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Cross-presentation of antigens from dead tumor cells by type 1 conventional dendritic cells (cDC1s) is thought to underlie priming of anti-cancer CD8 + T cells. cDC1 express high levels of DNGR-1 (a.k.a. CLEC9A), a receptor that binds to F-actin exposed by dead cell debris and promotes cross-presentation of associated antigens. Here, we show that secreted gelsolin (sGSN), an extracellular protein, decreases DNGR-1 binding to F-actin and cross-presentation of dead cell-associated antigens by cDC1s. Mice deficient in sGsn display increased DNGR-1-dependent resistance to transplantable tumors, especially ones expressing neoantigens associated with the actin cytoskeleton, and exhibit greater responsiveness to cancer immunotherapy. In human cancers, lower levels of intratumoral sGSN transcripts, as well as presence of mutations in proteins associated with the actin cytoskeleton, are associated with signatures of anti-cancer immunity and increased patient survival. Our results reveal a natural barrier to cross-presentation of cancer antigens that dampens anti-tumor CD8 + T cell responses., Competing Interests: Declaration of interests E.G., O.S., K.H.J.L., N.S., O.G., S.Z., S.S., P.C., and C.R.S. are named as inventors on a patent application on the use of sGSN for immunotherapies. C.R.S. owns stock options and/or is a paid consultant for Bicara Therapeutics, Montis Biosciences, Oncurious NV, Bicycle Therapeutics, and Sosei Heptares. C.R.S. holds a professorship at Imperial College London and honorary professorships at University College London and King’s College London. None of these activities are related to this work., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Skin melanocytes reside on the basement membrane (BM), which is mainly composed of laminin, collagen type IV, and proteoglycans. For melanoma cells, in order to invade into the skin, melanocytes must cross the BM. It has been reported that changes in the composition of the BM accompany melanocytes tumorigenesis. Previously, we reported high gelsolin (GSN)-an actin-binding protein-levels in melanoma cell lines and GSN's importance for migration of A375 cells. Here we investigate whether melanoma cells migrate differently depending on the type of fibrous extracellular matrix protein. We obtained A375 melanoma cells deprived of GSN synthesis and tested their migratory properties on laminin, collagens type I and IV, fibronectin, and Matrigel, which resembles the skin's BM. We applied confocal and structured illuminated microscopy (SIM), gelatin degradation, and diverse motility assays to assess GSN's influence on parameters associated with cells' ability to protrude. We show that GSN is important for melanoma cell migration, predominantly on laminin, which is one of the main components of the skin's BM.

Gelsolin (GSN) variants have been implicated in amyloidosis of the Finnish type. This case series reports a novel GSN:c.1477T>C,p.(Trp493Arg) variant in a family with ocular and systemic features consistent with Finnish Amyloidosis. Exome sequencing performed on affected individuals from two families manifesting cutis laxa and polymorphic corneal stromal opacities demonstrated the classic GSN:c.654G>A,p.Asp214Asn variant in single affected individual from one family, and a previously undocumented GSN:c.1477T>C variant in three affected first-degree relatives from a separate family. Immunohistochemical studies on corneal tissue from a proband with the c.1477T>C variant identified gelsolin protein within histologically defined corneal amyloid deposits. This study reports a novel association between the predicted pathogenic GSN:c.1477T>C variant and amyloidosis of the Finnish type, and is the first to provide functional evidence of a pathological GSN variant at a locus distant to the critical G2 calcium-binding region, resulting in the phenotype of amyloidosis of the Finnish type., (© 2021 Wiley Periodicals LLC.)

