Protein-lipid interactions: correlation of a predictive algorithm for lipid-binding sites with three-dimensional structural data

BACKGROUND: Over the past decade our laboratory has focused on understanding how soluble cytoskeleton-associated proteins interact with membranes and other lipid aggregates. Many protein domains mediating specific cell membrane interactions appear by fluorescence microscopy and other precision techniques to be partially inserted into the lipid bilayer. It is unclear whether these protein-lipid-interactions are dependent on shared protein motifs or unique regional physiochemistry, or are due to more global characteristics of the protein. RESULTS: We have developed a novel computational program that predicts a protein's lipid-binding site(s) from primary sequence data. Hydrophobic labeling, Fourier transform infrared spectroscopy (FTIR), film balance, T-jump, CD spectroscopy and calorimetry experiments confirm that the interfaces predicted for several key cytoskeletal proteins (alpha-actinin, Arp2, CapZ, talin and vinculin) partially insert into lipid aggregates. The validity of these predictions is supported by an analysis of the available three-dimensional structural data. The lipid interfaces predicted by our algorithm generally contain energetically favorable secondary structures (e.g., an amphipathic alpha-helix flanked by a flexible hinge or loop region), are solvent-exposed in the intact protein, and possess favorable local or global electrostatic properties. CONCLUSION: At present, there are few reliable methods to determine the region of a protein that mediates biologically important interactions with lipids or lipid aggregates. Our matrix-based algorithm predicts lipid interaction sites that are consistent with the available biochemical and structural data. To determine whether these sites are indeed correctly identified, and whether use of the algorithm can be safely extended to other classes of proteins, will require further mapping of these sites, including genetic manipulation and/or targeted crystallography

CapZ-lipid membrane interactions: a computer analysis

BACKGROUND: CapZ is a calcium-insensitive and lipid-dependent actin filament capping protein, the main function of which is to regulate the assembly of the actin cytoskeleton. CapZ is associated with membranes in cells and it is generally assumed that this interaction is mediated by polyphosphoinositides (PPI) particularly PIP(2), which has been characterized in vitro. RESULTS: We propose that non-PPI lipids also bind CapZ. Data from computer-aided sequence and structure analyses further suggest that CapZ could become partially buried in the lipid bilayer probably under mildly acidic conditions, in a manner that is not only dependent on the presence of PPIs. We show that lipid binding could involve a number of sites that are spread throughout the CapZ molecule i.e., alpha- and beta-subunits. However, a beta-subunit segment between residues 134–151 is most likely to be involved in interacting with and inserting into lipid membrane due to a slighly higher ratio of positively to negatively charged residues and also due to the presence of a small hydrophobic helix. CONCLUSION: CapZ may therefore play an essential role in providing a stable membrane anchor for actin filaments

Protocol for Translabial 3D-Ultrasonography for diagnosing levator defects (TRUDIL): a multicentre cohort study for estimating the diagnostic accuracy of translabial 3D-ultrasonography of the pelvic floor as compared to MR imaging

