Structures and interactions of the leukocyte-specific integrins cytoplasmic tails

Integrins are devoid of enzymatic activity but yet are involved in almost all aspects of mammalian physiology. Their unique bidirectional signal transduction property can enable them to continuously sense changes in their extracellular environment and respond rapidly through conformational changes that modulates their affinity for extracellular ligands. This continuous sense and response system mediated by integrin molecules is a quintessential property of the mammalian immune system, which requires leukocytes circulating in the high pressure enclosed blood vascular system to respond to minuscule changes in the release of cytokines during an immunological event. This process requires leukocytes to rapidly change from non-adhesive, symmetrical cells to highly adhesive asymmetric cells to attach to the vascular walls amid the high pressure blood flow, followed by progressive migration via adept modulation of specific integrins towards the site of injury.DOCTOR OF PHILOSOPHY (SBS

NMR Characterization and Membrane Interactions of the Loop Region of Kindlin-3 F1 Subdomain.

Kindlins-1,2 and 3 are FERM domain-containing cytosolic proteins involved in the activation and regulation of integrin-mediated cell adhesion. Apart from binding to integrin β cytosolic tails, kindlins and the well characterized integrin-activator talin bind membrane phospholipids. The ubiquitin-like F1 sub-domain of the FERM domain of talin contains a short loop that binds to the lipid membrane. By contrast, the F1 sub-domain of kindlins contains a long loop demonstrated binding to the membrane. Here, we report structural characterization and lipid interactions of the 83-residue F1 loop of kindlin-3 using NMR and optical spectroscopy methods. NMR studies demonstrated that the F1 loop of kindlin-3 is globally unfolded but stretches of residues assuming transient helical conformations could be detected in aqueous solution. We mapped membrane binding interactions of the F1 loop with small unilamellar vesicles (SUVs) containing either zwitterionic lipids or negatively charged lipids using 15N-1H HSQC titrations. These experiments revealed that the F1 loop of kindlin-3 preferentially interacted with the negatively charged SUVs employing almost all of the residues. By contrast, only fewer residues appeared to be interacted with SUVs containing neutral lipids. Further, CD and NMR data suggested stabilization of helical conformations and predominant resonance perturbations of the F1 loop in detergent containing solutions. Conformations of an isolated N-terminal peptide fragment, or EK21, of the F1 loop, containing a poly-Lys sequence motif, important for membrane interactions, were also investigated in detergent solutions. EK21 adopted a rather extended or β-type conformations in complex with negatively charged SDS micelles. To our knowledge, this is the first report describing the conformations and residue-specific interactions of kindlin F1 loop with lipids. These data therefore provide important insights into the interactions of kindlin FERM domain with membrane lipids that contribute toward the integrin activating property

NMR Structure of Integrin α4 Cytosolic Tail and Its Interactions with Paxillin

Integrins are a group of transmembrane signaling proteins that are important in biological processes such as cell adhesion, proliferation and migration. Integrins are α/β hetero-dimers and there are 24 different integrins formed by specific combinations of 18 α and 8 β subunits in humans. Generally, each of these subunits has a large extracellular domain, a single pass transmembrane segment and a cytosolic tail (CT). CTs of integrins are important in bidirectional signal transduction and they associate with a large number of intracellular proteins.H HSQC NMR experiments reveal interactions of the α4 CT C-terminal region with a fragment of paxillin (residues G139-K277) that encompassed LD2-LD4 repeats. Residues of these LD repeats including their adjoining linkers showed α4 CT binding-induced chemical shift changes. Furthermore, NMR studies using LD-containing peptides showed predominant interactions between LD3 and LD4 of paxillin and α4 CT. Docked structures of the α4 CT with these LD repeats suggest possible polar and/or salt-bridge and non-polar packing interactions.The current study provides molecular insights into the structural diversity of α CTs of integrins and interactions of integrin α4 CT with the adaptor protein paxillin

Bridging the audit expectation gap in Singapore.

