Intermediate Filament Binding Research Articles

Proteomics analysis on aortic smooth muscle cells reveals new potential targets for the treatment of Marfan syndrome-associated thoracic aortic aneurysm

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministry of Health Background Thoracic aortic aneurysm (TAA) is characterized by the abnormal enlargement of the initial segment of the aorta. Patients with genetic Marfan syndrome (MFS) develop TAA in young age, since mutations of fibrillin-1 contribute to thoracic aortic wall detrimental weakening. Presently, effective treatment options for this life-threatening condition are lacking, and existing pharmacological approaches are merely palliative, by postponing the need for surgical aortic replacement. To identify new treatments or MFS-related TAA (MFS-TAA), current research is concentrated on pinpointing specific biological targets and mechanisms underlying this aortic disorder. Purpose This study is aimed to unveil potential therapeutic targets that could be exploited for treating MFS-TAA by adopting an advanced proteomics technique. Material and Methods A label-free quantitative mass spectrometry technique was employed, utilizing a hybrid quadrupole-time of flight mass spectrometer coupled with a UPLC Mclass system and a nano-source, to analyze and compare the protein expression levels in vascular smooth muscle cells (VSMC) isolated from the aortas of MFS patients (MFS-VSMC) and healthy subjects (HC-VSMC). The proteomics findings were subsequently validated through qRT-PCR and Western blot assays. Results The proteomics investigation yielded 16 proteins displaying statistically significant differences. Among these, five proteins were notably more abundant in MFS-VSMC compared to HC-VSMC: nestin (NES, p<0.01), filamin B (FLNB, p<0.05), A-kinase anchor protein 12 (AKAP12, p<0.05), catenin delta-1 (CTNND1, p<0.05), and heparan sulfate proteoglycan core protein (HSPG2, p<0.05). The Gene Ontology (GO) analysis performed on cellular components of these 5 items using the clusterProfiler R package highlighted the enrichment of GO terms generally related to transmembrane proteins and anchoring junctions. Specifically, enriched GO terms included cell cortex (p<0.002), focal adhesion (p<0.002), cell-substrate junction (p<0.002), and intermediate filament binding (p<0.03). To verify these findings, two out of the five proteins with increased expression in MFS-VSMC (i.e., nestin and filamin B) were subjected to validation experiments. Results confirmed the elevated gene expression and protein levels of both nestin (p<0.05) and filamin B (p<0.05) in MFS-VSMC samples when compared with HC-VSMC. Conclusions Taken together, these results suggest that proteins with increased expression in MFS-VSMC may represent valuable biological target of MFS-TAA. This may encompass their potential both as diagnostic tools and/or mechanistic mediators of TAA in MFS subjects. In details, the pharmacological targeting of nestin and filamin B may represent a novel and exploitable therapeutic option for limiting/inhibiting TAA onset or progression in MFS pathological scenario.

Reconstitution of cytolinker-mediated crosstalk between actin and vimentin

Cell shape and motility are determined by the cytoskeleton, an interpenetrating network of actin filaments, microtubules, and intermediate filaments. The biophysical properties of each filament type individually have been studied extensively by cell-free reconstitution. By contrast, the interactions between the three cytoskeletal networks are relatively unexplored. They are coupled via crosslinkers of the plakin family such as plectin. These are challenging proteins for reconstitution because of their giant size and multidomain structure. Here we engineer a recombinant actin-vimentin crosslinker protein called ‘ACTIF’ that provides a minimal model system for plectin, recapitulating its modular design with actin-binding and intermediate filament-binding domains separated by a coiled-coil linker for dimerisation. We show by fluorescence and electron microscopy that ACTIF has a high binding affinity for vimentin and actin and creates mixed actin-vimentin bundles. Rheology measurements show that ACTIF-mediated crosslinking strongly stiffens actin-vimentin composites. Finally, we demonstrate the modularity of this approach by creating an ACTIF variant with the intermediate filament binding domain of Adenomatous Polyposis Coli. Our protein engineering approach provides a new cell-free system for the biophysical characterization of intermediate filament-binding crosslinkers and for understanding the mechanical synergy between actin and vimentin in mesenchymal cells.

Comprehensive Analysis of GLUT1 Immune Infiltrates and ceRNA Network in Human Esophageal Carcinoma.