Contains fulltext : 96237.pdf (publisher's version ) (Open Access)BACKGROUND: Pelvic organ prolapse (POP) is a condition affecting more than half of the women above age 40. The estimated lifetime risk of needing surgical management for POP is 11%. In patients undergoing POP surgery of the anterior vaginal wall, the re-operation rate is 30%. The recurrence risk is especially high in women with a levator ani defect. Such defect is present if there is a partially or completely detachment of the levator ani from the inferior ramus of the symphysis. Detecting levator ani defects is relevant for counseling, and probably also for treatment. Levator ani defects can be imaged with MRI and also with Translabial 3D ultrasonography of the pelvic floor. The primary aim of this study is to assess the diagnostic accuracy of translabial 3D ultrasonography for diagnosing levator defects in women with POP with Magnetic Resonance Imaging as the reference standard. Secondary goals of this study include quantification of the inter-observer agreement about levator ani defects and determining the association between levator defects and recurrent POP after anterior repair. In addition, the cost-effectiveness of adding translabial ultrasonography to the diagnostic work-up in patients with POP will be estimated in a decision analytic model. METHODS/DESIGN: A multicentre cohort study will be performed in nine Dutch hospitals. 140 consecutive women with a POPQ stage 2 or more anterior vaginal wall prolapse, who are indicated for anterior colporapphy will be included. Patients undergoing additional prolapse procedures will also be included. Prior to surgery, patients will undergo MR imaging and translabial 3D ultrasound examination of the pelvic floor. Patients will be asked to complete validated disease specific quality of life questionnaires before surgery and at six and twelve months after surgery. Pelvic examination will be performed at the same time points. Assuming a sensitivity and specificity of 90% of 3D ultrasound for diagnosing levator defects in a population of 120 women with POP, with a prior probability of levator ani defects of 40%, we will be able to estimate predictive values with good accuracy (i.e. confidence limits of at most 10% below or above the point estimates of positive and negative predictive values).Anticipating 3% unclassifiable diagnostic images because of technical reasons, and a further safety margin of 10% we plan to recruit 140 patients. TRIAL REGISTRATION: Nederlands trial register NTR2220

