Extracellular Domain Of Protein Research Articles

Insights into desmosome biology from inherited human skin disease and cardiocutaneous syndromes.

The importance of desmosomes in tissue homeostasis is highlighted by natural and engineered mutations in desmosomal genes, which compromise the skin or heart and in some instances both. Desmosomal gene mutations account for 45-50% of cases of arrhythmogenic right ventricular cardiomyopathy, and are mutated in an array of other disorders such as striate palmoplantar keratoderma, hypotrichosis with or without skin vesicles and lethal acantholytic epidermolysis bullosa. Recently, we reported loss-of-function mutations in the human ADAM17 gene, encoding for the 'sheddase' ADAM17, a transmembrane protein which cleaves extracellular domains of substrate proteins including TNF-α, growth factors and desmoglein (DSG) 2. Patients present with cardiomyopathy and an inflammatory skin and bowel syndrome with defective DSG processing. In contrast, the dominantly inherited tylosis with oesophageal cancer appears to result from gain-of-function in ADAM17 due to increased processing via iRHOM2. This review discusses the heterogeneity of mutations in desmosomes and their regulatory proteins.

Phenotypic Diversity of Breast Cancer-Related Mutations in Metalloproteinase-Disintegrin ADAM12

Six different somatic missense mutations in the human ADAM12 gene have been identified so far in breast cancer. Five of these mutations involve highly conserved residues in the extracellular domain of the transmembrane ADAM12-L protein. Two of these extracellular mutations, D301H and G479E, have been previously characterized in the context of mouse ADAM12. Three other mutations, T596A, R612Q, and G668A, have been reported more recently, and their effects on ADAM12-L protein structure/function are not known. Here, we show that ADAM12-L bearing the G668A mutation is largely retained in the endoplasmic reticulum in its nascent, full-length form, with an intact N-terminal pro-domain. The T596A and R612Q mutants are efficiently trafficked to the cell surface and proteolytically processed to remove their pro-domains. However, the T596A mutant shows decreased catalytic activity at the cell surface, while the R612Q mutant is fully active and comparable to the wild-type ADAM12-L. The D301H and G479E mutants, consistent with the corresponding D299H and G477E mutants of mouse ADAM12 described earlier, are not proteolytically processed and do not exhibit catalytic activity at the cell surface. Among all six breast cancer-associated mutations in ADAM12-L, mutations that preserve the activity - R612Q and L792F - occur in triple-negative breast cancers, while loss-of-function mutations - D301H, G479E, T596A, and G668A - are found in non-triple negative cancers. This apparent association between the catalytic activity of the mutants and the type of breast cancer supports a previously postulated role of an active ADAM12-L in the triple negative breast cancer disease.

WFS1 and non-syndromic low-frequency sensorineural hearing loss: A novel mutation in a Portuguese case

Low-frequency sensorineural hearing loss (LFSNHL) is an unusual type of HL in which frequencies at 2000Hz and below are predominantly affected. Most of the families with LFSNHL carry missense mutations in WFS1 gene, coding for wolframin.A Portuguese patient aged 49, reporting HL since her third decade of life, and also referring tinnitus, was shown to display bilateral moderate LFSNHL after audiological evaluation.Molecular analysis led to the identification of a novel mutation, c.511G>A (p.Asp171Asn), found in heterozygosity in the exon 5 of the WFS1 gene, and changing the aspartic acid at position 171 to an asparagine, in the extracellular N-terminus domain of the wolframin protein. This novel mutation wasn't present either in 200 control chromosomes analyzed or in the hearing proband's half-brother, and it had not been reported in 1000 Genomes, Exome Variant Server, HGMD or dbSNP databases. No mutations were found in GJB2 and GJB6 genes.Multi-alignment of 27 wolframin sequences from mammalian species, against the human wolframin sequence in ConSurf, indicated a conservation score corresponding to 7 in a 1–9 color scale where 9 is conserved and 1 is variable. In addition, the mutation p.Asp171Asn was predicted to be damaging and possibly damaging by SIFT and Polyphen-2, respectively.The auditory phenotype of this patient could thus be due to the novel mutation p.Asp171Asn. Further functional characterization might enable to elucidate in which way the change in the residue 171, as other changes introduced by LFSNHL-associated mutations previously described, leads to this type of HL.