BackgroundGlucose transporter 1 (GLUT1) is encoded by the solute carrier family 2A1 (SLC2A1) gene and is one of the glucose transporters with the greatest affinity for glucose. Abnormal expression of GLUT1 is associated with a variety of cancers. However, the biological role of GLUT1 in esophageal carcinoma (ESCA) remains to be determined.MethodsWe analyzed the expression of GLUT1 in pan-cancer and ESCA as well as clinicopathological analysis through multiple databases. Use R and STRING to perform GO/KEGG function enrichment and PPI analysis for GLUT1 co-expression. TIMER and CIBERSORT were used to analyze the relationship between GLUT1 expression and immune infiltration in ESCA. The TCGA ESCA cohort was used to analyze the relationship between GLUT1 expression and m6A modification in ESCA, and to construct a regulatory network in line with the ceRNA hypothesis.ResultsGLUT1 is highly expressed in a variety of tumors including ESCA, and is closely related to histological types and histological grade. GO/KEGG functional enrichment analysis revealed that GLUT1 is closely related to structural constituent of cytoskeleton, intermediate filament binding, cell-cell adheres junction, epidermis development, and P53 signaling pathway. PPI shows that GLUT1 is closely related to TP53, GIPC1 and INS, and these three proteins all play an important role in tumor proliferation. CIBERSORT analysis showed that GLUT1 expression is related to the infiltration of multiple immune cells. When GLUT1 is highly expressed, the number of memory B cells decreases. ESCA cohort analysis found that GLUT1 expression was related to 7 m6A modifier genes. Six possible crRNA networks in ESCA were constructed by correlation analysis, and all these ceRNA networks contained GLUT1.ConclusionGLUT1 can be used as a biomarker for the diagnosis and treatment of ESCA, and is related to tumor immune infiltration, m6A modification and ceRNA network.

Structure of the Intermediate Filament-Binding Region of Desmoplakin.

Desmoplakin (DP) is a cytoskeletal linker protein that connects the desmosomal cadherin/plakoglobin/plakophilin complex to intermediate filaments (IFs). The C-terminal region of DP (DPCT) mediates IF binding, and contains three plakin repeat domains (PRDs), termed PRD-A, PRD-B and PRD-C. Previous crystal structures of PRDs B and C revealed that each is formed by 4.5 copies of a plakin repeat (PR) and has a conserved positively charged groove on its surface. Although PRDs A and B are linked by just four amino acids, B and C are separated by a 154 residue flexible linker, which has hindered crystallographic analysis of the full DPCT. Here we present the crystal structure of a DPCT fragment spanning PRDs A and B, and elucidate the overall architecture of DPCT by small angle X-ray scattering (SAXS) analysis. The structure of PRD-A is similar to that of PRD-B, and the two domains are arranged in a quasi-linear arrangement, and separated by a 4 amino acid linker. Analysis of the B-C linker region using secondary structure prediction and the crystal structure of a homologous linker from the cytolinker periplakin suggests that the N-terminal ~100 amino acids of the linker form two PR-like motifs. SAXS analysis of DPCT indicates an elongated but non-linear shape with Rg = 51.5 Å and Dmax = 178 Å. These data provide the first structural insights into an IF binding protein containing multiple PRDs and provide a foundation for studying the molecular basis of DP-IF interactions.

Abstract 4299: Poor prognosis in smokers with non-small cell lung cancer correlates with the presence of a reparative progenitor cell population

Abstract Cigarette smoking is the most significant risk factor for the initiation and development of lung cancer. Tobacco exposure often results in chronic inflammation, tissue injury, and airway repair. A recent hypothesis demonstrates the presence of a stem/progenitor cell population that involved in airway epithelial repair. These cells may be tumor-initiating cells in lung cancer, and subsequently may be involved in tumor recurrence and metastasis. In the airway epithelium, cells that express keratin (K) 5 and its intermediate filament binding partner K14 are considered to be progenitor cells in the adult large airways at steady state and during airway epithelial repair. In order to identify subpopulations of airway epithelial stem/progenitor cells under steady state conditions, normal repair, aberrant repair with premalignant lesions, and lung cancer, and their respective correlation with injury and prognosis, we used immunostaining on mouse and human lung tissues, along with a human lung cancer tissue microarray. We found K5 and K14 expression in the steady state submucosal glands and gland ducts. In the basal layer of normal airway pseudostratified columnar epithelium, only K5 expression was prominent while K14 expression was absent. In a heterotopic, syngeneic, murine tracheal transplant model of repairing airway epithelium, we identified a population of K14+ progenitor epithelial cells that was temporally involved in normal repair after injury. Dysregulated repair resulted in persistence of K14+ cells in the airway epithelium of premalignant lesions. In human patients (n = 399), the presence of K14+ cells in non-small cell lung cancer (NSCLC) predicted poorer outcomes (P = 0.004) especially in patients who are smokers (P = 0.001). The predictive value was more pronounced in current smokers (P = 0.01) compared to former smokers (P = 0.04). In never smokers, the presence of K14+ cells had no predictive value for outcome. We found that the presence of K14+ cells in the primary tumors of current smokers was associated with metastatic disease (P = 0.02). We further found that non-adenocarcinoma NSCLCs from current smokers with metastases had a higher percentage of K14+ cells in the primary tumor sites. (P = 0.004). Examination of K14 expression in distant metastatic sites revealed a significant increase in the number of K14-expressing cells in metastases compared to the primary sites in squamous lung cancer, but not in other histologic subtypes. Our data propose that dysregulated repair after injury leads to a self-renewing K14+ progenitor cell population in premalignant lesions. The presence of K14+ progenitor airway epithelial cells in NSCLC predicted a poor prognosis with the strongest predictive value in smokers, where it correlated with metastasis. This suggests that reparative K14+ progenitor cells are the putative tumor-initiating cells in this subgroup of smokers with NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4299.

