Biomedical subjects

Gerhard Wiche

Publications and source records attributed to Gerhard Wiche.

At least 19 recordsLinked to original sources

PubMed2007

Oxidation and nitrosylation of cysteines proximal to the intermediate filament (IF)-binding site of plectin: effects on structure and vimentin binding and involvement in IF collapse.

As an intermediate filament (IF)-based cytolinker protein, plectin plays a key role in the maintenance of cellular cytoarchitecture and serves at the same time as a scaffolding platform for signaling cascades. Consisting of six structural repeats (R1-6) and harboring binding sites for different IF proteins and proteins involved in signaling, the plectin C-terminal domain is of strategic functional importance. Depending on the species, it contains at least 13 cysteines, 4 of which reside in the R5 domain. To investigate the structural and biological functions of R5 cysteines, we used cysteine-to-serine mutagenesis and spectroscopic, biochemical, and functional analyses. Urea-induced unfolding experiments indicated that wild-type R5 in the oxidized, disulfide bond-mediated conformation was more stable than its cysteine-free mutant derivative. The binding affinity of R5 for vimentin was significantly higher, however, when the protein was in the reduced, more relaxed conformation. Of the four R5 cysteines, one (Cys4) was particularly reactive as reflected by its ability to form disulfide bridges with R5 Cys1 and to serve as a target for nitrosylation in vitro. Using immortalized endothelial cell cultures from mice, we show that endogenous plectin is nitrosylated in vivo, and we found that NO donor-induced IF collapse proceeds dramatically faster in plectin-deficient compared with wild-type cells. Our data suggest an antagonistic role of plectin in nitrosylation (oxidative stress)-mediated alterations of IF cytoarchitecture and a possible role of R5 Cys4 as a regulatory switch.

Amino Acid Sequence↗

PubMed2006

Plectin-controlled keratin cytoarchitecture affects MAP kinases involved in cellular stress response and migration.

Plectin is a major intermediate filament (IF)-based cytolinker protein that stabilizes cells and tissues mechanically, regulates actin filament dynamics, and serves as a scaffolding platform for signaling molecules. In this study, we show that plectin deficiency is a cause of aberrant keratin cytoskeleton organization caused by a lack of orthogonal IF cross-linking. Keratin networks in plectin-deficient cells were more susceptible to osmotic shock-induced retraction from peripheral areas, and their okadaic acid-induced disruption (paralleled by stress-activated MAP kinase p38 activation) proceeded faster. Basal activities of the MAP kinase Erk1/2 and of the membrane-associated upstream protein kinases c-Src and PKCdelta were significantly elevated, and increased migration rates, as assessed by in vitro wound-closure assays and time-lapse microscopy, were observed. Forced expression of RACK1, which is the plectin-binding receptor protein for activated PKCdelta, in wild-type keratinocytes elevated their migration potential close to that of plectin-null cells. These data establish a link between cytolinker-controlled cytoarchitecture/scaffolding functions of keratin IFs and specific MAP kinase cascades mediating distinct cellular responses.

Animals↗

PubMed2006

Plectin scaffolds recruit energy-controlling AMP-activated protein kinase (AMPK) in differentiated myofibres.

Plectin, a cytolinker protein greater than 500 kDa in size, has an important role as a mechanical stabiliser of cells. It interlinks the various cytoskeletal filament systems and anchors intermediate filaments to peripheral junctional complexes. In addition, there is increasing evidence that plectin acts as a scaffolding platform that controls the spatial and temporal localisation and interaction of signaling proteins. In this study we show that, in differentiated mouse myotubes, plectin binds to the regulatory gamma1 subunit of AMP-activated protein kinase (AMPK), the key regulatory enzyme of energy homeostasis. No interaction was observed in undifferentiated myoblasts, and plectin-deficient myotubes showed altered positioning of gamma1-AMPK. In addition we found that plectin affects the subunit composition of AMPK, because isoform alpha1 of the catalytic subunit decreased in proportion to isoform alpha2 during in vitro differentiation of plectin(-/-) myotubes. In plectin-deficient myocytes we could also detect a higher level of activated (Thr172-phosphorylated) AMPK, compared with wild-type cells. Our data suggest a differentiation-dependent association of plectin with AMPK, where plectin selectively stabilises alpha1-gamma1 AMPK complexes by binding to the gamma1 regulatory subunit. The distinct plectin expression patterns in different fibre types combined with its involvement in the regulation of isoform compositions of AMPK complexes could provide a mechanism whereby cytoarchitecture influences energy homeostasis.

