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Robert Robinson - One of the best experts on this subject based on the ideXlab platform.
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Insights into the evolution of regulated actin dynamics via characterization of primitive Gelsolin/cofilin proteins from Asgard archaea
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Caner Akıl, Linh Tran, Magali Orhant-prioux, Yohendran Baskaran, Edward Manser, Laurent Blanchoin, Robert RobinsonAbstract:Asgard archaea genomes contain potential eukaryotic-like genes that provide intriguing insight for the evolution of eukaryotes. The eukaryotic actin polymerization/depolymerization cycle is critical for providing force and structure in many processes, including membrane remodeling. In general, Asgard genomes encode two classes of actin-regulating proteins from sequence analysis, profilins and Gelsolins. Asgard profilins were demonstrated to regulate actin filament nucleation. Here, we identify actin filament severing, capping, annealing and bundling, and monomer sequestration activities by Gelsolin proteins from Thorarchaeota (Thor), which complete a eukaryotic-like actin depolymerization cycle, and indicate complex actin cytoskeleton regulation in Asgard organisms. Thor Gelsolins have homologs in other Asgard archaea and comprise one or two copies of the prototypical Gelsolin domain. This appears to be a record of an initial preeukaryotic gene duplication event, since eukaryotic Gelsolins are generally comprise three to six domains. X-ray structures of these proteins in complex with mammalian actin revealed similar interactions to the first domain of human Gelsolin or cofilin with actin. Asgard two-domain, but not one-domain, Gelsolins contain calcium-binding sites, which is manifested in calcium-controlled activities. Expression of two-domain Gelsolins in mammalian cells enhanced actin filament disassembly on ionomycin-triggered calcium release. This functional demonstration, at the cellular level, provides evidence for a calcium-controlled Asgard actin cytoskeleton, indicating that the calcium-regulated actin cytoskeleton predates eukaryotes. In eukaryotes, dynamic bundled actin filaments are responsible for shaping filopodia and microvilli. By correlation, we hypothesize that the formation of the protrusions observed from Lokiarchaeota cell bodies may involve the Gelsolin-regulated actin structures.
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the role of Gelsolin domain 3 in familial amyloidosis finnish type
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Robert Robinson, Jan Gettemans, Habiba Zorgati, Marten Larsson, Weitong Ren, Adelene Y L Sim, Jonathan M GrimesAbstract:In the disease familial amyloidosis, Finnish type (FAF), also known as AGel amyloidosis (AGel), the mechanism by which point mutations in the calcium-regulated actin-severing protein Gelsolin lead to furin cleavage is not understood in the intact protein. Here, we provide a structural and biochemical characterization of the FAF variants. X-ray crystallography structures of the FAF mutant Gelsolins demonstrate that the mutations do not significantly disrupt the calcium-free conformations of Gelsolin. Small-angle X-ray–scattering (SAXS) studies indicate that the FAF calcium-binding site mutants are slower to activate, whereas G167R is as efficient as the wild type. Actin-regulating studies of the Gelsolins at the furin cleavage pH (6.5) show that the mutant Gelsolins are functional, suggesting that they also adopt relatively normal active conformations. Deletion of Gelsolin domains leads to sensitization to furin cleavage, and nanobody-binding protects against furin cleavage. These data indicate instability in the second domain of Gelsolin (G2), since loss or gain of G2-stabilizing interactions impacts the efficiency of cleavage by furin. To demonstrate this principle, we engineered non-FAF mutations in G3 that disrupt the G2-G3 interface in the calcium-activated structure. These mutants led to increased furin cleavage. We carried out molecular dynamics (MD) simulations on the FAF and non-FAF mutant G2-G3 fragments of Gelsolin. All mutants showed an increase in the distance between the center of masses of the 2 domains (G2 and G3). Since G3 covers the furin cleavage site on G2 in calcium-activated Gelsolin, this suggests that destabilization of this interface is a critical step in cleavage.
