Podocyte apoptosis in diabetic nephropathy by BASP1 activation of the p53 pathway via WT1.

AIMS: Diabetic nephropathy (DN) is a leading cause of end-stage renal disease. BASP1 (brain acid-soluble protein) is up-regulated in podocyte-specific protein phosphatase 2A knockout mice (Pod-PP2A-KO) that develop kidney dysfunction. Here, we explore the role of BASP1 for podocytes in DN. METHODS: BASP1 was assessed in kidneys from DN patients and DN mouse models, podocyte specific BASP1 knockout mice (Pod-BASP1-KO mice) were generated and studied in vivo. Furthermore, podocyte injury and apoptosis were measured after BASP1 knockdown and overexpression in a mouse podocyte cell line (MPC5). Potential signalling pathways involved in podocyte apoptosis were detected. RESULTS: BASP1 expression was up-regulated in DN patients compared to normal controls. BASP1 specific deletion in podocytes protected against podocyte injury by reducing the loss of expression of slit diaphragm molecules and foot process effacement in the DN model. BASP1 promoted actin cytoskeleton rearrangements and apoptosis in the MPC5 podocyte line. Molecules involved in the p53 pathway were down-regulated in BASP1 knockdown podocytes treated with high glucose compared to controls. BASP1 promoted podocyte apoptosis and P53 pathway activation through co-repression with Wilms' tumour 1 transcription factor (WT1). CONCLUSION: BASP1 activates the p53 pathway through modulation of WT1 to induce podocyte apoptosis in diabetic nephropathy.

Oligopeptides for Immunotherapy Approaches in Ovarian Cancer Treatment.

BACKGROUND: Anti-ovarian cancer vaccines based on minimal immune determinants uniquely expressed in ovarian cancer biomarkers appear to promise a high level of sensitivity and specificity for ovarian cancer immunodiagnostics, immunoprevention, and immunotherapy. METHODS: Using the Pir Peptide Match program, three ovarian cancer biomarkers - namely, sperm surface protein Sp17, WAP four-disulfide core domain protein 2, and müllerian-inhibiting substance - were searched for unique peptide segments not shared with other human proteins. Then, the unique peptide segments were assembled to define oligopeptides potentially usable as synthetic ovarian cancer antigens. RESULTS AND CONCLUSION: This study describes a methodology for constructing ovarian cancer biomarkerderived oligopeptide constructs that might induce powerful, specific, and non-crossreactive immune responses against ovarian cancer.

Biophysical Characterization of SG2NA Variants and their Interaction with DJ-1 and Calmodulin in vitro.

SG2NA was first discovered as nuclear autoantigen in lung and bladder cancer patient. It was named SG2NA as its expression increases during S to G2 phase of cell cycle. SG2NA/Striatin3 was classified as a member of Striatin family along with Straitin and Zinedin due to its structural and functional relatedness. At the molecular level, SG2NA is characterized by the presence of multiple protein-protein interaction domains viz., a caveolin binding motif, a coiled coil structure, Ca2+-calmodulin binding domain and a large WD-40 repeat domain in the same order from amino to the carboxyl termini. Analysis of secondary structures of 87 and 78 kDa SG2NA isoforms showed characteristic combinations of α-helix, ß-structure, ß-turns and random coil; suggesting of effective refolding after denaturation. This study for the first time establishes the structural differences between the two prevalent isoforms of SG2NA. Recently we observed that DJ-1 interacts with variants of SG2NA both in vitro and in vivo. The SG2NA isoforms purified from inclusion bodies showed the different secondary structure conformations, stability and interaction pattern for their interacting partners (DJ-1 and calmodulin) which imparts functional diversity of SG2NA. The SG2NA isoforms showed significant differential binding affinity to DJ-1 and Calmodulin.

Identification of calmodulin binding proteins in the entomopathogenic fungus Beauveria bassiana.

