homeostasis. Here, we present the three-dimensional structure of a multiprotein complex that includes S100A10, annexin A2, and AHNAK, which along with dysferlin, functions in muscle and cardiac tissue repair. The 3.5 Å resolution X-ray structure shows that a single region from the AHNAK C terminus is recruited by an S100A10-annexin A2 heterotetramer, forming an asymmetric ternary complex. The AHNAK peptide adopts a coil conformation that arches across the heterotetramer contacting both annexin A2 and S100A10 protomers with tight affinity (∼30 nM) and establishing a structural rationale whereby both S100A10 and annexin proteins are needed in AHNAK recruitment. The structure evokes a model whereby AHNAK is targeted to the membrane surface through sandwiching of the binding region between the S100A10/annexin A2 complex and the phospholipid membrane

CAPS-DB: a structural classification of helix-capping motifs

The regions of the polypeptide chain immediately preceding or following an α-helix are known as Nt- and Ct cappings, respectively. Cappings play a central role stabilizing α-helices due to lack of intrahelical hydrogen bonds in the first and last turn. Sequence patterns of amino acid type preferences have been derived for cappings but the structural motifs associated to them are still unclassified. CAPS-DB is a database of clusters of structural patterns of different capping types. The clustering algorithm is based in the geometry and the (ϕ–ψ)-space conformation of these regions. CAPS-DB is a relational database that allows the user to search, browse, inspect and retrieve structural data associated to cappings. The contents of CAPS-DB might be of interest to a wide range of scientist covering different areas such as protein design and engineering, structural biology and bioinformatics. The database is accessible at: http://www.bioinsilico.org/CAPSDB

pH Dependent Antimicrobial Peptides and Proteins, Their Mechanisms of Action and Potential as Therapeutic Agents

Antimicrobial peptides (AMPs) are potent antibiotics of the innate immune system that have been extensively investigated as a potential solution to the global problem of infectious diseases caused by pathogenic microbes. A group of AMPs that are increasingly being reported are those that utilise pH dependent antimicrobial mechanisms and here, we review research into this area. This review shows that these antimicrobial molecules are produced by a diverse spectrum of creatures, including vertebrates and invertebrates, and are primarily cationic although a number of anionic examples are known. Some of these molecules exhibit high pH optima for their antimicrobial activity but in most cases, these AMPs show activity against microbes that present low pH optima, which reflects the acidic pH generally found at their sites of action, particularly the skin. The modes of action used by these molecules are based on a number of major structure / function relationships, which include metal ion binding, changes to net charge and conformational plasticity, and primarily involve the protonation of histidine, aspartic acid and glutamic acid residues at low pH. The pH dependent activity of pore forming antimicrobial proteins involves mechanisms that generally differ fundamentally to those used by pH dependent AMPs, which can be described by the carpet, toroidal pore and barrel-stave pore models of membrane interaction. A number of pH dependent AMPs and antimicrobial proteins have been developed for medical purposes and have successfully completed clinical trials, including kappacins, LL-37, histatins and lactoferrin, along with a number of their derivatives. Major examples of the therapeutic application of these antimicrobial molecules include wound healing as well as the treatment of multiple cancers and infections due to viruses, bacteria and fungi. In general, these applications involve topical administration, such as the use of mouth washes, cream formulations and hydrogel delivery systems. Nonetheless, many pH dependent AMPs and antimicrobial proteins have yet to be fully characterized and these molecules, as a whole, represent an untapped source of novel biologically active agents that could aid fulfillment of the urgent need for alternatives to conventional antibiotics, helping to avert a return to the pre-antibiotic era

Analysis of an ultrafiltration model: Influence of operational conditions

Ultrafiltration is a widely used technique to remove hazardous pollutants from wastewaters. As ultrafiltration is a dynamic process, non steady-state UF models have been widely investigated and described in the literature. The main equations of most dynamic models found in the literature can be rearranged and simplified into a linear equation. In this work, experimental results from UF tests were expressed in terms of TMP2/J(P)(2) as a function of time to check linearity. Transmembrane pressure (TMP), feed concentration (FC), temperature and crossflow velocity (CFV) were varied during the experiments. The feed consisted in an aqueous solution of polyethylene glycol of 35 kg/mol molecular weight. The experimental results confirmed a linear relationship when TMP2/J(P)(2) was plotted vs. time. For severe fouling conditions, the linearity found when plotting experimental values of initial membrane fouling resistance vs. TMP was very high. A linear correlation between the initial membrane fouling resistance and the temperature was also found for the experimental conditions tested. However, it was found that, above 25 degrees C, increasing temperature did not result in a noticeable reduction of the gel layer resistance. The quotient r(c)/r(g) as a function of TMP and CFV was also evaluated to check if it followed any pattern. (C) 2011 Elsevier B.V. All rights reserved.Vincent Vela, MC.; Cuartas Uribe, BE.; Alvarez Blanco, S.; Lora García, J. (2012). Analysis of an ultrafiltration model: Influence of operational conditions. Desalination. 284:14-21. doi:10.1016/j.desal.2011.08.030S142128