Charakterisierungsstudie der Lipid Anker Region des Vinkulin Proteins

The contact of the cell to the extracellular matrix components such as collagen and fibronectin is important for cell adhesion and migration. Most of the cell matrix contacts are linked to the actin filaments by the focal adhesions (FA). At the FA, heterodimeric transmembrane integrin receptors link the ECM to the cytoskeleton via adaptor proteins that are part of a sub membrane plaque. A variety of those adaptor proteins have been shown to associate with, and in some cases insert into, lipid bilayers. The focal adhesion protein vinculin (1066 residues), which can be separated into a 95 kDa head and a 30 kDa tail domain, shows such lipid binding sites. However, the function of vinculin’’s lipid binding is still an enigma. Two regions on the 30 kDa tail domain have been experimentally identified as candidates for lipid-binding: Helix 3 (residues 935–978) and the lipid anchor (residues 1052–1066). The alteration of helix 3 (residues 944-978) and the unstructured C-terminal arm (residues 1052-1066, so-called lipid anchor) resulted in impaired lipid vesicle interaction of the vinculin-tail. Pull-down assays with artificial lipid membranes revealed that in contrast to vinculin-tail (vt), a variant lacking the lipid anchor (VtdelC), does not interact with vesicles. To what extent the last 15 residues are involved in lipid interaction was not determined. In this study the lipid-binding ability of the lipid anchor of vinculin as well as the influence to cell mechanical behavior were determined. Differential scanning calorimetry (DSC) demonstrated that vinculin’’s C-terminal arm, which includes the lipid anchor, is directly involved in lipid binding. The peptide inserts into the lipid vesicle consisting of DMPC/ DMPG at various molar ratios. The secondary structure of the C-terminal arm was also explored under different ionic conditions which represent nominal basic, neutral and acidic pH’’s using molecular dynamics simulations. The generated trajectories predicted an antiparallel beta-sheet followed by an unstructured C-terminal end for the peptide representing vinculin’’s C-terminal arm under "basic" and "neutral" conditions. This conformational behavior was investigated in more detail in the presence/absence of DMPC/ DMPG vesicles using CD-spectroscopy. The results suggest direct association of vinculin’’s lipid-binding region (residues 1052–1066) with membranes whilst forming a beta-sheet. To determine the orientation of the lipid anchor during membrane interaction, solid state NMR measurements were performed using vinculins C-terminal arm peptide. Those results imply that in presence of POPC/POPG vesicles, the beta-sheet inserts into the lipid membrane. Furthermore, it was demonstrated that cells expressing vinculin without the lipid anchor (vindelC) showed a decreased focal adhesion turnover rate, which results in impaired cell adhesion and migration. In additional in vivo experiments, the influence of the lipid anchor region (residues 1052–1066) in terms of cell mechanical behavior was determined using vinculin deficient mouse embryonic fibroblasts, retransfected with EGFP-linked vinculin lacking the lipid anchor (MEF-vindelC). Magnetic tweezer experiments revealed that MEF-vindelC cells, incubated with fibronectin coated paramagnetic beads, were less stiff and more beads detached during these experiments compared to MEF-resc cells. Cells expressing vindelC formed fewer focal contacts as determined by confocal microscopy. 2D-traction measurements showed that MEF-vindelC cells generated less force compared to rescue cells. Attenuated traction forces were also found in cells that expressed vinculin with point mutations of the lipid anchor that either impaired lipid binding or prevented src-phosphorylation at site Y1065. However, traction generation was not diminished in cells that expressed vinculin with impaired lipid binding due to point mutations on helix 3. These results show that both the lipid binding and the src-phosphorylation of vinculin’’s C-terminus are important for cell mechanical behavior, but the lipid binding of helix 3 is not, suggesting that both the lipid anchor and the src-phosphorylation of Y1065 affect cell mechanical behavior.Die extrazelluläre Matrix (ECM) beeinflusst und kontrolliert die Adhäsion sowie die Migration von Zellen. Der fokale Adhäsionskontakt (FA) verbindet intrazellulär das Aktin-Zellskelett über den transmembranen Integrin-Rezeptor mit Komponenten der ECM, wie Kollagen und Fibronektin (FN). Der Auf- und Umbau dieser Adhäsionskontakte ist unter anderem nur durch die Wechselwirkung von einigen fokalen Proteinen mit der Zellmembran möglich. Das Vinkulin-Protein, welches in eine Kopf- (95 kDa) und eine Schwanzgruppe (30 kDa) unterteilt werden kann, zeigt solche Membran-bindende Strukturen. Es konnte experimentell bestätig werden, dass Helix 3 (Aminosäuren 935-978) sowie die letzten 15 Aminosäuren des C-terminus (Aminosäuren 1052-1066; Lipid Anker) der Vinkulin-Schwanz Gruppe mit Lipid-Vesikeln interagieren. Sogenannte Pull-down Experimente mit Lipidvesikeln, die mit der Vinkulin-Schwanz gruppe ohne den Lipid Anker (vtailΔC) inkubiert wurden, zeigten im Gegensatz zu den Versuchen mit dem gesamten Vinkulin-Schwanz (vtail) keine Interaktion mit der artifiziellen Membran. Ob allerdings der Lipid-Anker tatsächlich direkt an der Membran Wechselwirkung beteiligt ist, wurde im Rahmen dieser Experimente nicht geklärt. In dieser Arbeit wurde mittels „Differential scanning Calorimetry“ (DSC) versucht, die Frage der direkten Beteiligung des Lipid-Ankers (Aminosäuren 1052-1066) an der Membranbindung von Vinkulin zu klären. Diese Messungen haben gezeigt, dass der C-terminale Arm von Vinkulin (Aminosäuren 1045-1066) mit dem hydrophoben Bereich der Lipidvesikel in Kontakt tritt. Molekulardynamische Simulationen und Circular Dichroismus (CD) Messungen lassen vermuten, dass der Lipid-Anker eine für Lipid-Interaktionen günstige anti-parallele beta-Faltblatt Konformation einnimmt. Nuclear magnetic resonance (NMR)-Messungen in Anwesenheit von POPC/POPG Membranen bestätigten dies. Weiterhin ist bekannt, dass Zellen die Vinkulin exprimieren welches nicht mit Membranen wechselwirken kann, eine verminderte FA-Umbaurate aufweisen. Dies wirkt sich negativ auf die Adhäsion und Migration der jeweiligen Zellen aus. Basierend auf diesen Ergebnissen wurden im Rahmen dieser Arbeit in zusätzlichen in vivo Experimenten der Einfluss des Lipid-Ankers auf die mechanischen Eigenschaften der Zellen getestet. Zu diesem Zweck wurden MEF-Vinkulin(-/-) Zellen mit Vinkulin dessen Lipid-Anker fehlte (MEF-vindelC) retransfiziert. Diese MEF-vindelC Zellen wurden mit Fibronectin beschichteten paramagnetischen „beads“ inkubiert und mit einer „magnetischen Nadel“ untersucht. Im Vergleich zu wildtyp (MEF-wt) und rescue (MEF-resc) Zellen verhielten sich die MEF-vindelC Zellen weniger steif. Gleichzeitig rissen während der Messung von diesen Zellen mehr „beads“ ab. Dies ist ein Indiz für die verminderte Anbindung an ECM beschichteten Oberflächen von MEF-vindelC Zellen. 2D-traction microscopy Experimente zeigten weiter, dass diese MEF-vindelC Zellen im Vergleich zu MEF-wt bzw. MEF-resc Zellen während der Adhäsion geringere Kräfte und weniger fokale Kontakte ausbilden. Dies wird auf die verminderte Actin-myosin Aktivität zurückgeführt. Messungen der Kraftentwicklung von Zellen, die weitere Lipidbindedefiziente Varianten von Vinkulin exprimierten, zeigten, dass lediglich der Knock-out des Lipid-Ankers von Vinkulin zu einer Reduktion der Kräfte führt. Der Knock-out der src-Phosphorylierungstelle im Lipid-Anker (Y1065F) führt zu ähnlich verringerten Kräften. Diese lässt den Schluss zu, dass die Zellmechanik über die Membranbindung des Lipid-Ankers, sowie der src-abhängige Phosphorylierung von Vinkulin beeinflusst wird