Calmodulin (CaM) is a primary Ca2+ receptor and plays a pivotal role in a variety of cellular responses in eukaryotes. Even though a large number of CaM-binding proteins are well known in yeast, plants, and animals, little is known regarding CaM-targeted proteins in filamentous fungi. To identify CaM-binding proteins in filamentous fungi, we used a proteomics method coupled with co-immunoprecipitation (CoIP) and MALDI-TOF/TOF mass spectrometry (MS) in Beauveria bassiana. Through this method, we identified ten CaM-binding proteins in B. bassiana. One of the CaM-targeted proteins was the heat shock protein 70 (BbHSP70) in B. bassiana. Our biochemical study showed that ATP inhibits the molecular interaction between BbHSP70 and CaM, suggesting a regulatory mechanism between CaM and ATP for regulating BbHSP70.

Cancer-Specific Retargeting of BAF Complexes by a Prion-like Domain.

Alterations in transcriptional regulators can orchestrate oncogenic gene expression programs in cancer. Here, we show that the BRG1/BRM-associated factor (BAF) chromatin remodeling complex, which is mutated in over 20% of human tumors, interacts with EWSR1, a member of a family of proteins with prion-like domains (PrLD) that are frequent partners in oncogenic fusions with transcription factors. In Ewing sarcoma, we find that the BAF complex is recruited by the EWS-FLI1 fusion protein to tumor-specific enhancers and contributes to target gene activation. This process is a neomorphic property of EWS-FLI1 compared to wild-type FLI1 and depends on tyrosine residues that are necessary for phase transitions of the EWSR1 prion-like domain. Furthermore, fusion of short fragments of EWSR1 to FLI1 is sufficient to recapitulate BAF complex retargeting and EWS-FLI1 activities. Our studies thus demonstrate that the physical properties of prion-like domains can retarget critical chromatin regulatory complexes to establish and maintain oncogenic gene expression programs.

Adducin in tumorigenesis and metastasis.

Adducin is a membrane-skeletal protein localized at spectrin-actin junctions, involving in the formation of the network of cytoskeleton, cellular signal transduction, ionic transportation, cell motility and cell proliferation. While previous researches focused mainly on the relationship between adducin and hypertension, there are few studies focusing on the role of adducin in tumor. Previous studies showed that adducin played a role in the evolution and progression of neoplasm. This review makes a brief summary on the structure, function and mechanism of adducin and how adducin functions in tumorigenesis and metastasis.

RNA-binding proteins with prion-like domains in health and disease.

Approximately 70 human RNA-binding proteins (RBPs) contain a prion-like domain (PrLD). PrLDs are low-complexity domains that possess a similar amino acid composition to prion domains in yeast, which enable several proteins, including Sup35 and Rnq1, to form infectious conformers, termed prions. In humans, PrLDs contribute to RBP function and enable RBPs to undergo liquid-liquid phase transitions that underlie the biogenesis of various membraneless organelles. However, this activity appears to render RBPs prone to misfolding and aggregation connected to neurodegenerative disease. Indeed, numerous RBPs with PrLDs, including TDP-43 (transactivation response element DNA-binding protein 43), FUS (fused in sarcoma), TAF15 (TATA-binding protein-associated factor 15), EWSR1 (Ewing sarcoma breakpoint region 1), and heterogeneous nuclear ribonucleoproteins A1 and A2 (hnRNPA1 and hnRNPA2), have now been connected via pathology and genetics to the etiology of several neurodegenerative diseases, including amyotrophic lateral sclerosis, frontotemporal dementia, and multisystem proteinopathy. Here, we review the physiological and pathological roles of the most prominent RBPs with PrLDs. We also highlight the potential of protein disaggregases, including Hsp104, as a therapeutic strategy to combat the aberrant phase transitions of RBPs with PrLDs that likely underpin neurodegeneration.

Helical model of smooth muscle myosin filament and the ribbons made of caldesmon: history revisited.

