LYP regulates SLP76 and other adaptor proteins in T cells.

Background: The LYP tyrosine phosphatase presents a SNP (1858C > T) that increases the risk of developing autoimmune diseases such as type I diabetes and arthritis. It remains unclear how this SNP affects LYP function and promotes the development of these diseases. The scarce information about LYP substrates is in part responsible for the poor understanding of LYP function.

Pathogen-specific structural features of Ras1 activation complex: uncovering new antifungal drug targets.

An important feature associated with pathogenicity is its ability to switch between yeast and hyphal forms, a process in which CaRas1 plays a key role. CaRas1 is activated by the guanine nucleotide exchange factor (GEF) CaCdc25, triggering hyphal growth-related signaling pathways through its conserved GTP-binding (G)-domain. An important function in hyphal growth has also been proposed for the long hypervariable region downstream the G-domain, whose unusual content of polyglutamine stretches and Q/N repeats make CaRas1 unique within Ras proteins.

Crk proteins activate the Rap1 guanine nucleotide exchange factor C3G by segregated adaptor-dependent and -independent mechanisms.

Background: C3G is a guanine nucleotide exchange factor (GEF) that activates Rap1 to promote cell adhesion. Resting C3G is autoinhibited and the GEF activity is released by stimuli that signal through tyrosine kinases. C3G is activated by tyrosine phosphorylation and interaction with Crk adaptor proteins, whose expression is elevated in multiple human cancers.

Fetal and neonatal alloimmune thrombocytopenia: Current pathophysiological insights and perspectives for future diagnostics and treatment.

FNAIT is a pregnancy-associated condition caused by maternal alloantibodies against paternally-inherited platelet antigens, most frequently HPA-1a on integrin β3. The clinical effects range from no symptoms to fatal intracranial hemorrhage, but underlying pathophysiological determinants are poorly understood. Accumulating evidence suggests that differential antibody-Fc-glycosylation, activation of complement/effector cells, and integrin function-blocking effects contribute to clinical outcome.

Regulation of hemidesmosome dynamics and cell signaling by integrin α6β4.

Hemidesmosomes (HDs) are specialized multiprotein complexes that connect the keratin cytoskeleton of epithelial cells to the extracellular matrix (ECM). In the skin, these complexes provide stable adhesion of basal keratinocytes to the underlying basement membrane. Integrin α6β4 is a receptor for laminins and plays a vital role in mediating cell adhesion by initiating the assembly of HDs.

EGFR-dependent tyrosine phosphorylation of integrin β4 is not required for downstream signaling events in cancer cell lines.

In epithelial cancers, the epidermal growth factor receptor (EGFR) and integrin α6β4 are frequently overexpressed and found to synergistically activate intracellular signaling pathways that promote cell proliferation and migration. In cancer cells, the β4 subunit is phosphorylated at tyrosine residues not normally recognized as kinase substrates; however, the function of these phosphotyrosine residues in cancer cells is a subject of much debate. In EGFR-overexpressing carcinoma cells, we found that the Src family kinase (SFK) inhibitor PP2 reduces β4 tyrosine phosphorylation following the activation of EGFR.

C3G self-regulatory mechanism revealed: implications for hematopoietic malignancies.

Abnormally increased signaling by the GTPase RAP1 favors progression of diverse tumors. We have characterized the auto-regulation and activation of C3G (RAPGEF1), an activator of RAP1. This led us to discover mutations in non-Hodgkin's lymphomas that activate C3G-RAP1 constitutively, suggesting that deregulation of C3G may favor the dissemination of tumor cells.

Mechanisms of autoregulation of C3G, activator of the GTPase Rap1, and its catalytic deregulation in lymphomas.

C3G is a guanine nucleotide exchange factor (GEF) that regulates cell adhesion and migration by activating the GTPase Rap1. The GEF activity of C3G is stimulated by the adaptor proteins Crk and CrkL and by tyrosine phosphorylation. Here, we uncovered mechanisms of C3G autoinhibition and activation.

Analysis of gene variants in the GASH/Sal model of epilepsy.

Epilepsy is a complex neurological disorder characterized by sudden and recurrent seizures, which are caused by various factors, including genetic abnormalities. Several animal models of epilepsy mimic the different symptoms of this disorder. In particular, the genetic audiogenic seizure hamster from Salamanca (GASH/Sal) animals exhibit sound-induced seizures similar to the generalized tonic seizures observed in epileptic patients.

A mutation in p62 protein (p. R321C), associated to Paget's disease of bone, causes a blockade of autophagy and an activation of NF-kB pathway.

Paget's disease of bone (PDB) is a bone disorder characterized by an increase in bone turnover in a disorganized way with a large increase in bone resorption followed by bone formation. The most important known genetic factor predisposing to PDB is mutation in Sequestosome1 (SQSTM1) gene. We have studied the prevalence of SQSTM1 mutations and examined genotype-phenotype correlations in a Spanish cohort of PDB patients.

Integrin α6β4 Recognition of a Linear Motif of Bullous Pemphigoid Antigen BP230 Controls Its Recruitment to Hemidesmosomes.

Mechanical stability of epithelia requires firm attachment to the basement membrane via hemidesmosomes. Dysfunction of hemidesmosomal proteins causes severe skin-blistering diseases. Two plakins, plectin and BP230 (BPAG1e), link the integrin α6β4 to intermediate filaments in epidermal hemidesmosomes.

C3G, through its GEF activity, induces megakaryocytic differentiation and proplatelet formation.

