Antigen presentation by major histocompatibility complex (MHC) proteins is essential for adaptive immunity. Prior to presentation, peptides need to be generated from proteins that are either produced by the cell’s own translational machinery or that are funneled into the endo-lysosomal vesicular system. The prolonged interaction between a T cell receptor and specific pMHC complexes, after an extensive search process in secondary lymphatic organs, eventually triggers T cells to proliferate and to mount a specific cellular immune response. Once processed, the peptide repertoire presented by MHC proteins largely depends on structural features of the binding groove of each particular MHC allelic variant. Additionally, two peptide editors—tapasin for class I and HLA-DM for class II—contribute to the shaping of the presented peptidome by favoring the binding of high-affinity antigens. Although there is a vast amount of biochemical and structural information, the mechanism of the catalyzed peptide exchange for MHC class I and class II proteins still remains controversial, and it is not well understood why certain MHC allelic variants are more susceptible to peptide editing than others. Recent studies predict a high impact of protein intermediate states on MHC allele-specific peptide presentation, which implies a profound influence of MHC dynamics on the phenomenon of immunodominance and the development of autoimmune diseases. Here, we review the recent literature that describe MHC class I and II dynamics from a theoretical and experimental point of view and we highlight the similarities between MHC class I and class II dynamics despite the distinct functions they fulfill in adaptive immunity.

Regeneration of the myocardium by transplantation of cardiomyocytes is an emerging therapeutic strategy. Human embryonic stem cells (HESC) form cardiomyocytes readily but until recently at low efficiency, so that preclinical studies on transplantation in animals are only just beginning. Here, we show the results of the first long-term (12 weeks) analysis of the fate of HESC-derived cardiomyocytes transplanted intramyocardially into healthy, immunocompromised (NOD-SCID) mice and in NOD-SCID mice that had undergone myocardial infarction (MI). Transplantation of mixed populations of differentiated HESC containing 20-25% cardiomyocytes in control mice resulted in rapid formation of grafts in which the cardiomyocytes became organized and matured over time and the noncardiomyocyte population was lost. Grafts also formed in mice that had undergone MI. Four weeks after transplantation and MI, this resulted in significant improvement in cardiac function measured by magnetic resonance imaging. However, at 12 weeks, this was not sustained despite graft survival. This suggested that graft size was still limiting despite maturation and organization of the transplanted cells. More generally, the results argued for requiring a minimum of 3 months follow-up in studies claiming to observe improved cardiac function, independent of whether HESC or other (adult) cell types are used for transplantation.

Adhesion of T cells after stimulation of the T-cell receptor (TCR) is mediated via signaling processes that have collectively been termed inside-out signaling. The molecular basis for inside-out signalingis not yet completely understood. Here, we show that a signaling module comprising the cytosolic adapter proteins ADAP and SKAP55 is involved in TCR-mediated inside-out signaling and, moreover, that the interaction between ADAP and SKAP55 is mandatory for integrin activation. Disruption of the ADAP/SKAP55 module leads to displacement of the small GTPase Rap1 from the plasma membrane without influencing its GTPase activity. These findings suggest that the ADAP/SKAP55 complex serves to recruit activated Rap1 to the plasma membrane. In line with this hypothesis is the finding that membrane targeting of the ADAP/SKAP55 module induces T-cell adhesion in the absence of TCR-mediated stimuli. However, it appears as if the ADAP/SKAP55 module can exert its signaling function outside of the classical raft fraction of the cell membrane.Within the immune system, integrins play important roles in regulating the interaction of T cells with other cells and with proteins of the extracellular matrix. By mediating T-cell adhesion, integrins control the homing and the trafficking of T cells as well as the interaction between T cells and antigen-presenting cells (34, 41). The major integrins expressed on T cells are the ␤2-integrin LFA-1 (␣L␤2) as well as members of the ␤1-family of integrins (␣4␤1, ␣5␤1, ␣6␤1, and VLA) (25). The physiologic ligands of LFA-1 include the intercellular adhesion molecule 1 (ICAM-1), ICAM-2, and ICAM-3 (25), whereas ligands for ␤1-integrins are vascular cell adhesion molecule 1 (VCAM-1) or proteins of the extracellular matrix, such as fibronectin (13,54).On resting T cells, ␤1-and ␤2-integrins are expressed in an inactive state. However, ligation of the T-cell receptor (TCR) by antigen/major histocompatibility complexes results in a rapid increase in the activity of ␤1-and ␤2-integrins, thereby enhancing ligand binding (15,46,50). Two distinct mechanisms mediate the activation of integrins. First, the affinity of an integrin for its ligand is enhanced, and second, the lateral mobility becomes altered, which results in integrin clustering (avidity regulation) (14). The processes leading to the activation of integrins have collectively been termed inside-out signaling (14, 15, 28).Several molecules have been suggested to play critical roles during TCR-mediated activation of ␤1-and ␤2-integrins (14, 28). Among these is the small GTPase Rap1, whose role for integrin activation has been a matter of intense research during the last few years (4, 29). The mechanisms for how Rap1 becomes activated are not yet completely understood (4). Rap1 activation has been shown to be mediated by particular guanine nucleotide exchange factors (GEFs), such as C3G, and Epac (5,8,11). It has been proposed that Rap1 is associated with CalDAG-GEFI and that TCR-induced Rap1 activation is dependent upon the activation of phosphol...