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tropomyosins regulate the severing activity of Gelsolin in isoform dependent and independent manners
Biophysical Journal, 2018Co-Authors: Nikolett Kisbicskei, Beata Bugyi, Tamas Huber, Bálint Bécsi, Ferenc Erdődi, Miklós Nyitrai, Robert Robinson, Gabor TalianAbstract:Abstract The actin cytoskeleton fulfills numerous key cellular functions, which are tightly regulated in activity, localization, and temporal patterning by actin binding proteins. Tropomyosins and Gelsolin are two such filament-regulating proteins. Here, we investigate how the effects of tropomyosins are coupled to the binding and activity of Gelsolin. We show that the three investigated tropomyosin isoforms (Tpm1.1, Tpm1.12, and Tpm3.1) bind to Gelsolin with micromolar or submicromolar affinities. Tropomyosin binding enhances the activity of Gelsolin in actin polymerization and depolymerization assays. However, the effects of the three tropomyosin isoforms varied. The tropomyosin isoforms studied also differed in their ability to protect pre-existing actin filaments from severing by Gelsolin. Based on the observed specificity of the interactions between tropomyosins, actin filaments, and Gelsolin, we propose that tropomyosin isoforms specify which populations of actin filaments should be targeted by, or protected from, Gelsolin-mediated depolymerization in living cells.
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the calcium activation of Gelsolin insights from the 3a structure of the g4 g6 actin complex
Journal of Molecular Biology, 2002Co-Authors: Leslie D. Burtnick, Robert Robinson, Helen L Yin, Han Choe, Marisan Mejillano, Senyon ChoeAbstract:Abstract Gelsolin participates in the reorganization of the actin cytoskeleton that is required during such phenomena as cell movement, cytokinesis, and apoptosis. It consists of six structurally similar domains, G1–G6, which are arranged at resting intracellular levels of calcium ion so as to obscure the three actin-binding surfaces. Elevation of Ca 2+ concentrations releases latches within the constrained structure and produces large shifts in the relative positioning of the domains, permitting Gelsolin to bind to and sever actin filaments. How Ca 2+ is able to activate Gelsolin has been a major question concerning the function of this protein. We present the improved structure of the C-terminal half of Gelsolin bound to monomeric actin at 3.0 A resolution. Two classes of Ca 2+ -binding site are evident on Gelsolin: type 1 sites share coordination of Ca 2+ with actin, while type 2 sites are wholly contained within Gelsolin. This structure of the complex reveals the locations of two novel metal ion-binding sites in domains G5 and G6, respectively. We identify both as type 2 sites. The absolute conservation of the type 2 calcium-ligating residues across the six domains of Gelsolin suggests that this site exists in each of the domains. In total, Gelsolin has the potential to bind eight calcium ions, two type 1 and six type 2. The function of the type 2 sites is to facilitate structural rearrangements within Gelsolin as part of the activation and actin-binding and severing processes. We propose the novel type 2 site in G6 to be the critical site that initiates overall activation of Gelsolin by releasing the tail latch that locks calcium-free Gelsolin in a conformation unable to bind actin.
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the crystal structure of plasma Gelsolin implications for actin severing capping and nucleation
Cell, 1997Co-Authors: Leslie D. Burtnick, Edward K Koepf, J M Grimes, E Y Jones, D I Stuart, P J Mclaughlin, Robert RobinsonAbstract:The structure of Gelsolin has been determined by crystallography and comprises six structurally related domains that, in a Ca2+-free environment, pack together to form a compact globular structure in which the putative actin-binding sequences are not sufficiently exposed to enable binding to occur. We propose that binding Ca2+ can release the connections that join the N- and C-terminal halves of Gelsolin, enabling each half to bind actin relatively independently. Domain shifts are proposed in response to Ca2+ as bases for models of how Gelsolin acts to sever, cap, or nucleate F-actin filaments. The structure also invites discussion of polyphosphoinositide binding to segment 2 and suggests how mutation at Asp-187 could initiate a series of events that lead to deposition of amyloid plaques, as observed in victims of familial amyloidosis (Finnish type).
Renu Garg - One of the best experts on this subject based on the ideXlab platform.