A proteomic approach for the elucidation of the specificity of ectodomain shedding

Ectodomain shedding (shedding) is a posttranslational modification mechanism, which liberates extracellular domains of membrane proteins through juxtamembrane processing. Because shedding alters cell characteristics in a rapid and irreversible manner, it must be strictly regulated. However, the regulatory mechanisms of shedding in response to environmental changes remain obscure. To evaluate the regulatory mechanisms of endogenous shedding, we previously developed a proteomic screening system to identify shedding targets. This system revealed a comprehensive picture of membrane proteins shed under defined conditions. In this study, we have improved the screening system to compare the shedding patterns in a mouse macrophage cell line treated with two different shedding inducers, lipopolysaccharide (LPS) and 12-O-tetradecanoylphorbol 13-acetate (TPA). We show here that LPS simultaneously activates the shedding of multiple membrane proteins. We further show that TPA specifically activates the shedding of αM/β2 integrin (Mac-1), which was not shed upon LPS-stimulation of macrophages. These results clearly demonstrate that the regulation of endogenous membrane protein shedding is both stimulus- and substrate-specific. The shedding targets reported to date play pivotal roles in a variety of biological phenomena, including the immunological response, cell growth, cell adhesion and cell movement. In addition, several disease-related membrane proteins are shedding targets. Thus, understanding the regulation of shedding is important for the elucidation of pathogenesis and the development of therapeutic strategies. We submit that a comprehensive characterization of endogenous shedding is indispensable for understanding the regulatory mechanisms of shedding, and thus have developed a proteomic screening system to identify shedding targets. In this study, using our screening system, we demonstrate that different extracellular stimuli activate different types of shedding, even in a single cell. Our results prove that this proteomic approach is quite effective for the elucidation of the regulatory mechanisms of shedding.

Microneedle delivery of an M2e-TLR5 ligand fusion protein to skin confers broadly cross-protective influenza immunity

Influenza vaccines with broad cross-protection are urgently needed to prevent an emerging influenza pandemic. A fusion protein of the Toll-like receptor (TLR) 5-agonist domains from flagellin and multiple repeats of the conserved extracellular domain of the influenza matrix protein 2 (M2e) was constructed, purified and evaluated as such a vaccine. A painless vaccination method suitable for possible self-administration using coated microneedle arrays was investigated for skin-targeted delivery of the fusion protein in a mouse model. The results demonstrate that microneedle immunization induced strong humoral as well as mucosal antibody responses and conferred complete protection against homo- and heterosubtypic lethal virus challenges. Protective efficacy with microneedles was found to be significantly better than that seen with conventional intramuscular injection, and comparable to that observed with intranasal immunization. Because of its advantages for administration, safety and storage, microneedle delivery of M2e-flagellin fusion protein is a promising approach for an easy-to-administer universal influenza vaccine.