In a business environment of continuing change and increasing complexity, public expectations of the auditing profession seem to have increased. The audit expectation gap is defined as the difference between the public’s expectations of the scope and function of audits and the public’s perception of the auditors’ performance. The expectation gap comprises of two main components: the standards gap, which is further distinguished between reasonable expectations and unreasonable expectations; and the performance gap, which is subdivided into actual performance deficiency and perceived performance deficiency. Besides giving a review of past research, this research also reports the results of a survey and the findings of interviews to ascertain the perceptions of individuals about audit issues in Singapore. The research identifies the duties which contribute to the expectation gap’s components and provides details of each component’s contribution to the overall gap in Singapore. In addition, the research also recommends possible solutions to close the expectation gap and discusses their likelihood of success in reducing the gap.ACCOUNTANC

Secondary conformations and micelle interactions of the F1 loop of kindlin-3.

<p>(panel A) Far UV-CD spectra of F1 loop of kindlin-3 in aqueous buffer solution (in solid black line), in solutions containing 50 mM DPC (in black squares) and 50 mM SDS (in white squares). (panel B) Overlay of ¹⁵N-¹H HSQC spectra of the F1 loop of kinldin-3 in aqueous buffer solution (black contour), in solutions containing either in 50 mM (red contour) or 100 mM (green contour) DPC. (panel C) Overlay of ¹⁵N-¹H HSQC spectra of the F1 loop of kinldin-3 in aqueous buffer solution (black contour), in solutions containing either in 50 mM (red contour) or 100 mM (green contour) SDS.</p

Conformational characteristics of EK21 peptide fragment derived from N-terminus of the F1 loop.

<p>(panel A) Far UV CD spectra of EK21 peptide in free buffer solution (solid line) and in solutions containing either 50 mM DPC (solid line with black squares) or 50 mM SDS (solid line with white squares). (penal B) Bar diagram showing secondary chemical shifts of αH resonances of residues of EK21 in solutions containing SDS micelles. (panel C) Selected section of ¹H-¹H two-dimensional NOESY spectrum of EK21 peptide obtained in aqueous solutions containing 200 mM perdeuterated SDS.</p

Localization of the FERM domain of kindlin in membrane.

<p>A hypothetical cartoon showing proximity of the FERM domain of kindlin to the lipid membrane and interactions with the beta cytosolic tail. The FERM domain interacts with the phospholipid lipid bilayer (shown as black stick with round head) through F0 subdomain (in blue), PH (in purple) and potentially with split F2 (in green) and F3 (in pink) subdomains by ionic interactions with exposed positively charged surface. The unfolded F1 loop of the F1 subdomain (in red) might insert into the lipid membrane using N-terminal positive charged residues of poly-lys and sidechain of hydrophobic residues. Such a membrane localization of the FERM domain of kindlin might bring the F3 subdomain for optimal interactions with the beta cytosolic tail attached with its TM (in light purple) for activation of integrins.</p

Domain organization of talin and kindlin and primary structures of the F1 loops.

<p>The FERM domain of talin, also called talin head, and kindlin is sub-divided into several subdomains F0, F1, F2 and F3. The FERM domain of kindlin also contains a PH domain inserted within the F2 subdomain. The full-length talin also contains a rod domain which is absent in kindlins. The F1 domains of talin and kindlin contain a loop insert for membrane interactions. The primary structures of F1 loop of kindlin-1, kindlin-2 and kindlin-3 and talin are shown.</p

Lipid specific interactions of SUVs with the F1 loop of kindlin-3.

<p>Overlay of ¹⁵N-¹H HSQC spectra of F1 loop at different concentrations, 0 mM (black contour), 4 mM (red contour) and 6 mM, of POPC (panel A), POPS (panel B) and POPG (panel C) SUVs. Bar diagrams showing attenuations of ¹⁵N-¹H HSQC cross-peak intensity of F1 loop of kindlin-3 after additions of 2 mM (black bar) and 4 mM (white bar) of POPC (panel D), POPS (panel E) and POPG (panel F) SUVs, respectively.</p