In early studies on smooth muscle, I described a crude myosin fraction (CMF) in which self-assembly of myosin filaments was observed. For the first time, the 14-nm periodicity stemming from regular arrangement of myosin heads on the filament surface was observed (Sobieszek in J Mol Biol 70:741-744, 1972). In this fraction, we also observed formation of long ribbon-shaped aggregates exhibiting a 5.6-nm periodicity, characteristic of tropomyosin (TM) paracrystals (Sobieszek and Small in Phil Trans R Soc Lond B 265:203-212, 1973). We therefore concluded that these ribbons were made of TM and they might be related to the myosin ribbons observed in electron micrographs (EM) of intact smooth muscle (Lowy and Small in Nature 227:46-51, 1970; Small and Squire in Mol Biol 67:117-149, 1972). Subsequently, Small (J Cell Sci 24:327-349, 1977) concluded that the ribbons observed in the EM sections were an artifact, but their observation in the CMF was not addressed. I have now revisited two aspects of the above studies. Firstly, based on my new multi-angle laser-scattering data and considering the length and stability of the building unit for the filament, a myosin trimer fit better to the previously proposed helical structure. Secondly, after two decades of systematic examinations of protein compositions in multiple smooth muscle extracts and isolated filaments, I concluded that the ribbons were made of caldesmon and not TM. Thirdly, actin-activated ATPase activity measurements indicated that modulation of this activity (by CaD and TM) was synergistic, cooperative and depended on myosin to actin ratio.

Getting Access to Low-Complexity Domain Modifications.

Low-complexity (LC) domains regulate the aggregation and phase transition of proteins in a modification-dependent manner. The study of LC domain modifications has now become feasible, as shown by genetic variants of the carboxy-terminal domain (CTD) of RNA Polymerase II (Pol II) that provide access to the type and position of modifications of a LC domain by mass spectrometry (MS).

STRIPAK complexes in cell signaling and cancer.

Striatin-interacting phosphatase and kinase (STRIPAK) complexes are striatin-centered multicomponent supramolecular structures containing both kinases and phosphatases. STRIPAK complexes are evolutionarily conserved and have critical roles in protein (de)phosphorylation. Recent studies indicate that STRIPAK complexes are emerging mediators and regulators of multiple vital signaling pathways including Hippo, MAPK (mitogen-activated protein kinase), nuclear receptor and cytoskeleton remodeling. Different types of STRIPAK complexes are extensively involved in a variety of fundamental biological processes ranging from cell growth, differentiation, proliferation and apoptosis to metabolism, immune regulation and tumorigenesis. Growing evidence correlates dysregulation of STRIPAK complexes with human diseases including cancer. In this review, we summarize the current understanding of the assembly and functions of STRIPAK complexes, with a special focus on cell signaling and cancer.

Folding and binding energy of a calmodulin-binding cell antiproliferative peptide.

We carry out a computational study of a calmodulin-binding peptide shown to be effective in reducing cell proliferation. We find several folded states for two short variants of different length of the peptide and determine the location of the binding site on calmodulin, the binding free energy for the different conformers and structural details that play a role in optimal binding. Binding to a hydrophobic pocket in calmodulin occurs via an anchoring phenylalanine residue of the natively disordered peptide, and is enhanced when a neighbouring hydrophobic residue acts as a co-anchor. The shorter sequence possesses better binding to calmodulin, which is encouraging in terms of the development of non-peptide analogues as therapeutic agents.

Primary pulmonary hyalinizing clear cell carcinoma of bronchial submucosal gland origin.