Upregulation of the Creatine Transporter Slc6A8 by Klotho

Background/Aims: The transmembrane Klotho protein contributes to inhibition of 1,25(OH)₂D₃ formation. The extracellular domain of Klotho protein could function as an enzyme with e.g. β-glucuronidase activity, be cleaved off and be released into blood and cerebrospinal fluid. Klotho regulates several cellular transporters. Klotho protein deficiency accelerates the appearance of age related disorders including neurodegeneration and muscle wasting and eventually leads to premature death. The main site of Klotho protein expression is the kidney. Klotho protein is also appreciably expressed in other tissues including chorioid plexus. The present study explored the effect of Klotho protein on the creatine transporter CreaT (Slc6A8), which participates in the maintenance of neuronal function and survival. Methods: To this end cRNA encoding Slc6A8 was injected into Xenopus oocytes with and without additional injection of cRNA encoding Klotho protein. Creatine transporter CreaT (Slc6A8) activity was estimated from creatine induced current determined by two-electrode voltage-clamp. Results: Coexpression of Klotho protein significantly increased creatine-induced current in Slc6A8 expressing Xenopus oocytes. Coexpression of Klotho protein delayed the decline of creatine induced current following inhibition of carrier insertion into the cell membrane by brefeldin A (5 µM). The increase of creatine induced current by coexpression of Klotho protein in Slc6A8 expressing Xenopus oocytes was reversed by β-glucuronidase inhibitor (DSAL). Similarly, treatment of Slc6A8 expressing Xenopus oocytes with recombinant human alpha Klotho protein significantly increased creatine induced current. Conclusion: Klotho protein up-regulates the activity of creatine transporter CreaT (Slc6A8) by stabilizing the carrier protein in the cell membrane, an effect requiring β-glucuronidase activity of Klotho protein.

Notch-ligand binding assays in Drosophila cells.

Activation of the Drosophila transmembrane receptor protein Notch is induced by association with its transmembrane ligands, Delta and Serrate. The ability to assay binding between Notch and its ligands has been essential for characterizing the influence of posttranslational modifications, such as glycosylation, as well as for characterizing structural motifs involved in receptor-ligand interactions. We describe here a simple, widely used method for assaying receptor-ligand binding. This method involves expression of soluble forms of either Notch or its ligands, comprising the extracellular domains fused to an easily assayed tag, the enzyme alkaline phosphatase. These soluble proteins are then incubated with their binding partners, either as transmembrane proteins expressed on the surface of cultured cells or as extracellular protein domains attached to agarose beads. After washing, the amount of bound protein can be readily assayed by measuring alkaline phosphatase activity.

Identification of Streptococcal M-Protein Cardiopathogenic Epitopes in Experimental Autoimmune Valvulitis

The M protein of rheumatogenic group A streptococci induces carditis and valvulitis in Lewis rats and may play a role in pathogenesis of rheumatic heart disease. To identify the epitopes of M5 protein that produce valvulitis, synthetic peptides spanning A, B, and C repeat regions contained within the extracellular domain of the streptococcal M5 protein were investigated. A repeat region peptides NT4, NT5/6, and NT7 induced valvulitis similar to the intact pepsin fragment of M5 protein. T cell lines from rats with valvulitis recognized M5 peptides NT5/6 and NT6. Passive transfer of an NT5/6-specific T cell line into naïve rats produced valvulitis characterized by infiltration of CD4+ cells and upregulation of VCAM-1, while an NT6-specific T cell line did not target the valve. Our new data suggests that M protein-specific T cells may be important mediators of valvulitis in the Lewis rat model of rheumatic carditis.

Abstract P2-08-05: Functional characterization of three novel breast cancer-related somatic mutations in metalloproteinase-disintegrin ADAM12

Abstract Six different somatic missense mutations in the human ADAM12 gene have been identified so far in breast cancer. Five of these mutations involve highly conserved residues in the extracellular domain of the transmembrane ADAM12-L protein. Two of these extracellular mutations, D301H and G479E, have been previously characterized in the context of mouse ADAM12. Three other mutations, T596A, R612Q, and G668A, have been reported more recently, and their effects on ADAM12-L protein structure/function are not known. Here, we show that ADAM12-L bearing the G668A mutation is largely retained in the endoplasmic reticulum in its nascent, full-length form, with an intact N-terminal pro-domain. The T596A and R612Q mutants are efficiently trafficked to the cell surface and proteolytically processed to remove their pro-domains. However, the T596A mutant shows decreased catalytic activity at the cell surface, while the R612Q mutant is fully active and comparable to the wild-type ADAM12-L. The D301H and G479E mutants, consistent with the effect of the corresponding D229H and G477E mutations in mouse ADAM12 protein described earlier, are not proteolytically processed and do not exhibit catalytic activity at the cell surface. Among all six breast cancer-associated mutations in ADAM12-L, mutations that preserve the activity - R612Q and L792F - occur in triple-negative breast cancers, while loss-of-function mutations - D301H, G479E, T596A, and G668A - are found in non-triple negative cancers. This apparent association between the catalytic activity of the mutants and the type of breast cancer supports a previously postulated role of ADAM12-L in the triple negative breast cancer disease. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-08-05.

