Renu Garg

Plasma gelsolin: A general prognostic marker of health

Plasma gelsolin (pGSN) is the only component of two member extracellular actin scavenger system capable of severing circulating actin microfilaments. Here, we put forth the hypothesis that pGSN level is an important and sensitive general prognostic biomarker for health and disease conditions in humans, urging the need for gelsolin replacement therapy to improve patients health status. Clinical significance and the therapeutic importance of this protein have been well illustrated in animal models as well as in patients with various diseases. Patients with decreased pGSN levels were observed to have higher mortality rate, longer hospital stay and longer ventilation time in intensive care units as compared to healthy controls. pGSN levels were found to be increasing in patients recovering from diseases; furthermore, it has been confirmed that repletion with exogenous recombinant pGSN increases the survival rate in animal models of different acute insults. To be used as a biomarker of health, however, establishing the accurate levels of gelsolin in human plasma and understanding its variance with age, race, gender and health status is a prerequisite. Upon establishing the accurate levels of pGSN in healthy individuals, this biomarker would predict the prognosis/disease progression in multiple health conditions and help in prioritizing the ones in-need of gelsolin replacement therapy.

Interactive Roles of Ets-1, Sp1, and Acetylated Histones in the Retinoic Acid-dependent Activation of Guanylyl Cyclase/Atrial Natriuretic Peptide Receptor-A Gene Transcription

Cardiac hormones atrial and brain natriuretic peptides activate guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), which plays a critical role in reduction of blood pressure and blood volume. Currently, the mechanisms responsible for regulating the Npr1 gene (coding for GC-A/NPRA) transcription are not well understood. The present study was conducted to examine the interactive roles of all-trans retinoic acid (ATRA), Ets-1, Sp1, and histone acetylation on the transcriptional regulation and function of the Npr1 gene. Deletion analysis of the Npr1 promoter and luciferase assays showed that ATRA enhanced a 16-fold Npr1 promoter activity and greatly stimulated guanylyl cyclase (GC) activity of the receptor protein in both atrial natriuretic peptide (ANP)-dependent and -independent manner. As confirmed by gel shift and chromatin immunoprecipitation assays, ATRA enhanced the binding of both Ets-1 and Sp1 to the Npr1 promoter. The retinoic acid receptor α (RARα) was recruited by Ets-1 and Sp1 to form a transcriptional activator complex with their binding sites in the Npr1 promoter. Interestingly, ATRA also increased the acetylation of histones H3 and H4 and enhanced their recruitment to Ets-1 and Sp1 binding sites within the Npr1 promoter. Collectively, the present results demonstrate that ATRA regulates Npr1 gene transcription and GC activity of the receptor by involving the interactive actions of Ets-1, Sp1, and histone acetylation.

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.

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. 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.

Analgesic and Anti-Inflammatory Properties of Gelsolin in Acetic Acid Induced Writhing, Tail Immersion and Carrageenan Induced Paw Edema in Mice.

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.

Subcellular trafficking of guanylyl cyclase/natriuretic peptide receptor-A with concurrent generation of intracellular cGMP.

Atrial natriuretic peptide (ANP) modulates blood pressure and fluid volume by activation of natriuretic peptide receptor-A (NPRA). Immunofluorescence (IF) studies reveal that NPRA is internalized and redistributed into subcellular compartments with concurrent production of cGMP.

Visualizing the elusive open shape of G-actin in solution by SAXS data analysis.

Though biochemical data upholds that ATP hydrolysis induces an opening of the nucleotide binding cleft, crystal structures of the G-actin in the absence of profillin represent the closed structure, regardless of the bound ATP/ADP. Analysis of small angle X-ray scattering (SAXS) intensities confirmed that ATP hydrolysis increases the radius of gyration (R(G)) and maximum linear dimension (D(max)) of G-actin molecules from 22.3 to 23.7 Ǻ and 70 to 78 Å, respectively. Kratky analysis confirmed that G-actin molecules behave like globular scattering particles regardless of the bound nucleotide state. Shape reconstruction using dummy residues and inertial axes overlay with known crystal structures confirmed that the ATP or AMP-PNP bound G-actin adopts a compact shape, and the nucleotide binding site opens up with ATP hydrolysis. Importantly, our ADP-state model resembled the open shape seen for β-actin and hexokinase.