Hyalinizing clear cell carcinoma (HCCC) has only been described in salivary glands of the head and neck. We report a 38-year-old man with a 2.6-cm lung tumor that was growing in a peribronchial location and had morphologic features of HCCC. The tumor cells expressed cytokeratin 7 and keratin AE1/AE3, and the vast majority of tumor cells marked also with p63 and p40. They were negative for cytokeratin 20, S-100, smooth muscle actin, napsin A, and thyroid transcription factor-1. Fluorescence in situ hybridization revealed Ewing Sarcoma Breakpoint Region 1 (EWSR1) rearrangement, and reverse-transcription polymerase chain reaction confirmed the presence of the EWSR1-Activating Transcription Factor 1 (ATF1) fusion transcript, which was subsequently sequenced. The morphologic, immunophenotypic, cytogenetic, and molecular findings together with the patient's history and location of the tumor support a diagnosis of primary pulmonary HCCC of bronchial submucosal gland origin. It is our understanding that this is the first report of HCCC arising as a primary tumor outside the head and neck region.

Nicotine exposure alters human vascular smooth muscle cell phenotype from a contractile to a synthetic type.

OBJECTIVE: Cigarette smoking is a known risk factor for arteriosclerosis. In atheromatous plaques, vascular smooth muscle cells (VSMCs) display a phenotype that is different from the contractile type under normal conditions. Nicotine is the major pharmacological agent in cigarette smoke. However, any direct effect of nicotine on VSMCs remains uncertain. Because nicotine promotes VSMC migration, its phenotype may change due to nicotine. APPROACH AND RESULTS: We used human aorta primary smooth muscle cells (HuAoSMCs), differentiated with transforming growth factor-ß, to investigate changes in the protein levels of differentiation markers and in the activity of mitogen-activated protein kinases (MAPKs) after exposure to 0.1 µM of nicotine for 48 h. After nicotine exposure, the protein levels of myosin II 10 (2.93-fold) and ß-actin (1.66-fold), synthetic type markers, were increased. In contrast, the levels of the contractile type markers, myosin II 11 (0.63-fold), high-molecular-weight caldesmon (0.40-fold) and SM22 (0.66-fold), which concern differentiated VSMC, were decreased. Moreover, nicotine exposure induced enhanced activation of p38 MAPK (1.30-fold) and extracellular signal-regulated kinase (1.91-fold). These results indicated that the phenotype of HuAoSMCs had changed to a synthetic-like type because of nicotine exposure. Thus, nicotine is one factor that can alter protein expression of differentiation markers in VSMCs. Besides, the increase of intracellular Ca(2+) levels suggested that these effects of nicotine were mediated through nicotinic acetylcholine receptors. CONCLUSION: Nicotine has already been reported to promote VSMC migration from the tunica media to atheromatous plaques in the vascular intima. This phenomenon may occur because nicotine directly induces VSMC transformation from contractile type to synthetic-like type via nicotinic acetylcholine receptors and G protein-coupled receptors.

Biosensor-based approach identifies four distinct calmodulin-binding domains in the G protein-coupled estrogen receptor 1.

The G protein-coupled estrogen receptor 1 (GPER) has been demonstrated to participate in many cellular functions, but its regulatory inputs are not clearly understood. Here we describe a new approach that identifies GPER as a calmodulin-binding protein, locates interaction sites, and characterizes their binding properties. GPER coimmunoprecipitates with calmodulin in primary vascular smooth muscle cells under resting conditions, which is enhanced upon acute treatment with either specific ligands or a Ca(2+)-elevating agent. To confirm direct interaction and locate the calmodulin-binding domain(s), we designed a series of FRET biosensors that consist of enhanced cyan and yellow fluorescent proteins flanking each of GPER's submembrane domains (SMDs). Responses of these biosensors showed that all four submembrane domains directly bind calmodulin. Modifications of biosensor linker identified domains that display the strongest calmodulin-binding affinities and largest biosensor dynamics, including a.a. 83-93, 150-175, 242-259, 330-351, corresponding respectively to SMDs 1, 2, 3, and the juxta-membranous section of SMD4. These biosensors bind calmodulin in a strictly Ca(2+)-dependent fashion and with disparate affinities in the order SMD2>SMD4>SMD3>SMD1, apparent K d values being 0.44 ± 0.03, 1.40 ± 0.16, 8.01 ± 0.29, and 136.62 ± 6.56 µM, respectively. Interestingly, simultaneous determinations of biosensor responses and suitable Ca(2+) indicators identified separate Ca(2+) sensitivities for their interactions with calmodulin. SMD1-CaM complexes display a biphasic Ca(2+) response, representing two distinct species (SMD1 sp1 and SMD1 sp2) with drastically different Ca(2+) sensitivities. The Ca(2+) sensitivities of CaM-SMDs interactions follow the order SMD1sp1>SMD4>SMD2>SMD1sp2>SMD3, EC50(Ca(2+)) values being 0.13 ± 0.02, 0.75 ± 0.05, 2.38 ± 0.13, 3.71 ± 0.13, and 5.15 ± 0.25 µM, respectively. These data indicate that calmodulin may regulate GPER-dependent signaling at the receptor level through multiple interaction sites. FRET biosensors represent a simple method to identify unknown calmodulin-binding domains in G protein-coupled receptors and to quantitatively assess binding properties.