Inhibitory Mechanism of an Allosteric Antibody Targeting the Glucagon Receptor

Elevated glucagon levels and increased hepatic glucagon receptor (GCGR) signaling contribute to hyperglycemia in type 2 diabetes. We have identified a monoclonal antibody that inhibits GCGR, a class B G-protein coupled receptor (GPCR), through a unique allosteric mechanism. Receptor inhibition is mediated by the binding of this antibody to two distinct sites that lie outside of the glucagon binding cleft. One site consists of a patch of residues that are surface-exposed on the face of the extracellular domain (ECD) opposite the ligand-binding cleft, whereas the second binding site consists of residues in the αA helix of the ECD. A docking model suggests that the antibody does not occlude the ligand-binding cleft. We solved the crystal structure of GCGR ECD containing a naturally occurring G40S mutation and found a shift in the register of the αA helix that prevents antibody binding. We also found that alterations in the αA helix impact the normal function of GCGR. We present a model for the allosteric inhibition of GCGR by a monoclonal antibody that may form the basis for the development of allosteric modulators for the treatment of diabetes and other class B GPCR-related diseases.

Expression and purification of the extracellular domain of the human follicle‐stimulating hormone receptor using Escherichia coli

Although much is known about the structure and biological functions of follicle-stimulating hormone (FSH) receptor (FSHR), the interaction of FSHR and FSH has been challenging to characterize due to the limited quantity of active FSHR protein produced by simple methods. The goal of this study was to express and purify the extracellular domain (ECD) of human FSHR (hFSHR). Total RNA was isolated from normal human ovary tissue. cDNA for hFSHR ECD were amplified and subsequently ligated into the pET32a(+) vector. The plasmid vector construct was confirmed by polymerase chain reaction and sequencing. Expression in Escherichia coli Rosetta (DE3) pLysS strain was induced by isopropyl-thio-β-D-thiogalactoside, and the recombinant products were purified by immuno-affinity chromatography using an Ni-NTA and High-Q column. The recombinant protein was confirmed by western blotting. Following induction, E. coli expressed a recombinant protein of approximately 65 kDa in size, whereas the non-induced E. coli did not express the recombinant protein. The recombinant fragments purified using a High-Q column demonstrated a single band and an abundant yield. The recombinant protein was soluble and specifically recognized by an antibody for hFSHR. Additionally, four mutation sites were detected that resulted in amino acid shifts at position 112 Asn/Thr, 197 Glu/Ala, 198 Leu/Val and 307 Ala/Thr. The recombinant hFSHR ECD protein was expressed and purified. This method could be easily scaled for increased production and may facilitate additional applications utilizing FSHR in assisted reproductive technology, a contraceptive FSH vaccine and FSHR-targeted therapeutic agents used to treat ovarian cancer.

Influenza Virus M2e with Additional Cysteine Residues Shows Enhanced Immunogenicity and Protection Against Lethal Virus Challenge

The amino acid sequence of the extracellular domain of matrix protein 2 (M2e) is conserved among all subtypes of influenza A viruses. Therefore, the M2e peptide can be considered as a target antigen for the development of a universal influenza vaccine. We evaluated the effects of adding cysteine residues to a peptide of amino acids 2-24 of M2e. Mice immunized with some of these peptides containing one, two, three, four, or five extra cysteines displayed enhanced antibody titers to M2e. In addition, immunization with a peptide containing three extra cysteines, along with an aluminum adjuvant, protected mice more effectively against a lethal influenza virus challenge than the original M2e peptide. These results indicated that an M2e peptide containing additional cysteine residues could be a universal influenza vaccine candidate even without the addition of strong adjuvants.