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analgesic and anti inflammatory properties of Gelsolin in acetic acid induced writhing tail immersion and carrageenan induced paw edema in mice
PLOS ONE, 2015Co-Authors: Ashok Kumar Gupta, Amin Sagar, Vikas Choudhary, Renu Garg, Bhupinder Singh Chopra, Devraj Parasar, Neeraj KhatriAbstract:Plasma Gelsolin levels significantly decline in several disease conditions, since Gelsolin gets scavenged when it depolymerizes and caps filamentous actin released in the circulation following tissue injury. It is well established that our body require/implement inflammatory and analgesic responses to protect against cell damage and injury to the tissue. This study was envisaged to examine analgesic and anti-inflammatory activity of exogenous Gelsolin (8 mg/mouse) in mice models of pain and acute inflammation. Administration of Gelsolin in acetic acid-induced writhing and tail immersion tests not only demonstrated a significant reduction in the number of acetic acid-induced writhing effects, but also exhibited an analgesic activity in tail immersion test in mice as compared to placebo treated mice. Additionally, anti-inflammatory function of Gelsolin (8 mg/mouse) compared with anti-inflammatory drug diclofenac sodium (10 mg/kg)] was confirmed in the carrageenan injection induced paw edema where latter was measured by vernier caliper and fluorescent tomography imaging. Interestingly, results showed that plasma Gelsolin was capable of reducing severity of inflammation in mice comparable to diclofenac sodium. Analysis of cytokines and histo-pathological examinations of tissue revealed administration of Gelsolin and diclofenac sodium significantly reduced production of pro-inflammatory cytokines, TNF-α and IL-6. Additionally, carrageenan groups pretreated with diclofenac sodium or Gelsolin showed a marked decrease in edema and infiltration of inflammatory cells in paw tissue. Our study provides evidence that administration of Gelsolin can effectively reduce the pain and inflammation in mice model.
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global shapes of f actin depolymerization competent minimal Gelsolins insight into the role of g2 g3 linker in ph ca2 insensitivity of the first half
Journal of Biological Chemistry, 2013Co-Authors: Nagesh Peddada, Amin Sagar, Yogendra S Rathore, Vikas Choudhary, Bharat U K Pattnaik, Neeraj Khatri, Renu GargAbstract:Because of its ability to rapidly depolymerize F-actin, plasma Gelsolin has emerged as a therapeutic molecule in different disease conditions. High amounts of exogenous Gelsolin are, however, required to treat animal models of different diseases. Knowing that the F-actin depolymerizing property of Gelsolin resides in its N terminus, we made several truncated versions of plasma Gelsolin. The smaller versions, particularly the one composed of the first 28–161 residues, depolymerized the F-actin much faster than the native Gelsolin and other truncates at the same molar ratios. Although G1-G3 loses its dependence on Ca2+ or low pH for the actin depolymerization function, interestingly, G1-G2 and its smaller versions were found to regain this requirement. Small angle x-ray scattering-based shape reconstructions revealed that G1-G3 adopts an open shape in both the presence and the absence of Ca2+ as well as low pH, whereas G1-G2 and residues 28–161 prefer collapsed states in Ca2+-free conditions at pH 8. The mutations in the g2-g3 linker resulted in the calcium sensitivity of the mutant G1-G3 for F-actin depolymerization activity, although the F-actin-binding sites remained exposed in the mutant G1-G3 as well as in the smaller truncates even in the Ca2+-free conditions at pH 8. Furthermore, unlike wild type G1-G3, calcium-sensitive mutants of G1-G3 acquired closed shapes in the absence of free calcium, implying a role of g2-g3 linker in determining the open F-actin depolymerizing-competent shape of G1-G3 in this condition. We demonstrate that the mobility of the G1 domain, essential for F-actin depolymerization, is indirectly regulated by the Gelsolin-like sequence of g2-g3 linker.