A conserved N-terminal motif is required for complex formation between FUS, EWSR1, TAF15 and their oncogenic fusion proteins.

The three FET (FUS, EWSR1, and TAF15) family RNA binding proteins are expressed in all tissues and almost all cell types. The disordered N-terminal parts are always present in FET fusion oncoproteins of sarcomas and leukemia. Mutations in FUS and TAF15 cause aggregation of FET proteins in neurological disorders. Here we used recombinant proteins in pulldown experiments and mass spectrometry to identify major interaction partners of the FET N-terminal parts. We report that FUS, EWSR1, and TAF15 form homo- and heterocomplexes as major binding partners and identify an evolutionarily conserved N-terminal motif (FETBM1) that is required for this interaction. The binding is RNA and DNA independent and robust up to 1 M of NaCl. The localization of FETBM1 and its target sequences supports a simple model for FET protein aggregation as reported in neurological disorders such as amyotrophic lateral sclerosis, frontotemporal dementia, and essential tremor. The FETBM1 localization also explains the binding of normal full-length FET proteins to their oncogenic fusion proteins.

Amino acid mutations in the caldesmon COOH-terminal functional domain increase force generation in bladder smooth muscle.

Caldesmon (CaD), a component of smooth muscle thin filaments, binds actin, tropomyosin, calmodulin, and myosin and inhibits actin-activated ATP hydrolysis by smooth muscle myosin. Internal deletions of the chicken CaD functional domain that spans from amino acids (aa) 718 to 731, which corresponds to aa 512-530 including the adjacent aa sequence in mouse CaD, lead to diminished CaD-induced inhibition of actin-activated ATP hydrolysis by myosin. Transgenic mice with mutations of five aa residues (Lys(523) to Gln, Val(524) to Leu, Ser(526) to Thr, Pro(527) to Cys, and Lys(529) to Ser), which encompass the ATPase inhibitory determinants located in exon 12, were generated by homologous recombination. Homozygous (-/-) animals did not develop, but heterozygous (+/-) mice carrying the expected mutations in the CaD ATPase inhibitory domain (CaD mutant) matured and reproduced normally. The peak force produced in response to KCl and electrical field stimulation by the detrusor smooth muscle from the CaD mutant was high compared with that of the wild type. CaD mutant mice revealed nonvoiding contractions during bladder filling on awake cystometry, suggesting that the CaD ATPase inhibitory domain suppresses force generation during the filling phase and this suppression is partially released by mutations in 50% of CaD in heterozygous. Our data show for the first time a functional phenotype, at the intact smooth muscle tissue and in vivo organ levels, following mutation of a functional domain at the COOH-terminal region of CaD.

The C-terminal linker of Escherichia coli FtsZ functions as an intrinsically disordered peptide.