Purpose: AGel amyloidosis is systemic amyloidosis caused by pathogenic variants in the GSN gene. In this study, we sought to characterize the clinical and brain magnetic resonance image (MRI) features of Korean patients with AGel amyloidosis., Materials and Methods: We examined 13 patients with AGel amyloidosis from three unrelated families. Brain MRIs were performed in eight patients and eight age- and sex-matched healthy controls. Therein, we analyzed gray and white matter content using voxel-based morphometry (VBM), tract-based spatial statistics (TBSS), and FreeSurfer., Results: The median age at examination was 73 (interquartile range: 64-76) years. The median age at onset of cutis laxa was 20 (interquartile range: 15-30) years. All patients over that age of 60 years had dysarthria, cutis laxa, dysphagia, and facial palsy. Two patients in their 30s had only mild cutis laxa. The median age at dysarthria onset was 66 (interquartile range: 63.5-70) years. Ophthalmoparesis was observed in three patients. No patient presented with muscle weakness of the limbs. Axial fluid-attenuated inversion recovery images of the brain showed no significant differences between the patient and control groups. Also, analysis of VBM, TBSS, and FreeSurfer revealed no significant differences in cortical thickness between patients and healthy controls at the corrected significance level., Conclusion: Our study outlines the clinical manifestations of prominent bulbar palsy and early-onset cutis laxa in 13 Korean patients with AGel amyloidosis and confirms that AGel amyloidosis mainly affects the peripheral nervous system rather than the central nervous system., Competing Interests: The authors have no potential conflicts of interest to disclose., (© Copyright: Yonsei University College of Medicine 2021.)

Background: Colorectal cancer (CRC) is the most common type of gastrointestinal malignant tumour. Colorectal adenocarcinoma (COAD) - the most common type of CRC - is particularly dangerous. The role of the immune system in the development of tumour-associated inflammation and cancer has received increasing attention recently., Methods: In the present study, we compiled the expression profiles of 262 patients with complete follow-up data from The Cancer Genome Atlas (TCGA) database as an experimental group and selected 65 samples from the Gene Expression Omnibus (GEO) dataset (of which 46 samples were with M0) as a verification group. First, we screened the immune T helper 17 (Th17) cells related to the prognosis of COAD. Subsequently, we identified Th17 cells-related hub genes by utilising Weighted Gene Co-expression Network Analysis (WGCNA) and Least Absolute Shrinkage and Selector Operation (LASSO) regression analysis. Six genes associated with the prognosis in patients with COAD were identified, including: KRT23, ULBP2, ASRGL1, SERPINA1, SCIN, and SLC28A2. We constructed a clinical prediction model and analysed its predictive power., Results: The identified hub genes are involved in developing many diseases and closely linked to digestive disorders. Our results suggested that the hub genes could influence the prognosis of COAD by regulating Th17 cells' infiltration., Conclusions: These newly discovered hub genes contribute to clarifying the mechanisms of COAD development and metastasis. Given that they promote COAD development, they may become new therapeutic targets and biomarkers of COAD., (© 2021 The Author(s).)

Competing Interests: The authors declare that they have no conflict of interest.