Animals↗

PubMed2006

Epiplakin is dispensable for skin barrier function and for integrity of keratin network cytoarchitecture in simple and stratified epithelia.

Epiplakin, a giant epithelial protein of >700 kDa, belongs to the plakin family of cytolinker proteins. It represents an atypical family member, however, as it consists entirely of plakin repeat domains but lacks any of the other domains commonly shared by plakins. Hence, its putative function as a cytolinker protein remains to be shown. To investigate epiplakin's biological role, we generated epiplakin-deficient mice by gene targeting in embryonic stem cells. Epiplakin-deficient mice were viable and fertile, without developing any discernible phenotype. Ultrastructurally, their epidermis revealed no differences compared to wild-type littermates, and cornified envelopes isolated from skin showed no alterations in shape or stability. Furthermore, neither embryonal formation nor later function of the epithelial barrier was affected. In primary cultures of epiplakin-deficient keratinocytes, the organization of actin filaments, microtubules, and keratin networks was found to be normal. Similarly, no alterations in keratin network organization were observed in simple epithelia of small intestine and liver or in primary hepatocytes. We conclude that, despite epiplakin's abundant and highly specific expression in stratified and simple epithelia, its absence in mice does not lead to severe skin dysfunctions, nor has it detectable consequences for keratin filament organization and cytoarchitecture of cells.

Animals↗

PubMed2006

Plectin regulates the organization of glial fibrillary acidic protein in Alexander disease.

Alexander disease (AxD) is a rare but fatal neurological disorder caused by mutations in the astrocyte-specific intermediate filament protein glial fibrillary acidic protein (GFAP). Histologically, AxD is characterized by cytoplasmic inclusion bodies called Rosenthal fibers (RFs), which contain GFAP, small heat shock proteins, and other undefined components. Here, we describe the expression of the cytoskeletal linker protein plectin in the AxD brain. RFs displayed positive immunostaining for plectin and GFAP, both of which were increased in the AxD brain. Co-localization, co-immunoprecipitation, and in vitro overlay analyses demonstrated direct interaction of plectin and GFAP. GFAP with the most common AxD mutation, R239C (RC GFAP), mainly formed abnormal aggregates in human primary astrocytes and murine plectin-deficient fibroblasts. Transient transfection of full-length plectin cDNA converted these aggregates to thin filaments, which exhibited diffuse cytoplasmic distribution. Compared to wild-type GFAP expression, RC GFAP expression lowered plectin levels in astrocytoma-derived stable transfectants and plectin-positive fibroblasts. A much higher proportion of total GFAP was found in the Triton X-insoluble fraction of plectin-deficient fibroblasts than in wild-type fibroblasts. Taken together, our results suggest that insufficient amounts of plectin, due to RC GFAP expression, promote GFAP aggregation and RF formation in AxD.

Alexander Disease↗

PubMed2005

Targeted ablation of plectin isoform 1 uncovers role of cytolinker proteins in leukocyte recruitment.

Plectin, a typical cytolinker protein, is essential for skin and skeletal muscle integrity. It stabilizes cells mechanically, regulates cytoskeleton dynamics, and serves as a scaffolding platform for signaling molecules. A variety of isoforms expressed in different tissues and cell types account for this versatility. To uncover the role of plectin 1, the major isoform expressed in tissues of mesenchymal origin, against the background of all other variants, we raised plectin isoform 1-specific antibodies and generated isoform-deficient mice. In contrast to plectin-null mice (lacking all plectin isoforms), which die shortly after birth because of severe skin blistering, plectin isoform 1-deficient mice were viable at birth, had a normal lifespan, and did not display the skin blistering phenotype. However, dermal fibroblasts isolated from plectin 1-deficient mice exhibited abnormalities in their actin cytoskeleton and impaired migration potential. Similarly, plectin 1-deficient T cells isolated from nymph nodes showed diminished chemotactic migration in vitro. Most strikingly, in vivo we found that leukocyte infiltration during wound healing was reduced in the mutant mice. These data show a specific role of a cytolinker protein in immune cell motility. Single isoform-deficient mice thus represent a powerful tool to unravel highly specific functions of plectin variants.