The Desmosome

Desmosomes are intercellular junctions that tether intermediate filaments to the plasma membrane. Desmogleins and desmocollins, members of the cadherin superfamily, mediate adhesion at desmosomes. Cytoplasmic components of the desmosome associate with the desmosomal cadherin tails through a series of protein interactions, which serve to recruit intermediate filaments to sites of desmosome assembly. These desmosomal plaque components include plakoglobin and the plakophilins, members of the armadillo gene family. Linkage to the cytoskeleton is mediated by the intermediate filament binding protein, desmoplakin, which associates with both plakoglobin and plakophilins. Although desmosomes are critical for maintaining stable cell-cell adhesion, emerging evidence indicates that they are also dynamic structures that contribute to cellular processes beyond that of cell adhesion. This article outlines the structure and function of the major desmosomal proteins, and explores the contributions of this protein complex to tissue architecture and morphogenesis.

Making Connections: Desmoplakin as an Intermediate Filament-Binding Protein

Early work on desmosome morphology and biochemical composition laid the groundwork for the utilization of molecular biological approaches in the 1990s. Important breakthroughs included the cloning and sequencing of the desmoplakin gene, the identification of desmoplakin as an intermediate filament-binding protein and the realization that desmoplakin was part of a family of filamentbinding proteins now known as the plakin family of cytolinkers (reviewed in Kowalczyk et al., 1999; Leung et al., 2002). Collectively, these studies form an important foundation for our understanding of how intermediate filaments associate with the plasma membrane and other cytoskeletal networks. Green et al. (1988), at Northwestern University in Chicago, first isolated and analyzed the desmoplakin coding sequence, which allowed for structural analysis of the amino-acid sequence of the desmoplakin protein (Green et al., 1990). From this work, desmoplakin was predicted to harbor a central alpha-helical coiledcoil domain similar to that found in intermediate filaments. This region of desmoplakin would thus encode a rod-like domain connecting globular aminoand carboxy-terminal domains. In addition, the carboxyterminal domain harbored several interesting features, including the presence of a 38-amino-acid repeated homology domain that was also present in the 230-kDa bullous pemphigoid antigen (BPAG1). The periodicity of charged amino acids within these carboxy-terminal repeats was similar to that found in the 1B rod domain of intermediate filament proteins, leading to the prediction that the desmoplakin carboxyl terminal domain might interact with intermediate filaments. More recent studies have revealed several different means of desmoplakin–intermediate filament interactions, but this early study by Green and colleagues made two major predictions that were substantiated by further studies. First, that desmoplakin and BPAG1 were part of a gene family, and second, that the carboxy-terminal region of this family of proteins might confer intermediate filament binding activity. Using the desmoplakin cDNA and structural information from the 1990 study as a foundation, along with emerging information about BPAG1 and plectin as intermediate filamentassociated proteins, Stappenbeck and Green used molecular biological approaches to define desmoplakin domains functionally (Stappenbeck and Green, 1992; Stappenbeck et al., 1993). The most striking finding of their studies was that the desmoplakin carboxy-terminal domain aligned along intermediate filament networks when expressed transiently in cultured cells. These studies demonstrated that the desmoplakin carboxy-terminal domain was both necessary and sufficient for interacting with intermediate filaments, and, when overexpressed, could alter intermediate filament organization. Subsequent studies by the Fuchs laboratory demonstrated that the desmoplakin intermediate filament interactions were direct (Kouklis et al., 1994). These findings thereby complemented the structural studies of the desmoplakin gene and were followed by numerous additional biochemical, structural, and yeast genetic approaches (two-hybrid), confirming that the desmoplakin carboxyl terminus is an intermediate filament-binding domain (reviewed in Kowalczyk et al., 1999). Subsequent studies in the late 1990s as well as current studies continue to build upon the foundation initiated by the pioneering discoveries of desmoplakin and plakin family structure. The desmoplakin amino-terminal domain was found to target desmoplakin to desmosomes through associations with armadillo proteins such as plakoglobin and plakophilins. Collectively, these studies revealed the modular nature of desmoplakin and the mechanisms by which it couples filaments to sites of tight cell–cell adhesion. Current studies are focused on the regulation of these interactions and the dynamic properties of desmoplakin in the assembly of adhesive structures (Godsel et al., 2005).