Understanding and control of structures and rates involved in protein ligand binding are essential for drug design. Unfortunately, atomistic molecular dynamics (MD) simulations cannot directly sample the excessively long residence and rearrangement times of tightly binding complexes. Here we exploit the recently developed multi-ensemble Markov model framework to compute full protein-peptide kinetics of the oncoprotein fragment 25–109Mdm2 and the nano-molar inhibitor peptide PMI. Using this system, we report, for the first time, direct estimates of kinetics beyond the seconds timescale using simulations of an all-atom MD model, with high accuracy and precision. These results only require explicit simulations on the sub-milliseconds timescale and are tested against existing mutagenesis data and our own experimental measurements of the dissociation and association rates. The full kinetic model reveals an overall downhill but rugged binding funnel with multiple pathways. The overall strong binding arises from a variety of conformations with different hydrophobic contact surfaces that interconvert on the milliseconds timescale.

Background-Pluripotent stem cells (PSCs) offer a new paradigm for modeling genetic cardiac diseases, but it is unclear whether mouse and human PSCs can truly model both gain-and loss-of-function genetic disorders affecting the Na ϩ current (I Na ) because of the immaturity of the PSC-derived cardiomyocytes. To address this issue, we generated multiple PSC lines containing a Na ϩ channel mutation causing a cardiac Na ϩ channel overlap syndrome. Method and Results-Induced PSC (iPSC) lines were generated from mice carrying the Scn5a 1798insD/ϩ (Scn5a-het) mutation. These mouse iPSCs, along with wild-type mouse iPSCs, were compared with the targeted mouse embryonic stem cell line used to generate the mutant mice and with the wild-type mouse embryonic stem cell line. Patch-clamp experiments showed that the Scn5a-het cardiomyocytes had a significant decrease in I Na density and a larger persistent I Na compared with Scn5a-wt cardiomyocytes. Action potential measurements showed a reduced upstroke velocity and longer action potential duration in Scn5a-het myocytes. These characteristics recapitulated findings from primary cardiomyocytes isolated directly from adult Scn5a-het mice. Finally, iPSCs were generated from a patient with the equivalent SCN5A 1795insD/ϩ mutation. Patch-clamp measurements on the derivative cardiomyocytes revealed changes similar to those in the mouse PSC-derived cardiomyocytes. Conclusion-Here, we demonstrate that both embryonic stem cell-and iPSC-derived cardiomyocytes can recapitulate the characteristics of a combined gain-and loss-of-function Na ϩ channel mutation and that the electrophysiological immaturity of PSC-derived cardiomyocytes does not preclude their use as an accurate model for cardiac Na ϩ channel disease. (Circulation. 2012;125:3079-3091.) Key Words: cell differentiation Ⅲ disease models, animal Ⅲ electrophysiology Ⅲ sodium channels Ⅲ pluripotent stem cells M ultiple cardiac arrhythmia syndromes, including long-QT syndrome type 3 (LQT3), Brugada syndrome (BrS), progressive cardiac conduction disease, and sinus node dysfunction, have been linked to mutations in SCN5A, the gene encoding the ␣-subunit of the cardiac sodium (Na ϩ ) channel. 1,2 Most SCN5A mutations associated with LQT3 act by disrupting fast inactivation of the Na ϩ channel, resulting in a persistent inward Na ϩ current (I Na ) during the action potential (AP) plateau phase, subsequently delaying ventricular repolarization and prolonging the QT interval (gain-of-function mutations). 3 In contrast, SCN5A mutations underlying BrS and conduction disease are loss-of-function mutations and are believed to reduce the total amount of available I Na as a result of expression of nonfunctional channels, impaired intracellular trafficking, and decreased membrane surface channel expression or through altered channel gating properties. 1,2 Editorial see p 3055 Clinical Perspective on p 3091Initially, it was believed that these arrhythmia syndromes constituted separate clinical entities, with individual SCN5A Received September 9...