Actins↗

PubMed2005

Plectin rodless isoform expression and its detection in mouse brain.

The widely expressed cytolinker protein plectin shows extensive isoform diversity both at the N-terminus and in the central part of the molecule. Judged on mRNA data, plectin variants lacking the central rod domain are expressed at a approximately 20-fold lower level than full-length proteins and their detection on the protein level can be difficult. Here we present data on the expression of plectin rodless isoforms in mouse brain and in rat glioma C6 cells on RNA and protein levels. Our data indicate that among the rodless variants expressed in neuronal tissues, those starting with exon 1c (plectin 1c) seem to be the most prominent ones. In addition, we show that similar to other monoclonal antibodies reported in the literature, the widely used mAb 7A8 recognizes an epitope within plectin's rod domain and therefore is unsuited to detect rodless variants of plectin.

Alternative Splicing↗

PubMed2005

Overlap of the gene encoding the novel poly(ADP-ribose) polymerase Parp10 with the plectin 1 gene and common use of exon sequences.

We have recently identified PARP10 as a novel functional poly(ADP-ribose) polymerase. The gene encoding PARP10 is conserved in vertebrates but no orthologs were found in lower organisms. In addition to the poly(ADP-ribose) polymerase domain, PARP10 possesses several additional sequence motifs, including an RNA recognition motif and two ubiquitin interaction motifs. We characterized the murine genomic locus of the Parp10 gene. We noticed that 3' Parp10 sequences overlapped with the plectin 1 gene in a head-to-tail arrangement. Detailed analyses revealed that the two most 3' Parp10 exons (exons 10 and 11) are also used for plectin 1. While these two exons code for part of the poly(ADP-ribose) polymerase domain in Parp10, they are noncoding for plectin 1 due to the lack of appropriate start codons. Furthermore our findings suggest that at least one of the plectin 1 promoters is located within intron 9 of the Parp10 gene.

Amino Acid Sequence↗

PubMed2004

Plectin-RACK1 (receptor for activated C kinase 1) scaffolding: a novel mechanism to regulate protein kinase C activity.

Agonist-induced translocation of protein kinase C (PKC) isozymes is mediated by receptors for the activated form of the kinase, shuttling it from one intracellular site to another and enhancing its catalytic activity. It is however unknown whether the receptors themselves are anchored to certain intracellular structures prior to their engagement with PKC. We show here sequestering of receptor for activated C kinase 1 (RACK1) to the cytoskeleton through the cytoskeletal linker protein plectin during the initial stages of cell adhesion. We found that upon PKC activation, RACK1 was released from the cytoskeleton and transferred to the detergent-soluble cell compartment, where it formed an inducible triple complex with one of the PKC isozymes, PKCdelta, and with plectin. In plectin-deficient cells the cytoskeleton-associated RACK1 fraction was reduced, and the protein was found predominantly at sites to which it normally translocated upon PKC activation. Concomitantly, dislocation of PKCdelta and elevated enzymatic activity were observed in these cells. PKCdelta was also more rapidly degraded, likely due to its overactivation. We propose a previously unrecognized function of plectin as cytoskeletal regulator of PKC signaling, and possibly other signaling events, through sequestration of the scaffolding protein RACK1.

Animals↗

PubMed2004

Severe mucous membrane involvement in epidermolysis bullosa simplex with muscular dystrophy due to a novel plectin gene mutation.

UNLABELLED: Epidermolysis bullosa simplex with muscular dystrophy (OMIM 226670) is an autosomal recessive disorder caused by mutations of the human plectin gene on chromosome 8q24. Here, we report a 3-year-old girl, offspring of a consanguineous Lebanese family, who presented with skin blistering and recurrent episodes of severe respiratory distress necessitating tracheotomy at the age of 2 years. Repeated examination did not provide any evidence of muscle involvement. Indirect immunofluorescence analysis of a diagnostic skin biopsy with four different domain specific plectin antibodies showed a complete absence of plectin staining. Mutation analysis revealed a novel homozygous single guanine insertion mutation (5588insG/5588insG) residing in the N-terminal part of exon 31 of the plectin gene. CONCLUSION: The complete lack of protein expression, which may be attributed to a nonsense-mediated plectin mRNA decay, is likely to cause muscular dystrophy and other multisystem involvement later in life.