Actin is a major intracellular protein with key functions in cellular motility, signaling, and structural rearrangements. Its dynamic behavior, such as polymerization and depolymerization of actin filaments in response to intracellular and extracellular cues, is regulated by an abundance of actin binding proteins. Out of these, gelsolin is one of the most potent for filament severing. However, myosin motor activity also fragments actin filaments through motor-induced forces, suggesting that these two proteins could cooperate to regulate filament dynamics and motility. To test this idea, we used an in vitro motility assay, where actin filaments are propelled by surface-adsorbed heavy meromyosin (HMM) motor fragments. This allows studies of both motility and filament dynamics using isolated proteins. Gelsolin, at both nanomolar and micromolar Ca 2+ concentration, appreciably enhanced actin filament severing caused by HMM-induced forces at 1 mM MgATP, an effect that was increased at higher HMM motor density. This finding is consistent with cooperativity between actin filament severing by myosin-induced forces and by gelsolin. We also observed reduced sliding velocity of the HMM-propelled filaments in the presence of gelsolin, providing further support of myosin-gelsolin cooperativity. Total internal reflection fluorescence microscopy-based single molecule studies corroborated that the velocity reduction was a direct effect of gelsolin binding to the filament and revealed different filament severing pattern of stationary and HMM propelled filaments. Overall, the results corroborate cooperative effects between gelsolin-induced alterations in the actin filaments and changes due to myosin motor activity leading to enhanced F-actin severing of possible physiological relevance., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Gelsolin is a calcium-regulated actin binding protein that severs and caps actin filaments. Gelsolin's severing activity is important for regulating actin filament assembly dynamics that are required for cell motility as well as survival. The majority of in vitro studies of gelsolin have been performed in dilute buffer conditions which do not simulate the molecular interactions occurring in the crowded intracellular environment. We hypothesize that crowding results in greater gelsolin severing activity due to induced conformational changes in actin filaments and/or gelsolin. In this study, we evaluated the effects of crowding on gelsolin-mediated actin filament severing and gelsolin binding to actin filaments in crowded solutions, utilizing total internal reflection fluorescence (TIRF) microscopy and co-sedimentation assays. Our data indicates that the presence of crowders causes a decrease in the rate of gelsolin severing as well as a decrease in the amount of gelsolin bound to actin filaments, with greater effects caused by the polymeric crowder. Despite the severing rate decrease, gelsolin-mediated filament severing is increased in the presence of crowders. Understanding the crowding effect on gelsolin-mediated actin filament severing offers insight into the interactions between gelsolin and actin that occur inside the crowded cytoplasm., Competing Interests: Declaration of competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The emergent properties of the array arrangement of the molecular motor myosin II in the sarcomere of the striated muscle, the generation of steady force and shortening, can be studied in vitro with a synthetic nanomachine made of an ensemble of eight heavy-meromyosin (HMM) fragments of myosin from rabbit psoas muscle, carried on a piezoelectric nanopositioner and brought to interact with a properly oriented actin filament attached via gelsolin (a Ca 2+ -regulated actin binding protein) to a bead trapped by dual laser optical tweezers. However, the application of the original version of the nanomachine to investigate the Ca 2+ -dependent regulation mechanisms of the other sarcomeric (regulatory or cytoskeleton) proteins, adding them one at a time, was prevented by the impossibility to preserve [Ca 2+ ] as a free parameter. Here, the nanomachine is implemented by assembling the bead-attached actin filament with the Ca 2+ -insensitive gelsolin fragment TL40. The performance of the nanomachine is determined both in the absence and in the presence of Ca 2+ (0.1 mM, the concentration required for actin attachment to the bead with gelsolin). The nanomachine exhibits a maximum power output of 5.4 aW, independently of [Ca 2+ ], opening the possibility for future studies of the Ca 2+ -dependent function/dysfunction of regulatory and cytoskeletal proteins.

Cell migration requires reposition and reshaping of the cell nucleus. The nuclear lamina is highly important for migration of both primary and cancer cells. B-type lamins are important for proper migration of epicardial cells and neurons and increased lamin B to lamin A ratio accelerates cancer cell migration through confined spaces. Moreover, a positive association between lamin B1 levels and tumor formation and progression is found in various cancer types. Still, the molecular mechanism by which B-type lamins promote cell migration is not fully understood. To better understand this mechanism, we tested the effects of lamin B1 on perinuclear actin organization. Here we show that induction of melanoma cell migration leads to the formation of a cytosolic Linker of Nucleoskeleton and Cytoskeleton (LINC) complex-independent perinuclear actin rim, which has not been detected in migrating cells, yet. Significantly, increasing the levels of lamin B1 but not the levels of lamin A prevented perinuclear actin rim formation while accelerated the cellular migration rate. To interfere with the perinuclear actin rim, we generated a chimeric protein that is localized to the outer nuclear membrane and cleaves perinuclear actin filaments in a specific manner without disrupting other cytosolic actin filaments. Using this tool, we found that disruption of the perinuclear actin rim accelerated the cellular migration rate in a similar manner to lamin B1 over-expression. Taken together, our results suggest that increased lamin B1 levels can accelerate cell migration by inhibiting the association of the nuclear envelope with actin filaments that may reduce nuclear movement and deformability.

