Regulation of hemidesmosome dynamics and cell signaling by integrin α6ß4.

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 α6ß4 is a receptor for laminins and plays a vital role in mediating cell adhesion by initiating the assembly of HDs. In addition, α6ß4 has been implicated in signal transduction events that regulate diverse cellular processes, including proliferation and survival. In this Review, we detail the role of α6ß4 in HD assembly and beyond, and we discuss the molecular mechanisms that regulate its function.

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. However, the molecular details of C3G activation and the interplay between phosphorylation and Crk interaction are poorly understood. METHODS: We combined biochemical, biophysical, and cell biology approaches to elucidate the mechanisms of C3G activation. Binding of Crk adaptor proteins to four proline-rich motifs (P1 to P4) in C3G was characterized in vitro using isothermal titration calorimetry and sedimentation velocity, and in Jurkat and HEK293T cells by affinity pull-down assays. The nucleotide exchange activity of C3G over Rap1 was measured using nucleotide-dissociation kinetic assays. Jurkat cells were also used to analyze C3G translocation to the plasma membrane and the C3G-dependent activation of Rap1 upon ligation of T cell receptors. RESULTS: CrkL interacts through its SH3N domain with sites P1 and P2 of inactive C3G in vitro and in Jurkat and HEK293T cells, and these sites are necessary to recruit C3G to the plasma membrane. However, direct stimulation of the GEF activity requires binding of Crk proteins to the P3 and P4 sites. P3 is occluded in resting C3G and is essential for activation, while P4 contributes secondarily towards complete stimulation. Tyrosine phosphorylation of C3G alone causes marginal activation. Instead, phosphorylation primes C3G lowering the concentration of Crk proteins required for activation and increasing the maximum activity. Unexpectedly, optimal activation also requires the interaction of CrkL-SH2 domain with phosphorylated C3G. CONCLUSION: Our study revealed that phosphorylation of C3G by Src and Crk-binding form a two-factor mechanism that ensures tight control of C3G activation. Additionally, the simultaneous SH2 and SH3N interaction of CrkL with C3G, required for the activation, reveals a novel adaptor-independent function of Crk proteins relevant to understanding their role in physiological signaling and their deregulation in diseases. Video abstract.

PSTPIP1-LYP phosphatase interaction: structural basis and implications for autoinflammatory disorders.

Mutations in the adaptor protein PSTPIP1 cause a spectrum of autoinflammatory diseases, including PAPA and PAMI; however, the mechanism underlying these diseases remains unknown. Most of these mutations lie in PSTPIP1 F-BAR domain, which binds to LYP, a protein tyrosine phosphatase associated with arthritis and lupus. To shed light on the mechanism by which these mutations generate autoinflammatory disorders, we solved the structure of the F-BAR domain of PSTPIP1 alone and bound to the C-terminal homology segment of LYP, revealing a novel mechanism of recognition of Pro-rich motifs by proteins in which a single LYP molecule binds to the PSTPIP1 F-BAR dimer. The residues R228, D246, E250, and E257 of PSTPIP1 that are mutated in immunological diseases directly interact with LYP. These findings link the disruption of the PSTPIP1/LYP interaction to these diseases, and support a critical role for LYP phosphatase in their pathogenesis.

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 Clostridium pasteurianum [Hammel KE, Cornwell KL, Buchanan BB (1983) Proc Natl Acad Sci USA 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. We have recently reinvestigated the problem and confirmed that the enzyme, here designated ferredoxin-dependent flavin thioredoxin reductase (FFTR), is a flavoprotein. The enzyme differs from ferredoxin-thioredoxin reductase (FTR), which has a signature [4Fe-4S] cluster, but shows structural similarities to NADP-dependent thioredoxin reductase (NTR). Comparative amino acid sequence analysis showed that FFTR is present in a number of clostridial species, some of which lack both FTR and an archetypal NTR. We have isolated, crystallized, and determined the structural properties of FFTR from a member of this group, Clostridium acetobutylicum, both alone and in complex with Trx. The structures showed an elongated FFTR homodimer, each monomer comprising two Rossmann domains and a noncovalently bound FAD cofactor that exposes the isoalloxazine ring to the solvent. The FFTR structures revealed an alternative domain organization compared with NTR that enables the enzyme to accommodate Fdx rather than NADPH. The results suggest that FFTR exists in a range of conformations with varying degrees of domain separation in solution and that the stacking between the two redox-active groups for the transfer of reducing equivalents results in a profound structural reorganization. A mechanism in accord with the findings is proposed.