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global shapes of f actin depolymerization competent minimal Gelsolins insight into the role of g2 g3 linker in ph ca2 insensitivity of the first half
Journal of Biological Chemistry, 2013Co-Authors: Nagesh Peddada, Amin Sagar, Yogendra S Rathore, Vikas Choudhary, Bharat U K Pattnaik, Neeraj Khatri, Renu GargAbstract:Because of its ability to rapidly depolymerize F-actin, plasma Gelsolin has emerged as a therapeutic molecule in different disease conditions. High amounts of exogenous Gelsolin are, however, required to treat animal models of different diseases. Knowing that the F-actin depolymerizing property of Gelsolin resides in its N terminus, we made several truncated versions of plasma Gelsolin. The smaller versions, particularly the one composed of the first 28–161 residues, depolymerized the F-actin much faster than the native Gelsolin and other truncates at the same molar ratios. Although G1-G3 loses its dependence on Ca2+ or low pH for the actin depolymerization function, interestingly, G1-G2 and its smaller versions were found to regain this requirement. Small angle x-ray scattering-based shape reconstructions revealed that G1-G3 adopts an open shape in both the presence and the absence of Ca2+ as well as low pH, whereas G1-G2 and residues 28–161 prefer collapsed states in Ca2+-free conditions at pH 8. The mutations in the g2-g3 linker resulted in the calcium sensitivity of the mutant G1-G3 for F-actin depolymerization activity, although the F-actin-binding sites remained exposed in the mutant G1-G3 as well as in the smaller truncates even in the Ca2+-free conditions at pH 8. Furthermore, unlike wild type G1-G3, calcium-sensitive mutants of G1-G3 acquired closed shapes in the absence of free calcium, implying a role of g2-g3 linker in determining the open F-actin depolymerizing-competent shape of G1-G3 in this condition. We demonstrate that the mobility of the G1 domain, essential for F-actin depolymerization, is indirectly regulated by the Gelsolin-like sequence of g2-g3 linker. Background: Shape-function studies are necessary to design better therapeutic alternatives of the plasma Gelsolin. Results: N-terminal fragment 30–161 is the smallest segment with F-actin depolymerization potential, and G1-G3 can function independent of Ca2+ ions or low pH. Conclusion: The g2-g3 linker plays a role in imparting pH/Ca2+ insensitivity to G1-G3. Significance: We provide the first evidence that g2-g3 linker regulates mobility of the G1 domain.
Neeraj Khatri - One of the best experts on this subject based on the ideXlab platform.
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protective effects of Gelsolin in acute pulmonary thromboembolism and thrombosis in the carotid artery of mice
PLOS ONE, 2019Co-Authors: Ashok Kumar Gupta, Amin Sagar, Bhupinder Singh Chopra, Bhavna Vaid, Sachin Raut, Maulik D Badmalia, Neeraj KhatriAbstract:The present study provides first evidence on the role of plasma Gelsolin in protecting pulmonary thromboembolism and thrombosis in a mouse model. Gelsolin is the most abundant actin depolymerizing protein in plasma and its significantly depleted values have been reported in metabolic disorders including cardiovascular diseases and myocardial infarction. Though Gelsolin replacement therapy (GRT) has been shown to be effective in some animal models, no such study has been reported for thrombotic diseases that are acutely in need of bio-therapeutics for immediate and lasting relief. Here, using mice model and recombinant human Gelsolin (rhuGSN), we demonstrate the antithrombotic effect of Gelsolin in ferric chloride induced thrombosis in carotid artery and thrombin induced acute pulmonary thromboembolism. In thrombosis model, arterial occlusion time was significantly enhanced upon subcutaneous (SC) treatment with 8 mg of Gelsolin per mice viz. 15.83 minutes vs. 8 minutes in the placebo group. Pertinently, histopathological examination showed channel formation within the thrombi in the carotid artery following injection of Gelsolin. Fluorescence molecular tomography imaging further confirmed that administration of Gelsolin reduced thrombus formation following carotid artery injury. In thrombin-induced acute pulmonary thromboembolism, mice pretreated with aspirin or Gelsolin showed 100 and 83.33% recovery, respectively. In contrast, complete mortality of mice was observed in vehicle treated group within 5 minutes of thrombin injection. Overall, our studies provide conclusive evidence on the thrombo-protective role of plasma Gelsolin in mice model of pulmonary thromboembolism and thrombosis.
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analgesic and anti inflammatory properties of Gelsolin in acetic acid induced writhing tail immersion and carrageenan induced paw edema in mice
PLOS ONE, 2015Co-Authors: Ashok Kumar Gupta, Amin Sagar, Vikas Choudhary, Renu Garg, Bhupinder Singh Chopra, Devraj Parasar, Neeraj KhatriAbstract:Plasma Gelsolin levels significantly decline in several disease conditions, since Gelsolin gets scavenged when it depolymerizes and caps filamentous actin released in the circulation following tissue injury. It is well established that our body require/implement inflammatory and analgesic responses to protect against cell damage and injury to the tissue. This study was envisaged to examine analgesic and anti-inflammatory activity of exogenous Gelsolin (8 mg/mouse) in mice models of pain and acute inflammation. Administration of Gelsolin in acetic acid-induced writhing and tail immersion tests not only demonstrated a significant reduction in the number of acetic acid-induced writhing effects, but also exhibited an analgesic activity in tail immersion test in mice as compared to placebo treated mice. Additionally, anti-inflammatory function of Gelsolin (8 mg/mouse) compared with anti-inflammatory drug diclofenac sodium (10 mg/kg)] was confirmed in the carrageenan injection induced paw edema where latter was measured by vernier caliper and fluorescent tomography imaging. Interestingly, results showed that plasma Gelsolin was capable of reducing severity of inflammation in mice comparable to diclofenac sodium. Analysis of cytokines and histo-pathological examinations of tissue revealed administration of Gelsolin and diclofenac sodium significantly reduced production of pro-inflammatory cytokines, TNF-α and IL-6. Additionally, carrageenan groups pretreated with diclofenac sodium or Gelsolin showed a marked decrease in edema and infiltration of inflammatory cells in paw tissue. Our study provides evidence that administration of Gelsolin can effectively reduce the pain and inflammation in mice model.