Anchorage of Vinculin to Lipid Membranes Influences Cell Mechanical Properties

The focal adhesion protein vinculin (1066 residues) can be separated into a 95-kDa head and a 30-kDa tail domain. Vinculin's lipid binding sites localized on the tail, helix 3 (residues 944–978) and the unstructured C-terminal arm (residues 1052–1066, the so-called lipid anchor), influence focal adhesion turnover and are important for cell migration and adhesion. Using magnetic tweezers, we characterized the cell mechanical behavior in mouse embryonic fibroblast (MEF)-vin(−/−) cells transfected with EGFP-linked-vinculin deficient of the lipid anchor (vinΔC, residues 1–1051). MEF-vinΔC cells incubated with fibronectin-coated paramagnetic beads were less stiff, and more beads detached during these experiments compared to MEF-rescue cells. Cells expressing vinΔC formed fewer focal contacts as determined by confocal microscopy. Two-dimensional traction measurements showed that MEF-vinΔC cells generate less force compared to rescue cells. Attenuated traction forces were also found in cells that expressed vinculin with point mutations (R1060 and K1061 to Q) of the lipid anchor that impaired lipid binding. However, traction generation was not diminished in cells that expressed vinculin with impaired lipid binding caused by point mutations on helix 3. Mutating the src-phosphorylation site (Y1065 to F) resulted in reduced traction generation. These observations show that both the lipid binding and the src-phosphorylation of vinculin's C-terminus are important for cell mechanical behavior

Immunopathogenesis of type 1 diabetes in Western society

Type 1 diabetes is a multifactorial disease. Genetic predisposition and environmental factors favor the triggering of multiple autoimmune responses against pancreatic β cells; over time, chronic autoimmunity leads to severe β‐cell loss and an insulin‐dependent diabetic state for the life of the patient. Worldwide epidemiologic data show that the disease is more common in the Western world, especially among Caucasians of Northern European descent. There is a high level of clinical heterogeneity, both in disease progression, age of onset, and severity of clinical manifestations, possibly reflecting the interplay of diverse genetic and environmental factors on the severity and predominance of the pathogenic mechanisms leading to overt disease. Further studies to better assess the relative contributions of different pathogenic factors during disease progression are needed for a more integrated understanding of the disease pathogenesis