CD8‐β ADP‐ribosylation affects CD8+T‐cell function

The CD8αβ coreceptor is crucial for effective peptide: MHC-I recognition by the TCR of CD8(+) T cells. Adenosine diphosphate ribosyl transferase 2.2 (ART2.2) utilizes extracellular NAD(+) to transfer ADP-ribose to arginine residues of extracellular domains of surface proteins. Here, we show that in the presence of extracellular NAD(+) , ART2.2 caused ADP-ribosylation of CD8-β on murine CD8(+) T cells in vitro and in vivo. Treatment with NAD(+) prevented binding of anti-CD8-β mAb YTS156.7.7 but not of mAb H35-17.2, indicating that NAD(+) caused modification of certain epitopes and not a general loss of CD8-β. Loss of antibody binding was strictly dependent on ART2.2, because it was not observed on ART2-deficient T cells or in the presence of inhibitory anti-ART2.2 single-domain antibodies. ADP-ribosylation of CD8-β occurred during cell isolation, particularly when cells were isolated from CD38-deficient mice. Incubation of ART2-expressing, but not of ART2-deficient, OVA-specific CD8(+) T cells with NAD(+) interfered with binding of OVA257-264 :MHC-I tetramers. In line with this result, treatment of WT mice with NAD(+) resulted in reduced CD8(+) T-cell mediated cytotoxicity in vivo. We propose that ADP-ribosylation of CD8-β can regulate the coreceptor function of CD8 in the presence of elevated levels of extracellular NAD(+) .

Administration of IL-1 Trap prolongs survival of transplanted pancreatic islets to type 1 diabetic NOD mice

We previously reported that IL-1 Trap (a hybrid molecule consisting of the extracellular domain of IL-1 receptor accessory protein and IL-1 receptor type 1 arranged inline and fused to the Fc-portion of IgG1) can protect rat pancreatic islets in vitro against noxious effects induced by IL-1β. In this study we tested the effect of administration of a murine IL-1 Trap on the recurrence of disease (ROD) model in non-obese diabetic (NOD) mice. Spontaneously diabetic female NOD mice received implantation of a curative number (600) of syngeneic pancreatic islets beneath their left kidney capsule from young healthy NOD mouse donors. Once a day, the mice were injected subcutaneously with IL-1 Trap (30mg/kg bodyweight), or an equimolar dose Fc-control protein (8.4mg/kg bodyweight) or saline. The treatments were maintained until ROD (i.e. a blood glucose value ⩾11.1mM for 2 consecutive days) or until 5days after transplantation. 3 out of 11 mice treated with IL-1 Trap showed a significantly increased graft survival compared to all other mice, and analysis of relative cytokine mRNA levels in isolated spleen cells showed elevated IL-4 mRNA levels, but no differences in FoxP3 or iNOS staining of grafts, from mice treated with IL-1 Trap, at both endpoints, compared to both control groups. Administration of IL-1 Trap counteracts islet cell destruction in the NOD mouse model of type 1 diabetes. In part this could be due to a shift towards Th2 cytokine production seen in IL-1 Trap treated animals.

Linear synthetic peptides induce anti-HER-2 antibody responses with Trastuzumab- (Herceptin) like biological activity (P4308)