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global shapes of f actin depolymerization competent minimal Gelsolins insight into the role of g2 g3 linker in ph ca2 insensitivity of the first half
Journal of Biological Chemistry, 2013Co-Authors: Nagesh Peddada, Amin Sagar, Yogendra S Rathore, Vikas Choudhary, Bharat U K Pattnaik, Neeraj Khatri, Renu GargAbstract:Because of its ability to rapidly depolymerize F-actin, plasma Gelsolin has emerged as a therapeutic molecule in different disease conditions. High amounts of exogenous Gelsolin are, however, required to treat animal models of different diseases. Knowing that the F-actin depolymerizing property of Gelsolin resides in its N terminus, we made several truncated versions of plasma Gelsolin. The smaller versions, particularly the one composed of the first 28–161 residues, depolymerized the F-actin much faster than the native Gelsolin and other truncates at the same molar ratios. Although G1-G3 loses its dependence on Ca2+ or low pH for the actin depolymerization function, interestingly, G1-G2 and its smaller versions were found to regain this requirement. Small angle x-ray scattering-based shape reconstructions revealed that G1-G3 adopts an open shape in both the presence and the absence of Ca2+ as well as low pH, whereas G1-G2 and residues 28–161 prefer collapsed states in Ca2+-free conditions at pH 8. The mutations in the g2-g3 linker resulted in the calcium sensitivity of the mutant G1-G3 for F-actin depolymerization activity, although the F-actin-binding sites remained exposed in the mutant G1-G3 as well as in the smaller truncates even in the Ca2+-free conditions at pH 8. Furthermore, unlike wild type G1-G3, calcium-sensitive mutants of G1-G3 acquired closed shapes in the absence of free calcium, implying a role of g2-g3 linker in determining the open F-actin depolymerizing-competent shape of G1-G3 in this condition. We demonstrate that the mobility of the G1 domain, essential for F-actin depolymerization, is indirectly regulated by the Gelsolin-like sequence of g2-g3 linker.
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global shapes of f actin depolymerization competent minimal Gelsolins insight into the role of g2 g3 linker in ph ca2 insensitivity of the first half
Journal of Biological Chemistry, 2013Co-Authors: Nagesh Peddada, Amin Sagar, Yogendra S Rathore, Vikas Choudhary, Bharat U K Pattnaik, Neeraj Khatri, Renu GargAbstract:Because of its ability to rapidly depolymerize F-actin, plasma Gelsolin has emerged as a therapeutic molecule in different disease conditions. High amounts of exogenous Gelsolin are, however, required to treat animal models of different diseases. Knowing that the F-actin depolymerizing property of Gelsolin resides in its N terminus, we made several truncated versions of plasma Gelsolin. The smaller versions, particularly the one composed of the first 28–161 residues, depolymerized the F-actin much faster than the native Gelsolin and other truncates at the same molar ratios. Although G1-G3 loses its dependence on Ca2+ or low pH for the actin depolymerization function, interestingly, G1-G2 and its smaller versions were found to regain this requirement. Small angle x-ray scattering-based shape reconstructions revealed that G1-G3 adopts an open shape in both the presence and the absence of Ca2+ as well as low pH, whereas G1-G2 and residues 28–161 prefer collapsed states in Ca2+-free conditions at pH 8. The mutations in the g2-g3 linker resulted in the calcium sensitivity of the mutant G1-G3 for F-actin depolymerization activity, although the F-actin-binding sites remained exposed in the mutant G1-G3 as well as in the smaller truncates even in the Ca2+-free conditions at pH 8. Furthermore, unlike wild type G1-G3, calcium-sensitive mutants of G1-G3 acquired closed shapes in the absence of free calcium, implying a role of g2-g3 linker in determining the open F-actin depolymerizing-competent shape of G1-G3 in this condition. We demonstrate that the mobility of the G1 domain, essential for F-actin depolymerization, is indirectly regulated by the Gelsolin-like sequence of g2-g3 linker. Background: Shape-function studies are necessary to design better therapeutic alternatives of the plasma Gelsolin. Results: N-terminal fragment 30–161 is the smallest segment with F-actin depolymerization potential, and G1-G3 can function independent of Ca2+ ions or low pH. Conclusion: The g2-g3 linker plays a role in imparting pH/Ca2+ insensitivity to G1-G3. Significance: We provide the first evidence that g2-g3 linker regulates mobility of the G1 domain.