GRAF1 deficiency blunts sarcolemmal injury repair and exacerbates cardiac and skeletal muscle pathology in dystrophin-deficient mice

Background The plasma membranes of striated muscle cells are particularly susceptible to rupture as they endure significant mechanical stress and strain during muscle contraction, and studies have shown that defects in membrane repair can contribute to the progression of muscular dystrophy. The synaptotagmin-related protein, dysferlin, has been implicated in mediating rapid membrane repair through its ability to direct intracellular vesicles to sites of membrane injury. However, further work is required to identify the precise molecular mechanisms that govern dysferlin targeting and membrane repair. We previously showed that the bin–amphiphysin–Rvs (BAR)–pleckstrin homology (PH) domain containing Rho-GAP GTPase regulator associated with focal adhesion kinase-1 (GRAF1) was dynamically recruited to the tips of fusing myoblasts wherein it promoted membrane merging by facilitating ferlin-dependent capturing of intracellular vesicles. Because acute membrane repair responses involve similar vesicle trafficking complexes/events and because our prior studies in GRAF1-deficient tadpoles revealed a putative role for GRAF1 in maintaining muscle membrane integrity, we postulated that GRAF1 might also play an important role in facilitating dysferlin-dependent plasma membrane repair. Methods We used an in vitro laser-injury model to test whether GRAF1 was necessary for efficient muscle membrane repair. We also generated dystrophin/GRAF1 doubledeficient mice by breeding mdx mice with GRAF1 hypomorphic mice. Evans blue dye uptake and extensive morphometric analyses were used to assess sarcolemmal integrity and related pathologies in cardiac and skeletal muscles isolated from these mice. Results Herein, we show that GRAF1 is dynamically recruited to damaged skeletal and cardiac muscle plasma membranes and that GRAF1-depleted muscle cells have reduced membrane healing abilities. Moreover, we show that dystrophin depletion exacerbated muscle damage in GRAF1-deficient mice and that mice with dystrophin/GRAF1 double deficiency phenocopied the severe muscle pathologies observed in dystrophin/dysferlin-double null mice. Consistent with a model that GRAF1 facilitates dysferlin-dependent membrane patching, we found that GRAF1 associates with and regulates plasma membrane deposition of dysferlin. Conclusions Overall, our work indicates that GRAF1 facilitates dysferlin-dependent membrane repair following acute muscle injury. These findings indicate that GRAF1 might play a role in the phenotypic variation and pathological progression of cardiac and skeletal muscle degeneration in muscular dystrophy patients

Particle Fabrication Techniques for Pharmaceutical Applications

Ph.DDOCTOR OF PHILOSOPH

Simulation of electrohydrodynamic atomization for enhanced particle collection efficiency in an encapsulation chamber

AIChE Annual Meeting, Conference Proceeding

Space Cooling Energy Potential of Domestic Cold Water before Household Consumption in Cold-Climate Regions

Space cooling energy consumption in residential buildings has tripled globally over the past three decades, leading to a significant increase in greenhouse gas (GHG) emissions and building operating costs. To reduce building cooling energy consumption, cooling energy can be recovered from domestic cold water (DCW) for space cooling by circulating DCW through thermally massive walls (herein “DCW-wall”) before regular household consumption (e.g., showers). This approach is more effective in cold climate regions since the DCW is cooler in these regions, yet its engineering design and effectiveness have not been evaluated previously. This study evaluated the cooling potential of DCW-walls in different operation scenarios (e.g., inlet temperatures, zone temperatures, and piping configurations). A typical DCW usage pattern and a daily amount of 1200 L were selected for evaluation. Three-dimensional transient thermal simulations were used to obtain the water outlet temperatures, average wall surface temperatures, and cooling potentials. The results showed that a DCW wall with a spiral piping configuration and DCW inlet at 12 °C can deliver 21.92 MJ of cooling energy daily to a zone at 25 °C. This amount of free energy can cover up to approximately 11% of the annual cooling energy demand of a four-person dwelling in Toronto, Canada, which has a warm and humid summer