Accumulation of amyloid-beta (Aβ) in the brain is thought to derive from the impairment of Aβ clearance mechanisms rather than from its overproduction, which consequently contributes to the development of Alzheimer's disease. The choroid plexus epithelial cells constitute an important clearance route for Aβ, either by facilitating its transport from the cerebrospinal fluid to the blood, or by synthesizing and secreting various proteins involved in Aβ degradation. Impaired choroid plexus synthesis, secretion, and transport of these Aβ-metabolizing enzymes have been therefore associated with the disruption of Aβ homeostasis and amyloid load. Factors such as aging, female gender, and circadian rhythm disturbances are related to the decline of choroid plexus functions that may be involved in the modulation of Aβ-clearance mechanisms. In this study, we investigated the impact of age, sex hormones, and circadian rhythm on the expression of Aβ scavengers such as apolipoprotein J, gelsolin, and transthyretin at the rat choroid plexus. Our results demonstrated that mRNA expression and both intracellular and secreted protein levels of the studied Aβ scavengers are age-, sex-, and circadian-dependent. These data suggest that the Aβ-degradation and clearance pathways at the choroid plexus, mediated by the presence of Aβ scavengers, might be compromised as a consequence of aging and circadian disturbances. These are important findings that enhance the understanding of Aβ-clearance-regulating mechanisms at the blood-cerebrospinal fluid barrier.

Although initial treatment of ovarian cancer is successful, tumors typically relapse and become resistant to treatment. Because of poor infiltration of effector T cells, patients are mostly unresponsive to immunotherapy. Plasma gelsolin (pGSN) is transported by exosomes (small extracellular vesicle, sEV) and plays a key role in ovarian cancer chemoresistance, yet little is known about its role in immunosurveillance. Here, we report the immunomodulatory roles of sEV-pGSN in ovarian cancer chemoresistance. In chemosensitive conditions, secretion of sEV-pGSN was low, allowing for optimal CD8 + T-cell function. This resulted in increased T-cell secretion of IFNγ, which reduced intracellular glutathione (GSH) production and sensitized chemosensitive cells to cis-diaminedichloroplatinum (CDDP)-induced apoptosis. In chemoresistant conditions, increased secretion of sEV-pGSN by ovarian cancer cells induced apoptosis in CD8 + T cells. IFNγ secretion was therefore reduced, resulting in high GSH production and resistance to CDDP-induced death in ovarian cancer cells. These findings support our hypothesis that sEV-pGSN attenuates immunosurveillance and regulates GSH biosynthesis, a phenomenon that contributes to chemoresistance in ovarian cancer. SIGNIFICANCE: These findings provide new insight into pGSN-mediated immune cell dysfunction in ovarian cancer chemoresistance and demonstrate how this dysfunction can be exploited to enhance immunotherapy., (©2020 American Association for Cancer Research.)

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.

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., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

