Villin-type headpiece domains show a wide range of F-actin-binding affinities

2002 ◽  
Vol 52 (1) ◽  
pp. 9-21 ◽  
Author(s):  
D. Vardar ◽  
A. H. Chishti ◽  
B. S. Frank ◽  
E. J. Luna ◽  
A. A. Noegel ◽  
...  
Keyword(s):  
Actin Binding ◽  
Binding Affinities ◽  
Wide Range ◽  
Headpiece Domains

scholarly journals MAGI-1 interacts with Slo1 channel proteins and suppresses Slo1 expression on the cell surface

2009 ◽  
Vol 297 (1) ◽  
pp. C55-C65 ◽  
Author(s):  
Lon D. Ridgway ◽  
Eun Young Kim ◽  
Stuart E. Dryer
Keyword(s):  
Cell Surface ◽  
Outward Current ◽  
Cell Types ◽  
Surface Expression ◽  
Scaffolding Protein ◽  
Actin Binding ◽  
Differentiated Cells ◽  
Cell Surface Biotinylation ◽  
Bait Construct ◽  
Wide Range

Large conductance Ca2+-activated K+ (BKCa) channels encoded by the Slo1 gene (also known as KCNMA1) are physiologically important in a wide range of cell types and form complexes with a number of other proteins that affect their function. We performed a yeast two-hybrid screen to identify proteins that interact with BKCa channels using a bait construct derived from domains in the extreme COOH-terminus of Slo1. A protein known as membrane-associated guanylate kinase with inverted orientation protein-1 (MAGI-1) was identified in this screen. MAGI-1 is a scaffolding protein that allows formation of complexes between certain transmembrane proteins, actin-binding proteins, and other regulatory proteins. MAGI-1 is expressed in a number of tissues, including podocytes and the brain. The interaction between MAGI-1 and BKCa channels was confirmed by coimmunoprecipitation and glutathione S-transferase pull-down assays in differentiated cells of a podocyte cell line and in human embryonic kidneys (HEK)293T cells transiently coexpressing MAGI-1a and three different COOH-terminal Slo1 variants. Coexpression of MAGI-1 with Slo1 channels in HEK-293T cells results in a significant reduction in the surface expression of Slo1, as assessed by cell-surface biotinylation assays, confocal microscopy, and whole cell recordings. Partial knockdown of endogenous MAGI-1 expression by small interfering RNA (siRNA) in differentiated podocytes increased the surface expression of endogenous Slo1 as assessed by electrophysiology and cell-surface biotinylation assays, whereas overexpression of MAGI-1a reduced steady-state voltage-evoked outward current through podocyte BKCa channels. These data suggest that MAGI-1 plays a role in regulation of surface expression of BKCa channels in the kidney and possibly in other tissues.

The Role of Actin-Binding Protein Filamin A in Cellular Stiffness and Morphology Studied by Wide-Range Scanning Probe Microscopy

2006 ◽  
Vol 45 (3B) ◽  
pp. 2328-2332 ◽  
Author(s):  
Kosaku Kato ◽  
Yukiko Ohmori ◽  
Takeomi Mizutani ◽  
Hisashi Haga ◽  
Kazuyo Ohashi ◽  
...  
Keyword(s):  
Scanning Probe Microscopy ◽  
Binding Protein ◽  
Scanning Probe ◽  
Actin Binding ◽  
Filamin A ◽  
Probe Microscopy ◽  
Actin Binding Protein ◽  
Wide Range ◽  
Range Scanning

Actin Binding Proteins: Regulation of Cytoskeletal Microfilaments

2003 ◽  
Vol 83 (2) ◽  
pp. 433-473 ◽  
Author(s):  
C. G. Dos Remedios ◽  
D. Chhabra ◽  
M. Kekic ◽  
I. V. Dedova ◽  
M. Tsubakihara ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Binding Proteins ◽  
Complex Structure ◽  
Actin Binding ◽  
Cellular Functions ◽  
Actin Binding Proteins ◽  
Ancestral Gene ◽  
Wide Range ◽  
Human Disorders ◽  
And Function