Abstract The monoclonal antibody Trastuzumab, which recognizes a conformation-dependent epitope on the extracellular domain of HER-2, has revolutionized the treatment of HER-2pos breast cancers. High costs and short half-life of Trastuzumab spurs the search for ways to induce therapeutic anti-HER-2 antibodies in vivo. Linear T cell epitopes have already been discovered for HER-2, and these have shown promise in vaccine studies where strong cell-mediated responses have been generated. Identifying conformation-independent B cell epitopes would allow the construction of linear T-B vaccine constructs that would generate both cellular and humoral anti-HER-2 responses with potential anti-cancer activity. We produced a synthetic peptide library consisting of 62 overlapping 20-mers spanning the HER-2 extracellular domain. These were used to vaccinate Balb/c mice. Resulting antisera were screened for reactivity against peptide and recombinant extracellular domain protein via ELISA, and whole native protein by immuno-fluorescent microscopy and FACS analysis. Sera reactive by all these criteria were compared with Trastuzumab for ability to disrupt HER-2 and HER-2/HER-3 dimer signaling with and without heregulin. We identified linear peptides that induced antibodies capable of blocking AKT as well as HER-3 phosphorylation in breast cancer lines similar to Trastuzumab. This suggests that a linear epitope/vaccination approach may be feasible for generating effective anti-HER-2 antibody responses.

Siglec‐h on activated microglia for recognition and engulfment of glioma cells

Sialic-acid-binding immunoglobulin-like lectin-h (Siglec-h) is a recently identified mouse-specific CD33-related Siglec that signals via DAP12/TYROBP. Expression of Siglec-h has been observed on plasmacytoid dendritic cells and microglia, but the ligand and the function of Siglec-h remained elusive. Here, we demonstrate gene transcription and protein expression of Siglec-h by mouse microglia after interferon-γ treatment or polarization into a M1-subtype. Microglial Siglec-h acted as phagocytosis receptor since targeting of microsphere beads to Siglec-h triggered their uptake into the microglia. The extracellular domain of Siglec-h protein bound to mouse glioma lines, but not to astrocytes or other normal mouse cells. Microglial cells stimulated to express Siglec-h engulfed intact glioma cells without prior induction of apoptosis and slightly reduced glioma cell number in culture. Phagocytosis of glioma cells by activated microglia was dependent on Siglec-h and its adapter molecule DAP12. Thus, data show that M1-polarized microglial cells can engulf glioma cells via a DAP12-mediated Siglec-h dependent mechanism.

Secondary B Cell Receptor Diversification Is Necessary for T Cell Mediated Neuro-Inflammation during Experimental Autoimmune Encephalomyelitis

BackgroundClinical studies of B cell depletion in Multiple Sclerosis (MS) have revealed that B Lymphocytes are involved in the neuro-inflammatory process, yet it remains unclear how B cells can exert pro- and anti-inflammatory functions during MS. Experimental Autoimmune Encephalomyelitis (EAE) is an animal model of MS whereby myelin-specific T cells become activated and subsequently migrate to the Central Nervous System (CNS) where they perform pro-inflammatory functions such as cytokine secretion. Typically EAE is induced by immunization of mice of a susceptible genetic background with peptide antigen emulsified in Complete Freund's Adjuvant. However, novel roles for B-lymphocytes in EAE may also be explored by immunization with full-length myelin oligodendrocyte glycoprotein (MOG) that contains the B cell conformational epitope. Here we show that full length MOG immunization promotes a chronic disease in mice that depends on antigen-driven secondary diversification of the B cell receptor.MethodsActivation-Induced Deaminase (AID) is an enzyme that is essential for antigen-driven secondary diversification of the B cell receptor. We immunized AID−/− mice with the extracellular domain (amino acids 1–120) of recombinant human MOG protein (rhMOG) and examined the incidence and severity of disease in AID−/− versus wild type mice. Corresponding with these clinical measurements, we also evaluated parameters of T cell activation in the periphery and the CNS as well as the generation of anti-MOG antibodies (Ab).ConclusionsAID−/− mice exhibit reduced severity and incidence of EAE. This suggests that the secondary diversification of the B cell receptor is required for B cells to exert their full encephalogenic potential during rhMOG-induced EAE, and possibly also during MS.

Abstract 363: Activin receptor-like kinase 1 (Alk1) ligands modulate endothelial cell function in vitro and in vivo.