Gene Expression in Temporal Lobe Epilepsy is Consistent with Increased Release of Glutamate by Astrocytes

Patients with temporal lobe epilepsy (TLE) often have a shrunken hippocampus that is known to be the location in which seizures originate. The role of the sclerotic hippocampus in the causation and maintenance of seizures in temporal lobe epilepsy (TLE) has remained incompletely understood despite extensive neuropathological investigations of this substrate. To gain new insights and develop new testable hypotheses on the role of sclerosis in the pathophysiology of TLE, the differential gene expression profile was studied. To this end, DNA microarray analysis was used to compare gene expression profiles in sclerotic and non-sclerotic hippocampi surgically removed from TLE patients. Sclerotic hippocampi had transcriptional signatures that were different from non-sclerotic hippocampi. The differentially expressed gene set in sclerotic hippocampi revealed changes in several molecular signaling pathways, which included the increased expression of genes associated with astrocyte structure (glial fibrillary acidic protein, ezrin-moesin-radixin, palladin), calcium regulation (S100 calcium binding protein beta, chemokine (C-X-C motif) receptor 4) and blood-brain barrier function (Aquaaporin 4, Chemokine (C-C- motif) ligand 2, Chemokine (C-C- motif) ligand 3, Plectin 1, intermediate filament binding protein 55kDa) and inflammatory responses. Immunohistochemical localization studies show that there is altered distribution of the gene-associated proteins in astrocytes from sclerotic foci compared with non-sclerotic foci. It is hypothesized that the astrocytes in sclerotic tissue have activated molecular pathways that could lead to enhanced release of glutamate by these cells. Such glutamate release may excite surrounding neurons and elicit seizure activity.

Novel Mutation in Desmoplakin Causes Arrhythmogenic Left Ventricular Cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a familial heart muscle disease characterized by structural, electrical, and pathological abnormalities of the right ventricle (RV). Several disease loci have been identified. Mutations in desmoplakin have recently been isolated in both autosomal-dominant and autosomal-recessive forms of ARVC. Primary left ventricular (LV) variants of the disease are increasingly recognized. We report on a large family with autosomal-dominant left-sided ARVC. The proband presented with sudden cardiac death and fibrofatty replacement of the LV myocardium. The family was evaluated. Diagnosis was based on modified diagnostic criteria for ARVC. Seven had inferior and/or lateral T-wave inversion on ECG, LV dilatation, and ventricular arrhythmia, predominantly extrasystoles of LV origin. Three had sustained ventricular tachycardia; 7 had late potentials on signal-averaged ECG. Cardiovascular magnetic resonance imaging in 4 patients revealed wall-motion abnormalities of the RV and patchy, late gadolinium enhancement in the LV, suggestive of fibrosis. Linkage confirmed cosegregation to the desmoplakin intragenic marker D6S2975. A heterozygous, single adenine insertion (2034insA) in the desmoplakin gene was identified in affected individuals only. A frameshift introducing a premature stop codon with truncation of the rod and carboxy terminus of desmoplakin was confirmed by Western blot analysis. We have described a new dominant mutation in desmoplakin that causes left-sided ARVC, with arrhythmias of LV origin, lateral T-wave inversion, and late gadolinium enhancement in the LV on magnetic resonance images. Truncation of the carboxy terminus of desmoplakin and consequent disruption of intermediate filament binding may account for the predominant LV phenotype.