The actin cytoskeleton is a complex structure that performs a wide range of cellular functions. In 2001, significant advances were made to our understanding of the structure and function of actin monomers. Many of these are likely to help us understand and distinguish between the structural models of actin microfilaments. In particular, 1) the structure of actin was resolved from crystals in the absence of cocrystallized actin binding proteins (ABPs), 2) the prokaryotic ancestral gene of actin was crystallized and its function as a bacterial cytoskeleton was revealed, and 3) the structure of the Arp2/3 complex was described for the first time. In this review we selected several ABPs (ADF/cofilin, profilin, gelsolin, thymosin β4, DNase I, CapZ, tropomodulin, and Arp2/3) that regulate actin-driven assembly, i.e., movement that is independent of motor proteins. They were chosen because 1) they represent a family of related proteins, 2) they are widely distributed in nature, 3) an atomic structure (or at least a plausible model) is available for each of them, and 4) each is expressed in significant quantities in cells. These ABPs perform the following cellular functions: 1) they maintain the population of unassembled but assembly-ready actin monomers (profilin), 2) they regulate the state of polymerization of filaments (ADF/cofilin, profilin), 3) they bind to and block the growing ends of actin filaments (gelsolin), 4) they nucleate actin assembly (gelsolin, Arp2/3, cofilin), 5) they sever actin filaments (gelsolin, ADF/cofilin), 6) they bind to the sides of actin filaments (gelsolin, Arp2/3), and 7) they cross-link actin filaments (Arp2/3). Some of these ABPs are essential, whereas others may form regulatory ternary complexes. Some play crucial roles in human disorders, and for all of them, there are good reasons why investigations into their structures and functions should continue.

scholarly journals Altered Expression Ratio of Actin-Binding Gelsolin Isoforms Is a Novel Hallmark of Mitochondrial OXPHOS Dysfunction

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 1922
Author(s):  
Alberto García-Bartolomé ◽  
Ana Peñas ◽  
María Illescas ◽  
Verónica Bermejo ◽  
Sandra López-Calcerrada ◽  
...  
Keyword(s):  
Subcellular Location ◽  
Complex Iii ◽  
Actin Binding ◽  
Future Research ◽  
Inborn Errors ◽  
Expression Ratio ◽  
Altered Expression ◽  
Wide Range ◽  
Promote Cell Survival ◽  
Clinical Conditions

Mitochondrial oxidative phosphorylation (OXPHOS) defects are the primary cause of inborn errors of energy metabolism. Despite considerable progress on their genetic basis, their global pathophysiological consequences remain undefined. Previous studies reported that OXPHOS dysfunction associated with complex III deficiency exacerbated the expression and mitochondrial location of cytoskeletal gelsolin (GSN) to promote cell survival responses. In humans, besides the cytosolic isoform, GSN presents a plasma isoform secreted to extracellular environments. We analyzed the interplay between both GSN isoforms in human cellular and clinical models of OXPHOS dysfunction. Regardless of its pathogenic origin, OXPHOS dysfunction induced the physiological upregulation of cytosolic GSN in the mitochondria (mGSN), in parallel with a significant downregulation of plasma GSN (pGSN) levels. Consequently, significantly high mGSN-to-pGSN ratios were associated with OXPHOS deficiency both in human cells and blood. In contrast, control cells subjected to hydrogen peroxide or staurosporine treatments showed no correlation between oxidative stress or cell death induction and the altered levels and subcellular location of GSN isoforms, suggesting their specificity for OXPHOS dysfunction. In conclusion, a high mitochondrial-to-plasma GSN ratio represents a useful cellular indicator of OXPHOS defects, with potential use for future research of a wide range of clinical conditions with mitochondrial involvement.

Docking Investigation on Bis (Nitro Indazolyl) Methanes; Synthesis and Antimicrobial Activity Towards Breast Cancer Applications

2021 ◽  
Vol 18 ◽  
Author(s):  
S. Jagadeesan ◽  
S. Karpagam
Keyword(s):  
Breast Cancer ◽  
Antimicrobial Activity ◽  
Docking Studies ◽  
Klebsiella Pneumonia ◽  
Binding Affinities ◽  
Efficient Catalyst ◽  
Molegro Virtual Docker ◽  
Starting Point ◽  
Wide Range ◽  
Human Estrogen Receptor

: Bismuth (III) nitrate pentahydrate (BN) was found to be a mild and efficient catalyst for the electrophilic condensation of 5-nitroindazole with a wide range of aldehydes to obtain Bis (5-nitro indazolyl) methanes 3 (a-h) at ambient temperature. This was structurally confirmed from FTIR, NMR, and HR-MS technique. Molecular docking studies of all compounds was carried out using breast cancer-causing human estrogen receptor (ER) from Molegro Virtual Docker software. Hydroxy Bis (nitro indazolyl) methanes (3b) were shown better binding affinities and the score obtained was -150.146 Kcal/mol compared with Tamoxifen drug. The major H-bond interactions were observed with the compound 3f and the value was -5.679. The antimicrobial activity results revealed that compounds 3b and 3d showed promising activity against bacteria Escherichia coli, Klebsiella pneumonia, Staphylococcus aureus, and Pseudomonas aeruginosa and maximum inhibition against Aspergillus niger and Aspergillus flavus. Methoxy derivatives of Bis (nitro indazolyl methanes) (3e) have shown better antioxidant activity and low MIC (6.25 µg/ml) observed for the compounds 3a and 3b. The synthesized compounds have a very promising starting point for the development and improvement of anti-breast cancer drugs.

scholarly journals Indirect association of ezrin with F-actin: isoform specificity and calcium sensitivity.