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). However, our experimental data show that the protein does not transfer reducing equivalents from flavins to disulfides as in NTRs but functions in the opposite direction. High-resolution structures of the protein from Gloeobacter violaceus and Synechocystis sp. PCC6803 obtained by X-ray crystallography showed two juxtaposed FAD molecules per monomer in redox communication with an active disulfide bridge in a variant of the fold adopted by NTRs. We have tentatively named the flavoprotein "DDOR" (diflavin-linked disulfide oxidoreductase) and propose that its activity is linked to a thiol-based transfer of reducing equivalents in bacterial membranes. These findings expand the structural and mechanistic repertoire of flavoenzymes with oxidoreductase activity and pave the way to explore new protein engineering approaches aimed at designing redox-active proteins for diverse biotechnological applications.

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. The plakin domains of plectin and other plakins harbor binding sites for junctional proteins. We have combined x-ray crystallography with small angle x-ray scattering (SAXS) to elucidate the structure of the plakin domain of plectin, extending our previous analysis of the SR1 to SR5 region. Two crystal structures of the SR5-SR6 region allowed us to characterize its uniquely wide inter-repeat conformational variability. We also report the crystal structures of the SR7-SR8 region, refined to 1.8 Å, and the SR7-SR9 at lower resolution. The SR7-SR9 region, which is conserved in all other plakin domains, forms a rigid segment stabilized by uniquely extensive inter-repeat contacts mediated by unusually long helices in SR8 and SR9. Using SAXS we show that in solution the SR3-SR6 and SR7-SR9 regions are rod-like segments and that SR3-SR9 of plectin has an extended shape with a small central kink. Other plakins, such as bullous pemphigoid antigen 1 and microtubule and actin cross-linking factor 1, are likely to have similar extended plakin domains. In contrast, desmoplakin has a two-segment structure with a central flexible hinge. The continuous versus segmented structures of the plakin domains of plectin and desmoplakin give insight into how different plakins might respond to tension and transmit mechanical signals.

Exploiting tertiary structure through local folds for crystallographic phasing.

We describe an algorithm for phasing protein crystal X-ray diffraction data that identifies, retrieves, refines and exploits general tertiary structural information from small fragments available in the Protein Data Bank. The algorithm successfully phased, through unspecific molecular replacement combined with density modification, all-helical, mixed alpha-beta, and all-beta protein structures. The method is available as a software implementation: Borges.

Combination of X-ray crystallography, SAXS and DEER to obtain the structure of the FnIII-3,4 domains of integrin α6ß4.

Integrin α6ß4 is a major component of hemidesmosomes that mediate the stable anchorage of epithelial cells to the underlying basement membrane. Integrin α6ß4 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 ß4 mediate binding to the hemidesmosomal proteins BPAG1e and BPAG2, and participate in signalling. Here, it is demonstrated that X-ray crystallography, small-angle X-ray scattering and double electron-electron resonance (DEER) complement each other to solve the structure of the FnIII-3,4 region. The crystal structures of the individual FnIII-3 and FnIII-4 domains were solved and the relative arrangement of the FnIII domains was elucidated by combining DEER with site-directed spin labelling. Multiple structures of the interdomain linker were modelled by Monte Carlo methods complying with DEER constraints, and the final structures were selected against experimental scattering data. FnIII-3,4 has a compact and cambered flat structure with an evolutionary conserved surface that is likely to correspond to a protein-interaction site. Finally, this hybrid method is of general application for the study of other macromolecules and complexes.

Structural basis of the interaction between integrin alpha6beta4 and plectin at the hemidesmosomes.