Novel Alternative Splicings of BPAG1 (Bullous Pemphigoid Antigen 1) Including the Domain Structure Closely Related to MACF (Microtubule Actin Cross-linking Factor)

BPAG1 (bullous pemphigoid antigen 1) was originally identified as a 230-kDa hemidesmosomal protein and belongs to the plakin family, because it consists of a plakin domain, a coiled-coil rod domain and a COOH-terminal intermediate filament binding domain. To date, alternatively spliced products of BPAG1, BPAG1e, and BPAG1n are known. BPAG1e is expressed in epithelial tissues and localized to hemidesmosomes, on the other hand, BPAG1n is expressed in neural tissues and muscles and has an actin binding domain at the NH(2)-terminal of BPAG1e. BPAG1 is also known as a gene responsible for Dystonia musculorum (dt) neurodegeneration syndrome of the mouse. Another plakin family protein MACF (microtubule actin cross-linking factor) has also an actin binding domain and the plakin domain at the NH(2)-terminal. However, in contrast to its high homology with BPAG1 at the NH(2)-terminal, the COOH-terminal structure of MACF, including a microtubule binding domain, resembles dystrophin rather than plakins. Here, we investigated RNAs and proteins expressed from the BPAG1 locus and suggest novel alternative splicing variants, which include one consisting of the COOH-terminal domain structure homologous to MACF. The results indicate that BPAG1 has three kinds of cytoskeletal binding domains and seems to play an important role in linking the different types of cytoskeletons.

The BPAG1 locus

Bullous pemphigoid antigen 1 (BPAG1) is a member of the plakin family with cytoskeletal linker properties. Mutations in BPAG1 cause sensory neuron degeneration and skin fragility in mice. We have analyzed the BPAG1 locus in detail and found that it encodes different interaction domains that are combined in tissue-specific manners. These domains include an actin-binding domain (ABD), a plakin domain, a coiled coil (CC) rod domain, two different potential intermediate filament–binding domains (IFBDs), a spectrin repeat (SR)-containing rod domain, and a microtubule-binding domain (MTBD). There are at least three major forms of BPAG1: BPAG1-e (302 kD), BPAG1-a (615 kD), and BPAG1-b (834 kD). BPAG1-e has been described previously and consists of the plakin domain, the CC rod domain, and the first IFBD. It is the primary epidermal BPAG1 isoform, and its absence that is the likely cause of skin fragility in mutant mice. BPAG1-a is the major isoform in the nervous system and a homologue of the microtubule actin cross-linking factor, MACF. BPAG1-a is composed of the ABD, the plakin domain, the SR-containing rod domain, and the MTBD. The absence of BPAG1-a is the likely cause of sensory neurodegeneration in mutant mice. BPAG1-b is highly expressed in muscles, and has extra exons encoding a second IFBD between the plakin and SR-containing rod domains of BPAG1-a.

Binding of integrin alpha6beta4 to plectin prevents plectin association with F-actin but does not interfere with intermediate filament binding.

Hemidesmosomes are stable adhesion complexes in basal epithelial cells that provide a link between the intermediate filament network and the extracellular matrix. We have investigated the recruitment of plectin into hemidesmosomes by the alpha6beta4 integrin and have shown that the cytoplasmic domain of the beta4 subunit associates with an NH(2)-terminal fragment of plectin that contains the actin-binding domain (ABD). When expressed in immortalized plectin-deficient keratinocytes from human patients with epidermol- ysis bullosa (EB) simplex with muscular dystrophy (MD-EBS), this fragment is colocalized with alpha6beta4 in basal hemidesmosome-like clusters or associated with F-actin in stress fibers or focal contacts. We used a yeast two-hybrid binding assay in combination with an in vitro dot blot overlay assay to demonstrate that beta4 interacts directly with plectin, and identified a major plectin-binding site on the second fibronectin type III repeat of the beta4 cytoplasmic domain. Mapping of the beta4 and actin-binding sites on plectin showed that the binding sites overlap and are both located in the plectin ABD. Using an in vitro competition assay, we could show that beta4 can compete out the plectin ABD fragment from its association with F-actin. The ability of beta4 to prevent binding of F-actin to plectin explains why F-actin has never been found in association with hemidesmosomes, and provides a molecular mechanism for a switch in plectin localization from actin filaments to basal intermediate filament-anchoring hemidesmosomes when beta4 is expressed. Finally, by mapping of the COOH-terminally located binding site for several different intermediate filament proteins on plectin using yeast two-hybrid assays and cell transfection experiments with MD-EBS keratinocytes, we confirm that plectin interacts with different cytoskeletal networks.