1995 ◽  
Vol 128 (5) ◽  
pp. 837-848 ◽  
Author(s):  
C B Shuster ◽  
I M Herman
Keyword(s):  
Calcium Sensitivity ◽  
Cytochalasin D ◽  
Actin Binding ◽  
Free Calcium ◽  
Cell Extracts ◽  
Beta Actin ◽  
Wide Range ◽  
Sepharose 4B

Whereas it has been demonstrated that muscle and nonmuscle isoactins are segregated into distinct cytoplasmic domains, the mechanism regulating subcellular sorting is unknown (Herman, 1993a). To reveal whether isoform-specific actin-binding proteins function to coordinate these events, cell extracts derived from motile (Em) versus stationary (Es) cytoplasm were selectively and sequentially fractionated over filamentous isoactin affinity columns prior to elution with a KCl step gradient. A polypeptide of interest, which binds specifically to beta-actin filament columns, but not to muscle actin columns has been conclusively identified as the ERM family member, ezrin. We studied ezrin-beta interactions in vitro by passing extracts (Em) over isoactin affinity matrices in the presence of Ca(2+)-containing versus Ca(2+)-free buffers, with or without cytochalasin D. Ezrin binds and can be released from beta-actin Sepharose-4B in the presence of Mg2+/EGTA and 100 mM NaCl (at 4 degrees C and room temperature), but not when affinity fractionation of Em is carried out in the presence of 0.2 mM CaCl2 or 2 microM cytochalasin D. N-acetyl-(leucyl)2-norleucinal and E64, two specific inhibitors of the calcium-activated protease, calpain I, protect ezrin binding to beta actin in the presence of calcium. Moreover, biochemical analysis of endothelial lysates reveals that a calpain I cleavage product of ezrin emerges when cell locomotion is stimulated in response to monolayer injury. Immunofluorescence analysis of leading lamellae reveals that anti-ezrin and anti-beta-actin IgGs can be simultaneously co-localized, extending the results of isoactin affinity fractionation of Em-derived extracts and suggesting that ezrin and beta-actin interact in vivo. To test the hypothesis that ezrin binds directly to beta-actin, we performed three sets of studies under a wide range of physiological conditions (pH 7.0-8.5) using purified pericyte ezrin and either alpha- or beta-actin. These included co-sedimentation, isoactin affinity fractionation, and co-immunoprecipitation. Results of these experiments reveal that purified ezrin does not directly bind to beta-actin filaments, either in solution or while isoactins are covalently cross-linked to Sepharose-4B. This is in contrast to our finding that ezrin and beta-actin could be co-immunoprecipitated or co-sedimented from Em-derived cell lysates. To explore whether calcium transients occur in cellular domains enriched in ezrin and beta-actin, we mapped cellular free calcium in endothelial monolayers crawling in response to injury.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Evidence for functional homology in the F-actin binding domains of gelsolin and alpha-actinin: implications for the requirements of severing and capping.

1992 ◽  
Vol 119 (4) ◽  
pp. 835-842 ◽  
Author(s):  
M Way ◽  
B Pope ◽  
A G Weeds
Keyword(s):  
Calcium Ions ◽  
Actin Filaments ◽  
Tandem Repeats ◽  
Actin Binding ◽  
Binding Affinities ◽  
Binding Domains ◽  
Functional Homology ◽  
Actin Binding Domain ◽  
Extended Sequence ◽  
Actin Binding Site

The F-actin binding domains of gelsolin and alpha-actinin compete for the same site on actin filaments with similar binding affinities. Both contain tandem repeats of approximately 125 amino acids, the first of which is shown to contain the actin-binding site. We have replaced the F-actin binding domain in the NH2-terminal half of gelsolin by that of alpha-actinin. The hybrid severs filaments almost as efficiently as does gelsolin or its NH2-terminal half, but unlike the latter, requires calcium ions. The hybrid binds two actin monomers and caps the barbed ends of filaments in the presence or absence of calcium. The cap produced by the hybrid binds with lower affinity than that of gelsolin and is not stable: It dissociates from filament ends with a half life of approximately 15 min. Although there is no extended sequence homology between these two different F-actin binding domains, our experiments show that they are functionally equivalent and provide new insights into the mechanism of microfilament severing.