The interaction between the integrin alpha6beta4 and plectin is essential for the assembly and stability of hemidesmosomes, which are junctional adhesion complexes that anchor epithelial cells to the basement membrane. We describe the crystal structure at 2.75 A resolution of the primary alpha6beta4-plectin complex, formed by the first pair of fibronectin type III domains and the N-terminal region of the connecting segment of beta4 and the actin-binding domain of plectin. Two missense mutations in beta4 (R1225H and R1281W) linked to nonlethal forms of epidermolysis bullosa prevent essential intermolecular contacts. We also present two structures at 1.75 and 2.05 A resolution of the beta4 moiety in the absence of plectin, which reveal a major rearrangement of the connecting segment of beta4 on binding to plectin. This conformational switch is correlated with the way alpha6beta4 promotes stable adhesion or cell migration and suggests an allosteric control of the integrin.

Nesprin-3 augments peripheral nuclear localization of intermediate filaments in zebrafish.

The outer nuclear membrane protein nesprin-3 binds the cytoskeletal linker protein plectin, which are proposed to anchor the intermediate filaments to the nuclear envelope. To investigate the function of nesprin-3 in vivo, we used the zebrafish as a vertebrate model system. Zebrafish nesprin-3 is expressed at the nuclear envelope of epidermal and skeletal muscle cells during development. Unexpectedly, loss of nesprin-3 did not affect embryonic development, viability or fertility. However, nesprin-3-deficient zebrafish embryos showed a reduced concentration of intermediate filaments around the nucleus. Additional analysis revealed the presence of two nesprin-3 isoforms in zebrafish, nesprin-3α and nesprin-3ß. Nesprin-3ß is only expressed during early development and lacks seven amino acids in its first spectrin repeat that are crucial for plectin binding and recruitment to the nuclear envelope. These seven amino acids are highly conserved and we showed that residues R43 and L44 within this motif are required for plectin binding. Furthermore, several residues in the actin-binding domain of plectin that are crucial for binding to the integrin ß4 subunit are also important for the binding to nesprin-3α, indicating partial overlapping binding sequences for nesprin-3α and integrin ß4. All this shows that nesprin-3 is dispensable for normal development in zebrafish, but important for mediating the association of the intermediate filament system with the nucleus in vivo.

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

An important feature associated with Candida albicans 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. Despite its biological importance, both the structure of CaRas1 and the molecular basis of its activation by CaCdc25 remain unexplored. Here, we show that CaRas1 has an elongated shape and limited conformational flexibility and that its hypervariable region contains helical structural elements, likely forming an intramolecular coiled-coil. Functional assays disclosed that CaRas1-activation by CaCdc25 is highly efficient, with activities up to 2,000-fold higher than reported for human GEFs. The crystal structure of the CaCdc25 catalytic region revealed an active conformation for the α-helical hairpin, critical for CaRas1-activation, unveiling a specific region exclusive to CTG-clade species. Structural studies on CaRas1/CaCdc25 complexes also revealed an interaction surface clearly distinct from that of homologous human complexes. Furthermore, we identified an inhibitory synthetic peptide, prompting the proposal of a key regulatory mechanism for CaCdc25. To our knowledge, this is the first report of specific inhibition of the CaRas1-activation via targeting its GEF. This, together with their unique pathogen-structural features, disclose a set of novel strategies to specifically block this important virulence-related mechanism. IMPORTANCE Candida albicans is the main causative agent of candidiasis, the commonest fungal infection in humans. The eukaryotic nature of C. albicans and the rapid emergence of antifungal resistance raise the challenge of identifying novel drug targets to battle this prevalent and life-threatening disease. CaRas1 and CaCdc25 are key players in the activation of signaling pathways triggering multiple virulence traits, including the yeast-to-hypha interconversion. The structural similarity of the conserved G-domain of CaRas1 to those of human homologs and the lack of structural information on CaCdc25 has impeded progress in targeting these proteins. The unique structural and functional features for CaRas1 and CaCdc25 presented here, together with the identification of a synthetic peptide capable of specifically inhibiting the GEF activity of CaCdc25, open new possibilities to uncover new antifungal drug targets against C. albicans virulence.

The structure of the plakin domain of plectin reveals a non-canonical SH3 domain interacting with its fourth spectrin repeat.