Plasma Gelsolin Levels Decrease in Diabetic State and Increase upon Treatment with F-Actin Depolymerizing Versions of Gelsolin

The study aims to map plasma gelsolin (pGSN) levels in diabetic humans and mice models of type II diabetes and to evaluate the efficacy of gelsolin therapy in improvement of diabetes in mice. We report that pGSN values decrease by a factor of 0.45 to 0.5 in the blood of type II diabetic humans and mice models. Oral glucose tolerance test in mice models showed that subcutaneous administration of recombinant pGSN and its F-actin depolymerizing competent versions brought down blood sugar levels comparable to Sitagliptin, a drug used to manage hyperglycemic condition. Further, daily dose of pGSN or its truncated versions to diabetic mice for a week kept sugar levels close to normal values. Also, diabetic mice treated with Sitagliptin for 7 days, showed increase in their pGSN values with the decrease in blood glucose as compared to their levels at the start of treatment. Gelsolin helped in improving glycemic control in diabetic mice. We propose that gelsolin level monitoring and replacement of F-actin severing capable gelsolin(s) should be considered in diabetic care.

Pregnancy-related hormones, progesterone and human chorionic gonadotrophin, upregulate expression of maternal plasma gelsolin

Plasma gelsolin (pGSN), a protein primarily involved in clearance of circulating actin filaments, is an upcoming novel biomarker. Its level changes in multiple disease and injury conditions, attributable mainly to its consumption during actin clearance; the endogenous regulation of its expression, however, remains elusive as well as unexplored. Here, we are reporting the first isolation of the promoter region of pGSN gene and investigation of its transcriptional regulation during pregnancy (a natural process associated with a well-programmed injury course of parturition). Interestingly, two of the pregnancy-related hormones, human chorionic gonadotrophin (hCG) and progesterone, significantly upregulated pGSN promoter activity in muscle cells. This action of both hormones was found to mediate through their respective cellular receptors and involved a contribution of multiple signaling pathways including those of protein kinase A, protein kinase C, epidermal growth factor receptor and prostaglandin-endoperoxidase synthase 2 in the case of hCG-mediated upregulation. This novel upregulation was further supported by elevated levels of endogenous pGSN transcripts as well as secreted protein upon hormonal treatments of muscle cells compared with untreated controls. A participation of pGSN promoter cis-elements, capable of interacting with endogenous transcription factors, Ap1, Sp1, and p300, was also observed during this hormonal upregulation. Additionally, the augmented pGSN levels observed in pregnant mice compared with the control animals further supported an upregulation of this protein during pregnancy, implicating vital role(s) played by pGSN during this period in mammals.

Visualizing Temperature Mediated Activation of Gelsolin and Its Deactivation By Pip 2 : A Saxs Based Study

This is the first report describing temperature based initiation of gelsolin’s F-actin depolymerization activity, even in absence of free Ca2+ or low pH. Small angle X-ray scattering (SAXS) and circular dichroism (CD) studies revealed that temperature in the range of 30–40 °C is capable of opening the G1 domain alone, as remaining domains are held together by the Ca2+-sensitive C-tail latch without any loss in the secondary structural content. Full opening of all domains of tail-less gelsolin, and retention of closed shape for G2–G6 gelsolin merely by heating, further substantiated our findings. The Ca2+/pH independent activity of gelsolin near physiological temperature brought out a query: whether gelsolin is always active, and if not, what might deactivate it? Earlier, PIP2 has been reported to render gelsolin inactive with no structural insight. Reduction in shape parameters and modeling revealed that PIP2 reverses the temperature induced extension of g1-g2 linker leading to a compact shape seen for Ca2+-free gelsolin. Similar results for partially activated gelsolin (by low pH or Ca2+ ions below 0.1 μM) imply that inside cells, depolymerization, capping, and nucleation of F-actin by gelsolin is regulated by the culmination of local Ca2+ ion concentration, pH, temperature and PIP2 levels.

Bonsai Gelsolin Survives Heat Induced Denaturation by Forming β-Amyloids which Leach Out Functional Monomer

Here, we report that minimal functional gelsolin i.e. fragment 28–161 can display F-actin depolymerizing property even after heating the protein to 80 °C. Small angle X-ray scattering (SAXS) data analysis confirmed that under Ca2+-free conditions, 28–161 associates into monomer to dimer and tetramer, which later forms β-amyloids, but in presence of Ca2+, it forms dimers which proceed to non-characterizable aggregates. The dimeric association also explained the observed decrease in ellipticity in circular dichroism experiments with increase in temperature. Importantly, SAXS data based models correlated well with our crystal structure of dimeric state of 28–161. Characterization of higher order association by electron microscopy, Congo red and ThioflavinT staining assays further confirmed that only in absence of Ca2+ ions, heating transforms 28–161 into β-amyloids. Gel filtration and other experiments showed that β-amyloids keep leaching out the monomer, and the release rates could be enhanced by addition of L-Arg to the amyloids. F-actin depolymerization showed that addition of Ca2+ ions to released monomer initiated the depolymerization activity. Overall, we propose a way to compose a supramolecular assembly which releases functional protein in sustained manner which can be applied for varied potentially therapeutic interventions.