Structure-activity studies on the opening- and closure-mechanism of L-type calcium-channels

In der vorliegenden Arbeit wurden gating-sensitive Aminosäuren und Sequenzabschnitte in den S6-Segmenten des kardialen L-Typ Kanals Cav1.2 identifiziert und mit Hilfe von Homologiemodellen Hypothesen zu deren Funktion formuliert. Der Großteil dieser Arbeit befasst sich mit der Struktur-Funktionsanalyse des stark konservierten "G/A/G/A" Motivs in der "bundle crossing region" der S6-Segmente. In den Bereich dieses Motivs fällt eine bekannte, autosomal dominante, Punktmutation, die zu einer Kanalerkrankung, dem Timothy Syndrom, führt. Tritt diese Mutation G432S auf, so sind u.a. Arrhythmien, Autismus und neurologische Defekte die Folge. Dabei wird die Inaktivierung aufgehoben und durch die so verlängerte Kanalöffnungszeit strömt vermehrt Kalzium ein. Die Arbeit zeigt, dass Mutationen in allen analogen Positionen der Domänen II, III und IV starke Verschiebungen der Aktivierung auslösen. Eine Entkopplung des Aktivierungsprozesses vom Inaktivierungsprozess für Mutationen in Position 432 konnte nachgewiesen werden. Darüber hinaus wurde mittels Homologiemodell festgestellt, dass die Aminosäuren des "G/A/G/A"-Motivs wahrscheinlich einen räumlich dicht gepackten Ring in der geschlossenen Konformation des Kanals bilden. Es wird angenommen, dass dieser bei jeglicher Form der Mutation nicht mehr vollständig schließen kann. Um den Einfluss von Spannungssensoren auf das entdeckte "G/A/G/A" Motiv - und damit das gating-Verhalten - zu untersuchen, wurde jedes S4 Segment der vier Domänen neutralisiert. Durch die Neutralisierung der S4 Segmente in den Domänen I, III und IV wurde der Kanal funktionsunfähig. Interessanterweise konnte die Aktivierung des neutralisierten Segments in Domäne II (IIS4N) nicht von jener des Wildtypkanals unterschieden werden. Die Kombination von (IIS4N) mit allen "G/A/G/A" Mutanten verursachte ein Rescue-Verhalten, die Wildtyp-Aktivierung wurde also wiederhergestellt. Die Kombination von IIS4N mit den "G/A/G/A" umliegenden Positionen verursachte kein Rescue-Verhalten, was auf eine Schlüsselrolle von "G/A/G/A" für die Kanalöffnung schließen lässt. Thermodynamische Untersuchungen zeigten, dass der Spannungssensor IIS4 energetisch an den "G/A/G/A" Ring gekoppelt ist und diese Kopplung zu Konformationsänderungen der Domänen II, III und IV führt.The objective of the present study was to identify gating sensitive positions in the segments of the cardiac L-type Cav1.2 channel. This was supported by homology models that suggested structural details of certain amino acid regions. The main part of this thesis focuses on the highly conserved "G/A/G/A" motif of the bundle crossing region in the S6 segments. A well known autosomal dominant point mutation which causes a channelopathy called Timothy Syndrome is located inside this motif. This inherited disease leads to arrhythmias, autism and neurological defects. The source of this channelopathy is mutation G423S which dramatically reduces inactivation. The prolonged opening of the channel causes increased calcium influx. This work shows that mutations in all analogous positions in domains II, III and IV accelerate activation. Inactivation in Domains II-IV is not affected. Since inactivation is Domain I specific, a decoupling of the inactivation and the deactivation process can be shown. Homology modelling demonstrates that all amino acids of the "G/A/G/A" motif form a tightly packed ring in the closed conformation of the channel. We hypothesize that mutating this ring prevents complete channel closure. To investigate influence of voltage sensors on the identified "G/A/G/A" motif, all charged positions in each of the S4 segments were neutralized. Neutralization of the S4 segments in domains I, III and IV lead to a non-functional channel. Interestingly the activation shift of the neutralized segment in domain II (IIS4N) could not be distinguished from the wild-type channel. Combination of IIS4N with all "G/A/G/A" mutants caused a rescue behaviour with wild-type like activation. In contrast IIS4N in conjunction with amino acid mutations next to the "G/A/G/A" motif showed no rescue behaviour. This indicates the important function of the "G/A/G/A" motif. Thermodynamic analysis showed that the voltage sensor IIS4 is energetically coupled to the "G/A/G/A" ring. This coupling leads to conformational changes of domains II, II and IV