Abstract The TGF-ß type-1 receptor activin receptor-like kinase 1 (Alk1) is predominantly expressed on endothelial cells, regulates vascular morphogenesis, and has been implicated in pathological angiogenesis. Alk1 expression is increased in the vasculature of a broad range of tumours and agents that disrupt of Alk1 signalling in tumour endothelial cells may be potential therapeutics for the treatment of a variety of solid tumours. Alk1 has been described as a receptor for the bone morphogenic proteins, BMP9 and BMP10. In order to study the effects of Alk1 signalling in endothelial cells in vitro and in the vasculature in vivo we used a soluble antagonist Alk1-Fc protein consisting of the extracellular domain of the Alk1 protein fused to the Fc domain of IgG1. In vitro, Alk1-Fc inhibited BMP9-dependent Alk1 signalling via phosphorylation and nuclear translocation of SMAD1/5 in endothelial cells and modulated endothelial cell functions such as an ability to form tube- like structures. In mice, physiological levels of BMP9 could be detected in serum by ELISA, consistent with detectable levels of phosphorylated SMAD1/5 in the lung, a site of high Alk1 vascular expression. In mice, the administration of Alk1-Fc effectively blocked SMAD1/5 phosphorylation in the lung, suggesting that in this tissue Alk1 ligands are the predominant driver of lung SMAD1/5 activation and that signalling is efficiently antagonised by Alk1-Fc. Despite suppression of Alk1 signalling in the lung and expression of Alk1 on the vasculature associated with tumours, Alk1-Fc did not have profound effects on tumour size or microvessel density in mice bearing tumours, however administration of Alk1-Fc in neonatal mice did cause defects in angiogenesis of the retinal vasculature demonstrating that in some contexts Alk1 ligands play an important role in regulating the angiogenic process. Citation Format: Gareth C. Davies, James A. Harper, Richard C. Sainson, Lee Brown, Grace Opoku-Ansah, Alison Smith, Matthew McCourt, James W. Legg. Activin receptor-like kinase 1 (Alk1) ligands modulate endothelial cell function in vitro and in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 363. doi:10.1158/1538-7445.AM2013-363

Protein chemical shift assignments of the unbound and RNA-bound forms of the alternative splicing factor SUP-12 from C. elegans

The splicing factor SUP-12 from Caenorhabditis elegans binds to regulatory RNA elements in pre-mRNA in order to generate tissue-specific alternative splicing for genes such as the fibroblast growth factor receptor egl-15. In nematode muscle cells, SUP-12 promotes the use of a mutually exclusive exon to impart variant binding specificity to the EGL-15 extracellular protein domain. Here we report the side chain and backbone (1)H, (13)C and (15)N chemical shift assignments for the bacterially expressed RNA recognition motif domain from SUP-12, both in isolation as well as bound to a short RNA derived from the intron sequence between exon 4 and exon 5B of egl-15. Comparison of protein chemical shift values for both the backbone and side chain nuclei, coupled with secondary chemical shift analysis, reveal initial details of the RNA recognition.

Disease-Causing Mutation in Extracellular and Intracellular Domain of FGFR1 Protein: Computational Approach

In-depth computationally based structural analysis of human fibroblast growth factor type 1 (FGFR1) protein carrying disease-causing mutation was performed in this study. Gain or loss of function due to missense mutations in FGFR1 is responsible for a variety of disorders including Kallmann syndrome, Apert syndrome, Pfeiffer syndrome, Crouzon syndrome, etc. The mutant model of the human FGFR1 protein was subjected to various in silico analysis, and most deleterious SNPs were screened out. Furthermore, docking and long molecular dynamics simulations were carried out with an intention of studying the possible impact of these mutations on the protein structure and hence its function. Analysis of various structural properties-especially of those of the functionally important regions: the extracellular immunoglobulin domain and intracellular Tyrosine kinase domain-gave some insights into the possible structural characteristics of the disease mutant and the wild-type forms of the protein. In a nutshell, compared to the wild-type protein, the mutant structures V273M and S685F are associated with significant changes, and the functionally important regions seem to adopt such structures that are not conducive for the wild-type-like functionality.