Alan R Fersht - One of the best experts on this subject based on the ideXlab platform.
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loss of a metal binding site in Gelsolin leads to familial amyloidosis finnish type
Nature Structural & Molecular Biology, 2002Co-Authors: Steven L Kazmirski, Rivka L Isaacson, Ashley M Buckle, Christopher M Johnson, Valerie Daggett, Alan R FershtAbstract:Mutations in domain 2 (D2, residues 151-266) of the actin-binding protein Gelsolin cause familial amyloidosis-Finnish type (FAF). These mutations, D187N or D187Y, lead to abnormal proteolysis of plasma Gelsolin at residues 172-173 and a second hydrolysis at residue 243, resulting in an amyloidogenic fragment. Here we present the structure of human Gelsolin D2 at 1.65 A and find that Asp 187 is part of a Cd2+ metal-binding site. Two Ca2+ ions are required for a conformational transition of Gelsolin to its active form. Differential scanning calorimetry (DSC) and molecular dynamics (MD) simulations suggest that the Cd2+-binding site in D2 is one of these two Ca2+-binding sites and is essential to the stability of D2. Mutation of Asp 187 to Asn disrupts Ca2+ binding in D2, leading to instabilities upon Ca2+ activation. These instabilities make the domain a target for aberrant proteolysis, thereby enacting the first step in the cascade leading to FAF.
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elucidating the mechanism of familial amyloidosis finnish type nmr studies of human Gelsolin domain 2
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Steven L Kazmirski, Rivka L Isaacson, Mark J Howard, Alan R FershtAbstract:Familial amyloidosis-Finnish type (FAF) results from a single mutation at residue 187 (D187N or D187Y) within domain 2 of the actin-regulating protein Gelsolin. The mutation somehow allows a masked cleavage site to be exposed, leading to the first step in the formation of an amyloidogenic fragment. We have performed NMR experiments investigating structural and dynamic changes between wild-type (WT) and D187N Gelsolin domain 2 (D2). On mutation, no significant structural or dynamic changes occur at or near the cleavage site. Areas in conformational exchange are observed between beta-strand 4 and alpha-helix 1 and within the loop region following beta-strand 5. Chemical shift differences are noted along the face of alpha-helix 1 that packs onto the beta-sheet, suggesting an altered conformation. Conformational changes within these areas can have an effect on actin binding and may explain why D187N Gelsolin is inactive. [(1)H-(15)N] nuclear Overhauser effect and chemical shift data suggest that the C-terminal tail of D187N Gelsolin D2 is less structured than WT by up to six residues. In the crystal structure of equine Gelsolin, the C-terminal tail of D2 lies across a large cleft between domains 1 and 2 where the masked cleavage site sits. We propose that the D187N mutation destabilizes the C-terminal tail of D2 resulting in a more exposed cleavage site leading to the first proteolysis step in the formation of the amyloidogenic fragment.
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equilibria and kinetics of folding of Gelsolin domain 2 and mutants involved in familial amyloidosis finnish type
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Rivka L Isaacson, Alan G. Weeds, Alan R FershtAbstract:Mutations D187N and D187Y in domain 2 of the actin-regulating protein Gelsolin cause familial amyloidosis–Finnish type (FAF). We have constructed and expressed a recombinant version of Gelsolin domain 2 that is sufficiently stable for kinetic and equilibrium measurements. The wild-type domain and the two amyloidogenic mutants fold via simple two-state kinetics without the accumulation of an intermediate. Unfolding kinetics exhibits significant curvature with increasing urea concentration, indicating that the transition state for unfolding becomes more native-like under increasingly denaturing conditions in accordance with the Hammond postulate. Mutations D187N and D187Y destabilize Gelsolin domain 2 by 1.22 and 2.16 kcal⋅mol−1 (1 kcal = 4.18 kJ) respectively. The mutations do not prevent disulfide bond formation despite their direct contiguity with a cysteine residue involved in disulfide linkage. The destabilization conferred on Gelsolin domain 2 by the FAF mutations is sufficient to predict that an appreciable fraction is unfolded and, therefore, extremely susceptible to proteolysis at body temperature.