Base Sequence↗

PubMed2004

High-pressure cryoimmobilization of murine skin reveals novel structural features and prevents extraction artifacts.

Cryoimmobilization by high-pressure freezing (HPF) and subsequent freeze substitution has been proven as an effective method to preserve tissues. Here, we demonstrate for the first time that a comprehensive morphological and ultrastructural preservation of mouse skin throughout all its layers can be achieved in this way. Using conditions limiting tissue-extraction during freeze substitution, we could prevent the massive interdigitation of cell membranes, the loss of tubular structures of the Golgi complex, the aggregation of keratin to electron-dense bundles, the formation of round-shaped keratohyalin aggregates, the dispersion of locally organized ribosomes, the excessive aggregation of material at hemidesmosomal plaques, the massive extraction of material from the basement membrane and the adjacent dermal region, and the dissociation of components of the dermal matrix. Taken together, HPF in combination with freeze substitution emerges as a highly sensitive tool for morphological and ultrastructural analysis.

Animals↗

PubMed2004

Actin-binding domain of mouse plectin. Crystal structure and binding to vimentin.

Plectin, a large and widely expressed cytolinker protein, is composed of several subdomains that harbor binding sites for a variety of different interaction partners. A canonical actin-binding domain (ABD) comprising two calponin homology domains (CH1 and CH2) is located in proximity to its amino terminus. However, the ABD of plectin is unique among actin-binding proteins as it is expressed in the form of distinct, plectin isoform-specific versions. We have determined the three-dimensional structure of two distinct crystalline forms of one of its ABD versions (pleABD/2alpha) from mouse, to a resolution of 1.95 and 2.0 A. Comparison of pleABD/2alpha with the ABDs of fimbrin and utrophin revealed structural similarity between plectin and fimbrin, although the proteins share only low sequence identity. In fact, pleABD/2alpha has been found to have the same compact fold as the human plectin ABD and the fimbrin ABD, differing from the open conformation described for the ABDs of utrophin and dystrophin. Plectin harbors a specific binding site for intermediate filaments of various types within its carboxy-terminal R5 repeat domain. Our experiments revealed an additional vimentin-binding site of plectin, residing within the CH1 subdomain of its ABD. We show that vimentin binds to this site via the amino-terminal part of its rod domain. This additional amino-terminal intermediate filament protein binding site of plectin may have a function in intermediate filament dynamics and assembly, rather than in linking and stabilizing intermediate filament networks.

Actins↗

PubMed2004

Plectin.

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Animals↗

PubMed2003

Plectin 5'-transcript diversity: short alternative sequences determine stability of gene products, initiation of translation and subcellular localization of isoforms.

Plectin is a large cytoskeletal linker protein expressed as several different isoforms from a highly complex gene. This transcript diversity is mainly caused by short 5'-sequences contained in alternative first exons. To elucidate the influence of these sequence differences and to determine potential differential functionality of the resulting protein forms, we conducted a systematic investigation of plectin isoforms on transcript and protein levels. Isoform expression was highly dependent on the different 5' ends, largely due to effects of the 5'-untranslated regions. Initiation of translation downstream of the expected start site led to loss of actin- and integrin beta4-binding in some isoforms. The small alternative N-terminal sequences (5-180 residues) profoundly affected the subcelluar localization of this >500 kDa protein. Specifically, plectin 1f was concentrated at focal adhesion contacts and plectin 1b was exclusively targeted to mitochondria, providing a connection of these organelles to intermediate filaments. Thus, with plectin as a model, we demonstrate a role for 5'-untranslated regions and alternative 5'-splicing as an important regulatory mechanism of protein expression and protein function.

5' Untranslated Regions↗

PubMed2003

Epiplakin gene analysis in mouse reveals a single exon encoding a 725-kDa protein with expression restricted to epithelial tissues.

Based on cDNA cloning and sequencing, human epiplakin has been classified as a member of the plakin protein family of cytolinkers. We report here the characterization of the mouse epiplakin gene locus and the isolation of full-length mouse epiplakin cDNA using BAC vectors. We found that the protein is encoded by a single remarkably large exon (>20 kb) that consists of a series of 0.8-1.5-kb-long DNA repeats, eight of which are virtually identical. Consequently, mouse epiplakin contains 16 plakin repeat domains, three more than reported for the human protein and eight more than predicted for the mouse protein based on the contig characterized by the Mouse Genome Sequencing Consortium. Using antibodies raised to a highly conserved repeating epiplakin sequence domain, we show that the protein in cells is expressed in its full length (725 kDa), and we provide evidence that the size of human epiplakin previously may have been underestimated. In addition we show on transcript and protein levels that epiplakin is restricted to epithelial tissues and that its gene maps to mouse chromosome 15 (human chromosome 8). This study lays the groundwork for future genetic approaches aimed at defining the biological role of this unique protein.