Background: Megakaryopoiesis allows platelet formation, which is necessary for coagulation, also playing an important role in different pathologies. However, this process remains to be fully characterized. C3G, an activator of Rap1 GTPases, is involved in platelet activation and regulates several differentiation processes.

Ferredoxin-linked flavoenzyme defines a family of pyridine nucleotide-independent thioredoxin reductases.

Ferredoxin-dependent thioredoxin reductase was identified 35 y ago in the fermentative bacterium [Hammel KE, Cornwell KL, Buchanan BB (1983) 80:3681-3685]. The enzyme, a flavoprotein, was strictly dependent on ferredoxin as reductant and was inactive with either NADPH or NADH. This early work has not been further pursued.

Unprecedented pathway of reducing equivalents in a diflavin-linked disulfide oxidoreductase.

Flavoproteins participate in a wide variety of physiologically relevant processes that typically involve redox reactions. Within this protein superfamily, there exists a group that is able to transfer reducing equivalents from FAD to a redox-active disulfide bridge, which further reduces disulfide bridges in target proteins to regulate their structure and function. We have identified a previously undescribed type of flavin enzyme that is exclusive to oxygenic photosynthetic prokaryotes and that is based on the primary sequence that had been assigned as an NADPH-dependent thioredoxin reductase (NTR).

The nesprin-cytoskeleton interface probed directly on single nuclei is a mechanically rich system.

The cytoskeleton provides structure and plays an important role in cellular function such as migration, resisting compression forces, and transport. The cytoskeleton also reacts to physical cues such as fluid shear stress or extracellular matrix remodeling by reorganizing filament associations, most commonly focal adhesions and cell-cell cadherin junctions. These mechanical stimuli can result in genome-level changes, and the physical connection of the cytoskeleton to the nucleus provides an optimal conduit for signal transduction by interfacing with nuclear envelope proteins, called nesprins, within the LINC (linker of the nucleus to the cytoskeleton) complex.

A nucleotide-controlled conformational switch modulates the activity of eukaryotic IMP dehydrogenases.

Inosine-5'-monophosphate dehydrogenase (IMPDH) is an essential enzyme for nucleotide metabolism and cell proliferation. Despite IMPDH is the target of drugs with antiviral, immunosuppressive and antitumor activities, its physiological mechanisms of regulation remain largely unknown. Using the enzyme from the industrial fungus Ashbya gossypii, we demonstrate that the binding of adenine and guanine nucleotides to the canonical nucleotide binding sites of the regulatory Bateman domain induces different enzyme conformations with significantly distinct catalytic activities.

The Structure of the Plakin Domain of Plectin Reveals an Extended Rod-like Shape.

Plakins are large multi-domain proteins that interconnect cytoskeletal structures. Plectin is a prototypical plakin that tethers intermediate filaments to membrane-associated complexes. Most plakins contain a plakin domain formed by up to nine spectrin repeats (SR1-SR9) and an SH3 domain.

Purification and Structural Analysis of Plectin and BPAG1e.

Plectin and BPAG1e belong to the plakin family of high-molecular-weight proteins that interconnect the cytoskeletal systems and anchor them to junctional complexes. Plectin and BPAG1e are prototypical plakins with a similar tripartite modular structure. The N- and C-terminal regions are built of multiple discrete structural domains, while the central rod domain mediates dimerization by coiled-coil interactions.

Guanine nucleotide binding to the Bateman domain mediates the allosteric inhibition of eukaryotic IMP dehydrogenases.

Inosine-5'-monophosphate dehydrogenase (IMPDH) plays key roles in purine nucleotide metabolism and cell proliferation. Although IMPDH is a widely studied therapeutic target, there is limited information about its physiological regulation. Using Ashbya gossypii as a model, we describe the molecular mechanism and the structural basis for the allosteric regulation of IMPDH by guanine nucleotides.

Increased riboflavin production by manipulation of inosine 5'-monophosphate dehydrogenase in Ashbya gossypii.

Guanine nucleotides are the precursors of essential biomolecules including nucleic acids and vitamins such as riboflavin. The enzyme inosine-5'-monophosphate dehydrogenase (IMPDH) catalyzes the ratelimiting step in the guanine nucleotide de novo biosynthetic pathway and plays a key role in controlling the cellular nucleotide pools. Thus, IMPDH is an important metabolic bottleneck in the guanine nucleotide synthesis, susceptible of manipulation by means of metabolic engineering approaches.

The rod domain is not essential for the function of plectin in maintaining tissue integrity.

Epidermolysis bullosa simplex associated with late-onset muscular dystrophy (EBS-MD) is an autosomal recessive disorder resulting from mutations in the plectin gene. The majority of these mutations occur within the large exon 31 encoding the central rod domain and leave the production of a low-level rodless plectin splice variant unaffected. To investigate the function of the rod domain, we generated rodless plectin mice through conditional deletion of exon 31.

Combination of X-ray crystallography, SAXS and DEER to obtain the structure of the FnIII-3,4 domains of integrin α6β4.

Integrin α6β4 is a major component of hemidesmosomes that mediate the stable anchorage of epithelial cells to the underlying basement membrane. Integrin α6β4 has also been implicated in cell proliferation and migration and in carcinoma progression. The third and fourth fibronectin type III domains (FnIII-3,4) of integrin β4 mediate binding to the hemidesmosomal proteins BPAG1e and BPAG2, and participate in signalling.