Amin Sagar - One of the best experts on this subject based on the ideXlab platform.
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protective effects of Gelsolin in acute pulmonary thromboembolism and thrombosis in the carotid artery of mice
PLOS ONE, 2019Co-Authors: Ashok Kumar Gupta, Amin Sagar, Bhupinder Singh Chopra, Bhavna Vaid, Sachin Raut, Maulik D Badmalia, Neeraj KhatriAbstract:The present study provides first evidence on the role of plasma Gelsolin in protecting pulmonary thromboembolism and thrombosis in a mouse model. Gelsolin is the most abundant actin depolymerizing protein in plasma and its significantly depleted values have been reported in metabolic disorders including cardiovascular diseases and myocardial infarction. Though Gelsolin replacement therapy (GRT) has been shown to be effective in some animal models, no such study has been reported for thrombotic diseases that are acutely in need of bio-therapeutics for immediate and lasting relief. Here, using mice model and recombinant human Gelsolin (rhuGSN), we demonstrate the antithrombotic effect of Gelsolin in ferric chloride induced thrombosis in carotid artery and thrombin induced acute pulmonary thromboembolism. In thrombosis model, arterial occlusion time was significantly enhanced upon subcutaneous (SC) treatment with 8 mg of Gelsolin per mice viz. 15.83 minutes vs. 8 minutes in the placebo group. Pertinently, histopathological examination showed channel formation within the thrombi in the carotid artery following injection of Gelsolin. Fluorescence molecular tomography imaging further confirmed that administration of Gelsolin reduced thrombus formation following carotid artery injury. In thrombin-induced acute pulmonary thromboembolism, mice pretreated with aspirin or Gelsolin showed 100 and 83.33% recovery, respectively. In contrast, complete mortality of mice was observed in vehicle treated group within 5 minutes of thrombin injection. Overall, our studies provide conclusive evidence on the thrombo-protective role of plasma Gelsolin in mice model of pulmonary thromboembolism and thrombosis.
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analgesic and anti inflammatory properties of Gelsolin in acetic acid induced writhing tail immersion and carrageenan induced paw edema in mice
PLOS ONE, 2015Co-Authors: Ashok Kumar Gupta, Amin Sagar, Vikas Choudhary, Renu Garg, Bhupinder Singh Chopra, Devraj Parasar, Neeraj KhatriAbstract:Plasma Gelsolin levels significantly decline in several disease conditions, since Gelsolin gets scavenged when it depolymerizes and caps filamentous actin released in the circulation following tissue injury. It is well established that our body require/implement inflammatory and analgesic responses to protect against cell damage and injury to the tissue. This study was envisaged to examine analgesic and anti-inflammatory activity of exogenous Gelsolin (8 mg/mouse) in mice models of pain and acute inflammation. Administration of Gelsolin in acetic acid-induced writhing and tail immersion tests not only demonstrated a significant reduction in the number of acetic acid-induced writhing effects, but also exhibited an analgesic activity in tail immersion test in mice as compared to placebo treated mice. Additionally, anti-inflammatory function of Gelsolin (8 mg/mouse) compared with anti-inflammatory drug diclofenac sodium (10 mg/kg)] was confirmed in the carrageenan injection induced paw edema where latter was measured by vernier caliper and fluorescent tomography imaging. Interestingly, results showed that plasma Gelsolin was capable of reducing severity of inflammation in mice comparable to diclofenac sodium. Analysis of cytokines and histo-pathological examinations of tissue revealed administration of Gelsolin and diclofenac sodium significantly reduced production of pro-inflammatory cytokines, TNF-α and IL-6. Additionally, carrageenan groups pretreated with diclofenac sodium or Gelsolin showed a marked decrease in edema and infiltration of inflammatory cells in paw tissue. Our study provides evidence that administration of Gelsolin can effectively reduce the pain and inflammation in mice model.