Amino Acid Sequence↗

PubMed2003

A binding motif for Siah ubiquitin ligase.

The Drosophila SINA (seven in absentia) protein and its mammalian orthologs (Siah, seven in absentia homolog) are RING domain proteins that function in E3 ubiquitin ligase complexes and facilitate ubiquitination and degradation of a wide range of cellular proteins, including beta-catenin. Despite these diverse targets, the means by which SINASiah recognize substrates or binding proteins has remained unknown. Here we identify a peptide motif (RPVAxVxPxxR) that mediates the interaction of Siah protein with a range of protein partners. Sequence alignment and mutagenesis scanning revealed residues that are important to this interaction. This consensus sequence correctly predicted a high-affinity interaction with a peptide from the cytoskeletal protein plectin-1 (residues 95-117). The unusually high-affinity binding obtained with a 23-residue peptide (K(Dapp) = 29 nM with SINA) suggests that it may serve as a useful dominant negative reagent for SINASiah proteins.

Amino Acid Motifs↗

PubMed2003

Plectin-isoform-specific rescue of hemidesmosomal defects in plectin (-/-) keratinocytes.

The various plectin isoforms are among the major crosslinking elements of the cytoskeleton. The importance of plectin in epithelia is convincingly supported by the severe skin blistering observed in plectin-deficient humans and mice. Here, we identified plectin 1a (> 500 kDa), a full length plectin variant containing the sequence encoded by the alternative first exon 1a, as the isoform most prominently expressed in human and mouse keratinocytes. In skin sections and cultured keratinocytes, plectin 1a was shown to colocalize with hemidesmosomal structures. In contrast, a second isoform expressed in epithelia, plectin 1c, differing from 1a merely by a short N-terminal sequence, colocalized with microtubules. Expression of plectin 1a, but not of its N-terminal fragment alone, or of a third alternative full length isoform (plectin 1), restored the reduced number of hemidesmosome-like stable anchoring contacts in cultured plectin-null keratinocytes. Our results show for the first time that different isoforms of a cytolinker protein expressed in one cell type perform distinct functions. Moreover, the identification of plectin 1a as the isoform defects in which cause skin blistering in plectin-related genetic diseases, such as epidermolysis bullosa simplex MD and epidermolysis bullosa simplex Ogna, could have implications for the future development of clinical therapies for patients.

Alternative Splicing↗

PubMed2002

Primary longitudinal adhesion structures: plectin-containing precursors of costameres in differentiating human skeletal muscle cells.

Plectin is a high molecular mass protein (ca 530 kDa) that binds actin, intermediate filaments, and microtubules. Mutations of the human plectin gene cause epidermolysis bullosa simplex with muscular dystrophy. In mature human skeletal muscle, plectin is localized between neighboring myofibrils and between myofibrils and the sarcolemma, both at the level of Z-discs. In the present study we have analyzed plectin expression patterns with emphasis on its sarcolemmal localization during human skeletal muscle differentiation in vitro. In myoblasts plectin showed a cytoplasmic intermediate filament-like distribution, whereas in myotubes plectin is also found at the level of the sarcolemma. In particular, in early myotubes a specific plectin isoform colocalizes with the costameric proteins vinculin and beta1D integrin in longitudinally orientated structures which increased in number and longitudinal extension upon further maturation. In mature myotubes processes perpendicular to the parallel system of longitudinal structures became apparent. Subsequent to the occurrence of spontaneous myofibrillar contractions, the number of longitudinal streaks decreased, and plectin and other costameric proteins were found in an orderly cross-striated sarcolemmal lattice overlying myofibrillar Z-discs. Our study demonstrates that plectin is preassembled together with vinculin and beta1D integrin into primary longitudinal adhesion structures. After the occurrence of spontaneous contractions, these structures reorient and mature costameres are assembled.

Cell Differentiation↗