Tropomyosin-Based Regulation of the Actin Cytoskeleton in Time and Space

2008 ◽  
Vol 88 (1) ◽  
pp. 1-35 ◽  
Author(s):  
Peter Gunning ◽  
Geraldine O’neill ◽  
Edna Hardeman
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filaments ◽  
Striated Muscle ◽  
Coiled Coil ◽  
Cell Types ◽  
Actin Binding ◽  
Regulation Of Expression ◽  
Wide Range ◽  
Muscle Tropomyosin ◽  
The Many

Tropomyosins are rodlike coiled coil dimers that form continuous polymers along the major groove of most actin filaments. In striated muscle, tropomyosin regulates the actin-myosin interaction and, hence, contraction of muscle. Tropomyosin also contributes to most, if not all, functions of the actin cytoskeleton, and its role is essential for the viability of a wide range of organisms. The ability of tropomyosin to contribute to the many functions of the actin cytoskeleton is related to the temporal and spatial regulation of expression of tropomyosin isoforms. Qualitative and quantitative changes in tropomyosin isoform expression accompany morphogenesis in a range of cell types. The isoforms are segregated to different intracellular pools of actin filaments and confer different properties to these filaments. Mutations in tropomyosins are directly involved in cardiac and skeletal muscle diseases. Alterations in tropomyosin expression directly contribute to the growth and spread of cancer. The functional specificity of tropomyosins is related to the collaborative interactions of the isoforms with different actin binding proteins such as cofilin, gelsolin, Arp 2/3, myosin, caldesmon, and tropomodulin. It is proposed that local changes in signaling activity may be sufficient to drive the assembly of isoform-specific complexes at different intracellular sites.

An in vivo imaging-based assay for detecting protein interactions over a wide range of binding affinities

2009 ◽  
Vol 395 (2) ◽  
pp. 166-177 ◽  
Author(s):  
A. Nicole Edwards ◽  
Jason D. Fowlkes ◽  
Elizabeth T. Owens ◽  
Robert F. Standaert ◽  
Dale A. Pelletier ◽  
...  
Keyword(s):  
Protein Interactions ◽  
In Vivo Imaging ◽  
Binding Affinities ◽  
Wide Range

scholarly journals Cell-permeable transgelin-2 as a potent therapeutic for dendritic cell-based cancer immunotherapy

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Hye-Ran Kim ◽  
Jeong-Su Park ◽  
Jin-Hwa Park ◽  
Fatima Yasmin ◽  
Chang-Hyun Kim ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cell ◽  
Tumor Growth ◽  
Cancer Immunotherapy ◽  
Actin Binding ◽  
B16f10 Melanoma ◽  
Wide Range ◽  
Viral Transduction ◽  
Tumor Growth And Metastasis

Abstract Background Transgelin-2 is a 22 kDa actin-binding protein that has been proposed to act as an oncogenic factor, capable of contributing to tumorigenesis in a wide range of human malignancies. However, little is known whether this tiny protein also plays an important role in immunity, thereby keeping body from the cancer development and metastasis. Here, we investigated the functions of transgelin-2 in dendritic cell (DC) immunity. Further, we investigated whether the non-viral transduction of cell-permeable transgelin-2 peptide potentially enhance DC-based cancer immunotherapy. Methods To understand the functions of transgelin-2 in DCs, we utilized bone marrow-derived DCs (BMDCs) purified from transgelin-2 knockout (Tagln2−/−) mice. To observe the dynamic cellular mechanism of transgelin-2, we utilized confocal microscopy and flow cytometry. To monitor DC migration and cognate T–DC interaction in vivo, we used intravital two-photon microscopy. For the solid and metastasis tumor models, OVA+ B16F10 melanoma were inoculated into the C57BL/6 mice via intravenously (i.v.) and subcutaneously (s.c.), respectively. OTI TCR T cells were used for the adoptive transfer experiments. Cell-permeable, de-ubiquitinated recombinant transgelin-2 was purified from Escherichia coli and applied for DC-based adoptive immunotherapy. Results We found that transgelin-2 is remarkably expressed in BMDCs during maturation and lipopolysaccharide activation, suggesting that this protein plays a role in DC-based immunity. Although Tagln2−/− BMDCs exhibited no changes in maturation, they showed significant defects in their abilities to home to draining lymph nodes (LNs) and prime T cells to produce antigen-specific T cell clones, and these changes were associated with a failure to suppress tumor growth and metastasis of OVA+ B16F10 melanoma cells in mice. Tagln2−/− BMDCs had defects in filopodia-like membrane protrusion and podosome formation due to the attenuation of the signals that modulate actin remodeling in vitro and formed short, unstable contacts with cognate CD4+ T cells in vivo. Strikingly, non-viral transduction of cell-permeable, de-ubiquitinated recombinant transgelin-2 potentiated DC functions to suppress tumor growth and metastasis. Conclusion This work demonstrates that transgelin-2 is an essential protein for both cancer and immunity. Therefore, transgelin-2 can act as a double-edged sword depending on how we apply this protein to cancer therapy. Engineering and clinical application of this protein may unveil a new era in DC-based cancer immunotherapy. Our findings indicate that cell-permeable transgelin-2 have a potential clinical value as a cancer immunotherapy based on DCs.

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