The tubulin homologue FtsZ provides the cytoskeletal framework and constriction force for bacterial cell division. FtsZ has an 50-amino-acid (aa) linker between the protofilament-forming globular domain and the C-terminal (Ct) peptide that binds FtsA and ZipA, tethering FtsZ to the membrane. This Ct-linker is widely divergent across bacterial species and thought to be an intrinsically disordered peptide (IDP). We confirmed that the Ct-linkers from three bacterial species behaved as IDPs in vitro by circular dichroism and trypsin proteolysis. We made chimeras, swapping the Escherichia coli linker for Ct-linkers from other bacteria, and even for an unrelated IDP from human α-adducin. Most substitutions allowed for normal cell division, suggesting that sequence of the IDP did not matter. With few exceptions, almost any sequence appears to work. Length, however, was important: IDPs shorter than 43 or longer than 95 aa had compromised or no function. We conclude that the Ct-linker functions as a flexible tether between the globular domain of FtsZ in the protofilament, and its attachment to FtsA/ZipA at the membrane. Modelling the Ct-linker as a worm-like chain, we predict that it functions as a stiff entropic spring linking the bending protofilaments to the membrane.

γ-Adducin promotes process outgrowth and secretory protein exit from the Golgi apparatus.

α, ß, and γ adducins mediate F-actin remodeling of plasma membrane structures as heterotetramers. Here, we present two new functions of γ-adducin. (1) Overexpression of γ-adducin promoted formation of neurite-like processes in non-neuronal fibroblast COS7 cells. Conversely, overexpression of the C-terminal 38 amino acids of γ-adducin (γAdd(C38)) acting as a dominant negative inhibited formation of neurites/processes in Neuro2A cells and anterior pituitary AtT20 cells. (2) γ-Adducin appears to facilitate pro-opiomelanocortin (POMC) exit from the trans-Golgi network (TGN) by re-organizing the actin network around the Golgi complex. Filamentous actins (F-actins) which formed puncti around the Golgi complex in control cells were dispersed in AtT20 cells stably transfected with γAdd(C38). Furthermore, γAdd(C38)-transfectants showed significant accumulation of POMC/adrenocorticotropin (ACTH) in the Golgi complex and diminished POMC/ACTH vesicles in the cell processes. The C-terminal 38 amino acids of γ-adducin interacted with F-actins around the Golgi complex, to facilitate F-actin-mediated budding of POMC/ACTH vesicles from the TGN. Thus, we propose that γ-adducin, via its interaction with F-actins, plays a critical role in actin remodeling to facilitate process/neurite outgrowth, as well as budding of POMC/ACTH vesicles from the TGN via its interaction with peri-Golgi F-actins.

[Amyloid-like oligomers of presynaptic protein BASP1].

Brain protein BASP1 forms oligomers that resemble amyloid protein oligomers in some respects, including interaction with conformation-specific antibodies against amyloid oligomers. Aggregation-prone N-terminal myristoylated peptide myr-BASP1 (1-13) forms fibrillar aggregates of amyloid-like structure under physiological conditions in vitro, which is also the evidence of BASP1 similarity to amyloid proteins. Protein cross-linking by glutaraldehyde in situ demonstrated that BASP1 exists on the presynaptic membrane as lipid raft-associated oligomers. In addition, BASP1 is non-toxic to PC12 cells, when applied either as a monomer or oligomer. We conclude that BASP1 oligomer is a non-pathological physiologically relevant functional form of this protein.

RNA targets of wild-type and mutant FET family proteins.

FUS, EWSR1 and TAF15, constituting the FET protein family, are abundant, highly conserved RNA-binding proteins with important roles in oncogenesis and neuronal disease, yet their RNA targets and recognition elements are unknown. Using PAR-CLIP, we defined global RNA targets for all human FET proteins and two ALS-causing human FUS mutants. FET members showed similar binding profiles, whereas FUS mutants showed a drastically altered binding pattern, consistent with changes in subcellular localization.