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global shapes of f actin depolymerization competent minimal Gelsolins insight into the role of g2 g3 linker in ph ca2 insensitivity of the first half
Journal of Biological Chemistry, 2013Co-Authors: Nagesh Peddada, Amin Sagar, Yogendra S Rathore, Vikas Choudhary, Bharat U K Pattnaik, Neeraj Khatri, Renu GargAbstract:Because of its ability to rapidly depolymerize F-actin, plasma Gelsolin has emerged as a therapeutic molecule in different disease conditions. High amounts of exogenous Gelsolin are, however, required to treat animal models of different diseases. Knowing that the F-actin depolymerizing property of Gelsolin resides in its N terminus, we made several truncated versions of plasma Gelsolin. The smaller versions, particularly the one composed of the first 28–161 residues, depolymerized the F-actin much faster than the native Gelsolin and other truncates at the same molar ratios. Although G1-G3 loses its dependence on Ca2+ or low pH for the actin depolymerization function, interestingly, G1-G2 and its smaller versions were found to regain this requirement. Small angle x-ray scattering-based shape reconstructions revealed that G1-G3 adopts an open shape in both the presence and the absence of Ca2+ as well as low pH, whereas G1-G2 and residues 28–161 prefer collapsed states in Ca2+-free conditions at pH 8. The mutations in the g2-g3 linker resulted in the calcium sensitivity of the mutant G1-G3 for F-actin depolymerization activity, although the F-actin-binding sites remained exposed in the mutant G1-G3 as well as in the smaller truncates even in the Ca2+-free conditions at pH 8. Furthermore, unlike wild type G1-G3, calcium-sensitive mutants of G1-G3 acquired closed shapes in the absence of free calcium, implying a role of g2-g3 linker in determining the open F-actin depolymerizing-competent shape of G1-G3 in this condition. We demonstrate that the mobility of the G1 domain, essential for F-actin depolymerization, is indirectly regulated by the Gelsolin-like sequence of g2-g3 linker.
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global shapes of f actin depolymerization competent minimal Gelsolins insight into the role of g2 g3 linker in ph ca2 insensitivity of the first half
Journal of Biological Chemistry, 2013Co-Authors: Nagesh Peddada, Amin Sagar, Yogendra S Rathore, Vikas Choudhary, Bharat U K Pattnaik, Neeraj Khatri, Renu GargAbstract:Because of its ability to rapidly depolymerize F-actin, plasma Gelsolin has emerged as a therapeutic molecule in different disease conditions. High amounts of exogenous Gelsolin are, however, required to treat animal models of different diseases. Knowing that the F-actin depolymerizing property of Gelsolin resides in its N terminus, we made several truncated versions of plasma Gelsolin. The smaller versions, particularly the one composed of the first 28–161 residues, depolymerized the F-actin much faster than the native Gelsolin and other truncates at the same molar ratios. Although G1-G3 loses its dependence on Ca2+ or low pH for the actin depolymerization function, interestingly, G1-G2 and its smaller versions were found to regain this requirement. Small angle x-ray scattering-based shape reconstructions revealed that G1-G3 adopts an open shape in both the presence and the absence of Ca2+ as well as low pH, whereas G1-G2 and residues 28–161 prefer collapsed states in Ca2+-free conditions at pH 8. The mutations in the g2-g3 linker resulted in the calcium sensitivity of the mutant G1-G3 for F-actin depolymerization activity, although the F-actin-binding sites remained exposed in the mutant G1-G3 as well as in the smaller truncates even in the Ca2+-free conditions at pH 8. Furthermore, unlike wild type G1-G3, calcium-sensitive mutants of G1-G3 acquired closed shapes in the absence of free calcium, implying a role of g2-g3 linker in determining the open F-actin depolymerizing-competent shape of G1-G3 in this condition. We demonstrate that the mobility of the G1 domain, essential for F-actin depolymerization, is indirectly regulated by the Gelsolin-like sequence of g2-g3 linker. Background: Shape-function studies are necessary to design better therapeutic alternatives of the plasma Gelsolin. Results: N-terminal fragment 30–161 is the smallest segment with F-actin depolymerization potential, and G1-G3 can function independent of Ca2+ ions or low pH. Conclusion: The g2-g3 linker plays a role in imparting pH/Ca2+ insensitivity to G1-G3. Significance: We provide the first evidence that g2-g3 linker regulates mobility of the G1 domain.