Cold Tumors: A Therapeutic Challenge for Immunotherapy

Therapeutic monoclonal antibodies targeting immune checkpoints (ICPs) have changed the treatment landscape of many tumors. However, response rate remains relatively low in most cases. A major factor involved in initial resistance to ICP inhibitors is the lack or paucity of tumor T cell infiltration, characterizing the so-called “cold tumors.” In this review, we describe the main mechanisms involved in the absence of T cell infiltration, including lack of tumor antigens, defect in antigen presentation, absence of T cell activation and deficit of homing into the tumor bed. We discuss then the different therapeutic approaches that could turn cold into hot tumors. In this way, specific therapies are proposed according to their mechanism of action. In addition, ‘‘supra-physiological’’ therapies, such as T cell recruiting bispecific antibodies and Chimeric Antigen Receptor (CAR) T cells, may be active regardless of the mechanism involved, especially in MHC class I negative tumors. The determination of the main factors implicated in the lack of preexisting tumor T cell infiltration is crucial for the development of adapted algorithms of treatments for cold tumors

Clonal karyotype evolution involving ring chromosome 1 with myelodysplastic syndrome subtype RAEB-t progressing into acute leukemia

s Karyotypic evolution is a well-known phenomenon in patients with malignant hernatological disorders during disease progression. We describe a 50-year-old male patient who had originally presented with pancytopenia in October 1992. The diagnosis of a myelodysplastic syndrome (MDS) FAB subtype RAEB-t was established in April 1993 by histological bone marrow (BM) examination, and therapy with low-dose cytosine arabinoside was initiated. In a phase of partial hernatological remission, cytogenetic assessment in August 1993 revealed a ring chromosome 1 in 13 of 21 metaphases beside BM cells with normal karyotypes {[}46,XY,r(1)(p35q31)/46,XY]. One month later, the patient progressed to an acute myeloid leukemia (AML), subtype M4 with 40% BM blasts and cytogenetic examination showed clonal evolution by the appearance of additional numerical aberrations in addition to the ring chromosome{[}46,XY,r(1),+8,-21/45,XY,r(1),+8,-21,-22/46, XY]. Intensive chemotherapy and radiotherapy was applied to induce remission in preparation for allogeneic bone marrow transplantation (BMT) from the patient's HLA-compatible son. After BMT, complete remission was clinically, hematologically and cytogenetically (normal male karyotype) confirmed. A complete hematopoietic chimerism was demonstrated. A relapse in January 1997 was successfully treated using donor lymphocyte infusion and donor peripheral blood stem cells (PB-SC) in combination with GM-CSF as immunostimulating agent in April 1997, and the patient's clinical condition remained stable as of January 2005. This is an interesting case of a patient with AML secondary to MDS. With the ring chromosome 1 we also describe a rare cytogenetic abnormality that predicted the poor prognosis of the patient, but the patient could be cured by adoptive immunotherapy and the application of donor's PB-SC. This case confirms the value of cytogenetic analysis in characterizing the malignant clone in hernatological neoplasias, the importance of controlling the quality of an induced remission and of the detection of a progress of the disease. Copyright (c) 2006 S. Karger AG, Basel

Fighting viral infections and virus-driven tumors with cytotoxic CD4+ T cells

CD4+ T cells have been and are still largely regarded as the orchestrators of immune responses, being able to differentiate into distinct T helper cell populations based on differentiation signals, transcription factor expression, cytokine secretion, and specific functions. Nonetheless, a growing body of evidence indicates that CD4+ T cells can also exert a direct effector activity, which depends on intrinsic cytotoxic properties acquired and carried out along with the evolution of several pathogenic infections. The relevant role of CD4+ T cell lytic features in the control of such infectious conditions also leads to their exploitation as a new immunotherapeutic approach. This review aims at summarizing currently available data about functional and therapeutic relevance of cytotoxic CD4+ T cells in the context of viral infections and virus-driven tumors

Longitudinally Profiling Neutralizing Antibody Response to SARS Coronavirus with Pseudotypes

SARS-CoV spike protein pseudotypes are the basis of an in vitro microneutralization assay sensitive and specific for SARS-CoV neutralizing antibodies

Large-scale discovery of insertion hotspots and preferential integration sites of human transposed elements

Throughout evolution, eukaryotic genomes have been invaded by transposable elements (TEs). Little is known about the factors leading to genomic proliferation of TEs, their preferred integration sites and the molecular mechanisms underlying their insertion. We analyzed hundreds of thousands nested TEs in the human genome, i.e. insertions of TEs into existing ones. We first discovered that most TEs insert within specific ‘hotspots’ along the targeted TE. In particular, retrotransposed Alu elements contain a non-canonical single nucleotide hotspot for insertion of other Alu sequences. We next devised a method for identification of integration sequence motifs of inserted TEs that are conserved within the targeted TEs. This method revealed novel sequences motifs characterizing insertions of various important TE families: Alu, hAT, ERV1 and MaLR. Finally, we performed a global assessment to determine the extent to which young TEs tend to nest within older transposed elements and identified a 4-fold higher tendency of TEs to insert into existing TEs than to insert within non-TE intergenic regions. Our analysis demonstrates that TEs are highly biased to insert within certain TEs, in specific orientations and within specific targeted TE positions. TE nesting events also reveal new characteristics of the molecular mechanisms underlying transposition