Objective-Endothelial cells (ECs), pericytes, and vascular smooth muscle cells (vSMCs)

FGF2 is secreted from cells by an unconventional secretory pathway. This process is mediated by direct translocation across the plasma membrane. Here, we define the minimal molecular machinery required for FGF2 membrane translocation in a fully reconstituted inside-out vesicle system. FGF2 membrane translocation is thermodynamically driven by PI(4,5)P2-induced membrane insertion of FGF2 oligomers. The latter serve as dynamic translocation intermediates of FGF2 with a subunit number in the range of 8-12 FGF2 molecules. Vectorial translocation of FGF2 across the membrane is governed by sequential and mutually exclusive interactions with PI(4,5)P2 and heparan sulfates on opposing sides of the membrane. Based on atomistic molecular dynamics simulations, we propose a mechanism that drives PI(4,5)P2 dependent oligomerization of FGF2. Our combined findings establish a novel type of self-sustained protein translocation across membranes revealing the molecular basis of the unconventional secretory pathway of FGF2.DOI: http://dx.doi.org/10.7554/eLife.28985.001

Jervell and Lange-Nielsen syndrome (JLNS) is one of the most severe life-threatening cardiac arrhythmias. Patients display delayed cardiac repolarization, associated high risk of sudden death due to ventricular tachycardia, and congenital bilateral deafness. In contrast to the autosomal dominant forms of long QT syndrome, JLNS is a recessive trait, resulting from homozygous (or compound heterozygous) mutations in KCNQ1 or KCNE1. These genes encode the α and β subunits, respectively, of the ion channel conducting the slow component of the delayed rectifier K + current, I Ks . We used complementary approaches, reprogramming patient cells and genetic engineering, to generate human induced pluripotent stem cell (hiPSC) models of JLNS, covering splice site (c.478-2A>T) and missense (c.1781G>A) mutations, the two major classes of JLNS-causing defects in KCNQ1. Electrophysiological comparison of hiPSC-derived cardiomyocytes (CMs) from homozygous JLNS, heterozygous, and wild-type lines recapitulated the typical and severe features of JLNS, including pronounced action and field potential prolongation and severe reduction or absence of I Ks . We show that this phenotype had distinct underlying molecular mechanisms in the two sets of cell lines: the previously unidentified c.478-2A>T mutation was amorphic and gave rise to a strictly recessive phenotype in JLNS-CMs, whereas the missense c.1781G>A lesion caused a gene dosage-dependent channel reduction at the cell membrane. Moreover, adrenergic stimulation caused action potential prolongation specifically in JLNS-CMs. Furthermore, sensitivity to proarrhythmic drugs was strongly enhanced in JLNSCMs but could be pharmacologically corrected. Our data provide mechanistic insight into distinct classes of JLNS-causing mutations and demonstrate the potential of hiPSC-CMs in drug evaluation.Jervell and Lange-Nielsen syndrome | long QT syndrome | human induced pluripotent stem cells | disease modeling | KCNQ1