Plectin belongs to the plakin family of cytoskeletal crosslinkers, which is part of the spectrin superfamily. Plakins contain an N-terminal conserved region, the plakin domain, which is formed by an array of spectrin repeats (SR) and a Src-homology 3 (SH3), and harbors binding sites for junctional proteins. We have combined x-ray crystallography and small angle x-ray scattering (SAXS) to elucidate the structure of the central region of the plakin domain of plectin, which corresponds to the SR3, SR4, SR5, and SH3 domains. The crystal structures of the SR3-SR4 and SR4-SR5-SH3 fragments were determined to 2.2 and 2.95 Å resolution, respectively. The SH3 of plectin presents major alterations as compared with canonical Pro-rich binding SH3 domains, suggesting that plectin does not recognize Pro-rich motifs. In addition, the SH3 binding site is partially occluded by an intramolecular contact with the SR4. Residues of this pseudo-binding site and the SR4/SH3 interface are conserved within the plakin family, suggesting that the structure of this part of the plectin molecule is similar to that of other plakins. We have created a model for the SR3-SR4-SR5-SH3 region, which agrees well with SAXS data in solution. The three SRs form a semi-flexible rod that is not altered by the presence of the SH3 domain, and it is similar to those found in spectrins. The flexibility of the plakin domain, in analogy with spectrins, might contribute to the role of plakins in maintaining the stability of tissues subject to mechanical stress.

Current insights into the formation and breakdown of hemidesmosomes.

Hemidesmosomes are multiprotein adhesion complexes that promote epithelial stromal attachment in stratified and complex epithelia. Modulation of their function is of crucial importance in a variety of biological processes, such as differentiation and migration of keratinocytes during wound healing and carcinoma invasion, in which cells become detached from the substrate and acquire a motile phenotype. Although much is known about the signaling potential of the alpha6beta4 integrin in carcinoma cells, the events that coordinate the disassembly of hemidesmosomes during differentiation and wound healing remain unclear. The binding of alpha6beta4 to plectin has a central role in hemidesmosome assembly and it is becoming clear that disrupting this interaction is a crucial event in hemidesmosome disassembly. In addition, further insight into the functional interplay between alpha3beta1 and alpha6beta4 has contributed to our understanding of hemidesmosome disassembly and cell migration.

EGFR-dependent tyrosine phosphorylation of integrin ß4 is not required for downstream signaling events in cancer cell lines.

In epithelial cancers, the epidermal growth factor receptor (EGFR) and integrin α6ß4 are frequently overexpressed and found to synergistically activate intracellular signaling pathways that promote cell proliferation and migration. In cancer cells, the ß4 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 ß4 tyrosine phosphorylation following the activation of EGFR. However, siRNA mediated knockdown of the SFKs Src, Fyn, Yes and Lyn, individually or in combination, did not affect the EGF-induced phosphorylation of ß4. Using phospho-peptide affinity chromatography and mass spectrometry, we found that PLCγ1 binds ß4 at the phosphorylated residues Y1422/Y1440, but were unable to verify this interaction in A431 carcinoma cells that overexpress the EGFR. Furthermore, using A431 cells devoid of ß4 or reconstituted with phenylalanine specific mutants of ß4, the activation of several downstream signaling pathways, including PLCγ/PKC, MAPK and PI3K/Akt, were not substantially affected. We conclude that tyrosine-phosphorylated ß4 does not enhance EGFR-mediated signaling in EGFR-overexpressing cells, despite the fact that this integrin subunit is highly tyrosine phosphorylated in these cells.

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.