We have previously shown that gelsolin (GSN) levels are significantly lower in the blood of patients with glioblastoma (GBM) than in healthy controls. Here, we analyzed the function of GSN in GBM and examined its clinical significance. Furthermore, microRNAs involved in GSN expression were also identified. The expression of GSN was determined using western blot analysis and found to be significantly lower in GBM samples than normal ones. Gelsolin was mainly localized in normal astrocytes, shown using immunohistochemistry and immunofluorescence. Higher expression of GSN was correlated with more prolonged progression-free survival and overall survival. Gelsolin knockdown using siRNA and shRNA markedly accelerated cell proliferation and invasion in GBM in vitro and in vivo. The inactive form of glycogen synthase kinase-3β was dephosphorylated by GSN knockdown. In GBM tissues, the expression of GSN and microRNA (miR)-654-5p and miR-450b-5p showed an inverse correlation. The miR-654-5p and miR-450b-5p inhibitors enhanced GSN expression, resulting in reduced proliferation and invasion. In conclusion, GSN, which inhibits cell proliferation and invasion, is suppressed by miR-654-5p and miR-450b-5p in GBM, suggesting that these miRNAs can be targets for treating GBM., (© 2020 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Persistent infection with high-risk strains of human papillomavirus (HPV) is the primary cause of cervical cancer, the fourth most common cancer among women worldwide. Two oncoproteins encoded by the HPV genome, E6 and E7, are required for epigenetic modifications that promote cervical cancer development. We found that knockdown of HPV E6/E7 by siRNA reduced the levels of ubiquitin-like containing PHD and RING finger domain 1 (UHRF1) but increased the levels of gelsolin (GSN) in early stage cervical cancer cells. In addition, we found that UHRF1 levels were increased and GSN levels were decreased in early stage cervical cancer compared with those in normal cervical tissues, as shown by Western blot analysis, immunohistochemistry, and analysis of the Oncomine database. Moreover, knockdown of UHRF1 resulted in increased cell death in cervical cancer cell lines. Treatment of E6/E7-transformed HaCaT (HEK001) cells and HeLa cells with the DNA-hypomethylating agent 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor Trichostatin A increased GSN expression levels. UHRF1 knockdown in HEK001 cells by siRNA or the UHRF1 antagonist thymoquinone increased GSN levels, induced cell cycle arrest and apoptosis, and increased the levels of p27 and cleaved PARP. Those results indicate that upregulation of UHRF1 by HPV E6/E7 causes GSN silencing and a reduction of cell death in early stage cervical cancer, suggesting that GSN might be a useful therapeutic target in early stage cervical cancer., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The dynamics and organization of the actin cytoskeleton are crucial to many cellular events such as motility, polarization, cell shaping, and cell division. The intracellular and extracellular signaling associated with this cytoskeletal network is communicated through cell membranes. Hence the organization of membrane macromolecules and actin filament assembly are highly interdependent. Although the actin-membrane linkage is known to happen through many routes, the major class of interactions is through the direct interaction of actin-binding proteins with the lipid class containing poly-phosphatidylinositols (PPIs). Among the PPIs, phosphatidylinositol bisphosphate (PI(4,5)P 2 ) acts as a significant factor controlling actin polymerization in the proximity of the membrane by binding to actin-associated proteins. The molecular interactions between these actin-binding proteins and the membrane lipids remain elusive. Here, using molecular modeling, analytical theory, and experimental methods, we investigate the binding of three different actin-binding proteins, mDia2, NWASP, and gelsolin, to membranes containing PI(4,5)P 2 lipids. We perform molecular dynamics simulations on the protein-bilayer system and analyze the membrane binding in the form of hydrogen bonds and salt bridges at various PI(4,5)P 2 and cholesterol concentrations. Our experimental study with PI(4,5)P 2 -containing large unilamellar vesicles mimics the computational experiments. Using the multivalencies of the proteins obtained in molecular simulations and the cooperative binding mechanisms of the proteins, we also propose a multivalent binding model that predicts the actin filament distributions at various PI(4,5)P 2 and protein concentrations.

Background: Psoriatic arthritis (PsA) is an inflammatory disorder affecting the joints of psoriatic patients. Gelsolin regulated the actin assembly and disassembly. Reduction in plasma gelsolin levels was detected in tissue damages, including trauma, sepsis, and chronic inflammatory disorders., Objectives: The study aims to investigate the potential role of gelsolin in PsA and to determine the association between gelsolin and the disease activity., Methods: Plasma gelsolin levels were measured in 76 PsA patients in comparison with 40 patients having psoriasis only and 40 age- and sex -matched healthy controls., Results: Plasma gelsolin levels were decreased in PsA patients compared to controls and psoriasis-only patients (p ˂ 0.0001). The optimal cutoff point of gelsolin was 172.5 mg/L. Gelsolin showed 92.1% sensitivity and 95% specificity in detecting PsA. But, it had 92.1% sensitivity and 80% specificity in differentiating between psoriasis and PsA. Plasma gelsolin showed a significant negative correlation with inflammatory markers as C-reactive protein and erythrocyte sedimentation rate (p < 0.0001 and p = 0.039; respectively). A significant negative correlation between plasma gelsolin and PsA activity was detected (p < 0.0001). The PsA activity was defined by the Disease Activity for Psoriatic Arthritis Score and the Composite Psoriatic Disease Activity Index., Conclusions: The plasma gelsolin levels were decreased in PsA patients, suggesting that gelsolin may be implicated in the chronic joint inflammation process. Plasma gelsolin seems to be a useful predictive biomarker for diagnosing PsA and monitoring the disease activity.Key Points• This study introduces an unprecedented focus within which the relationship between the levels of plasma gelsolin and PsA is investigated• The study examines the potential role of gelsolin in PsA, and detects the association between gelsolin and the arthritis activity.• There were decreased plasma gelsolin levels in PsA patients. So, gelsolin can constitute a role in the chronic joint inflammation process.• Gelsolin may be a useful biomarker for diagnosing of PsA and monitoring the disease activity.