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. However, the genetic alterations underlying the audiogenic seizure susceptibility of the GASH/Sal model remain unknown. In addition, gene variations in the GASH/Sal might have a close resemblance with those described in humans with epilepsy, which is a prerequisite for any new preclinical studies that target genetic abnormalities. Here, we performed whole exome sequencing (WES) in GASH/Sal animals and their corresponding controls to identify and characterize the mutational landscape of the GASH/Sal strain. After filtering the results, moderate- and high-impact variants were validated by Sanger sequencing, assessing the possible impact of the mutations by "in silico" reconstruction of the encoded proteins and analyzing their corresponding biological pathways. Lastly, we quantified gene expression levels by RT-qPCR. In the GASH/Sal model, WES showed the presence of 342 variations, in which 21 were classified as high-impact mutations. After a full bioinformatics analysis to highlight the high quality and reliable variants, the presence of 3 high-impact and 15 moderate-impact variants were identified. Gene expression analysis of the high-impact variants of Asb14 (ankyrin repeat and SOCS Box Containing 14), Msh3 (MutS Homolog 3) and Arhgef38 (Rho Guanine Nucleotide Exchange Factor 38) genes showed a higher expression in the GASH/Sal than in control hamsters. In silico analysis of the functional consequences indicated that those mutations in the three encoded proteins would have severe functional alterations. By functional analysis of the variants, we detected 44 significantly enriched pathways, including the glutamatergic synapse pathway. The data show three high-impact mutations with a major impact on the function of the proteins encoded by these genes, although no mutation in these three genes has been associated with some type of epilepsy until now. Furthermore, GASH/Sal animals also showed gene variants associated with different types of epilepsy that has been extensively documented, as well as mutations in other genes that encode proteins with functions related to neuronal excitability, which could be implied in the phenotype of the GASH/Sal. Our findings provide valuable genetic and biological pathway data associated to the genetic burden of the audiogenic seizure susceptibility and reinforce the need to validate the role of each key mutation in the phenotype of the GASH/Sal model.

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

[This corrects the article DOI: 10.1371/journal.pone.0229953.].

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. Specifically, we found that two intramolecular interactions regulate the activity of C3G. First, an autoinhibitory region (AIR) within the central domain of C3G binds to and blocks the catalytic Cdc25H domain. Second, the binding of the protein's N-terminal domain to its Ras exchanger motif (REM) is required for its GEF activity. CrkL activated C3G by displacing the AIR/Cdc25HD interaction. Two missense mutations in the AIR found in non-Hodgkin's lymphomas, Y554H and M555K, disrupted the autoinhibitory mechanism. Expression of C3G-Y554H or C3G-M555K in Ba/F3 pro-B cells caused constitutive activation of Rap1 and, consequently, the integrin LFA-1. Our findings suggest that sustained Rap1 activation by deregulated C3G might promote progression of lymphomas and that designing therapeutics to target C3G might treat these malignancies.

Structure of the Calx-beta domain of the integrin beta4 subunit: insights into function and cation-independent stability.

The integrin alpha6beta4 is a receptor for laminins and provides stable adhesion of epithelial cells to the basement membranes. In addition, alpha6beta4 is important for keratinocyte migration during wound healing and favours the invasion of carcinomas into surrounding tissue. The cytoplasmic domain of the beta4 subunit is responsible for most of the intracellular interactions of the integrin; it contains four fibronectin type III domains and a Calx-beta motif. The crystal structure of the Calx-beta domain of beta4 was determined to 1.48 A resolution. The structure does not contain cations and biophysical data support the supposition that the Calx-beta domain of beta4 does not bind calcium. Comparison of the Calx-beta domain of beta4 with the calcium-binding domains of Na(+)/Ca(2+)-exchanger 1 reveals that in beta4 Arg1003 occupies a position equivalent to that of the calcium ions in the Na(+)/Ca(2+)-exchanger. By combining mutagenesis and thermally induced unfolding, it is shown that Arg1003 contributes to the stability of the Calx-beta domain. The structure of the Calx-beta domain is discussed in the context of the function and intracellular interactions of the integrin beta4 subunit and a putative functional site is proposed.

Integrin α6ß4 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 α6ß4 to intermediate filaments in epidermal hemidesmosomes. Here, we show that a linear sequence within the isoform-specific N-terminal region of BP230 binds to the third and fourth FnIII domains of ß4. The crystal structure of the complex and mutagenesis analysis revealed that BP230 binds between the two domains of ß4. BP230 induces closing of the two FnIII domains that are locked in place by an interdomain ionic clasp required for binding. Disruption of BP230-ß4 binding prevents recruitment of BP230 to hemidesmosomes in human keratinocytes, revealing a key role of this interaction for hemidesmosome assembly. Phosphomimetic substitutions in ß4 and BP230 destabilize the complex. Thus, our study provides insights into the architecture of hemidesmosomes and potential mechanisms of regulation.