AGel amyloidosis is a dominantly inherited systemic amyloidosis caused by mutations p.D214N or p.D214Y resulting in gelsolin amyloid (AGel) formation. AGel accumulates extracellularly in many tissues and alongside elastic fibres. AGel deposition associates with elastic fibre degradation leading to severe clinical manifestations, such as cutis laxa and angiopathic complications. We analysed elastic fibre pathology in dermal and vascular tissue and plasma samples from 35 patients with AGel amyloidosis and 40 control subjects by transmission electron microscopy, immunohistochemistry and ELISA methods. To clarify the pathomechanism(s) of AGel-related elastolysis, we studied the roles of MMP-2, -7, -9, -12 and -14, TIMP-1 and TGFβ. We found massive accumulation of amyloid fibrils along elastic fibres as well as fragmentation and loss of elastic fibres in all dermal and vascular samples of AGel patients. Fibrils of distinct types formed fibrous matrix. The degradation pattern of elastic fibres in AGel patients was different from the age-related degradation in controls. The elastin of elastic fibres in AGel patients was strongly decreased compared to controls. MMP-9 was expressed at lower and TGFβ at higher levels in AGel patients than in controls. The accumulation of amyloid fibrils with severe elastolysis characterises both dermal and vascular derangement in AGel amyloidosis.

Purpose: Familial amyloidosis of the Finnish type (FAF) is an inherited amyloidosis arising from mutations in the gelsolin protein (GSN). The disease includes facial paralysis, loose skin, and lattice corneal dystrophy. To date, FAF has been invariably associated with substitution of Asp214 in GSN. We describe the clinical, histopathological, and genetic features of a family with FAF due to a novel GSN mutation., Methods: Five affected adult individuals in a three-generation FAF pedigree were included in the study. Histopathological analysis was performed on an eyelid skin biopsy from one patient. Genetic analysis included next-generation sequencing (NGS) and Sanger sequencing for confirmation of the GSN variant. Several tools for in silico analysis of pathogenicity for the novel variant and to predict the effect of the amino acid replacement on protein stability were used., Results: Three older adult affected patients exhibited corneal lattice dystrophy, cutis laxa, and facultative peripheral neuropathy. Two younger adult individuals presented only with corneal amyloid deposits. NGS identified a heterozygous GSN c.1631T>G transversion, predicting a novel p.Met544Arg mutation. All in silico tools indicated that p.Met544Arg is deleterious for GSN functionality or stability., Conclusions: The results expand the molecular spectrum of GSN-linked systemic amyloidosis. The novel p.Met544Arg pathogenic variant is predicted to affect gelsolin function, presumably by impairing a potential calcium-sensitive, actin-binding region., (Copyright © 2020 Molecular Vision.)

Septum formation in fungi is equivalent to cytokinesis. It differs mechanistically in filamentous ascomycetes (Pezizomycotina) from that of ascomycete yeasts by the retention of a central septal pore in the former group. However, septum formation in both groups is accomplished by contractile actin ring (CAR) assembly and constriction. The specific components regulating septal pore organization during septum formation are poorly understood. In this study, a novel Pezizomycotina-specific actin regulatory protein GlpA containing gelsolin domains was identified using bioinformatics. A glpA deletion mutant exhibited increased distances between septa, abnormal septum morphology and defective regulation of septal pore closure. In glpA deletion mutant hyphae, overaccumulation of actin filament (F-actin) was observed, and the CAR was abnormal with improper assembly and failure in constriction. In wild-type cells, GlpA was found at the septum formation site similarly to the CAR. The N-terminal 329 residues of GlpA are required for its localization to the septum formation site and essential for proper septum formation, while its C-terminal gelsolin domains are required for the regular CAR dynamics during septum formation. Finally, in this study we elucidated a novel Pezizomycotina-specific actin modulating component, which participates in septum formation by regulating the CAR dynamics., (© 2020 John Wiley & Sons Ltd.)

Early studies indicated that estrogen receptor α (ERα) might impact the progression of hepatocellular carcinoma (HCC). However, the detailed mechanisms, especially its linkage to the gelsolin (GSN)-mediated cell invasion, remain unclear. Here we found that ERα could decrease HCC cell invasion via suppressing the circular RNA-SMG1.72 (circRNA-SMG1.72) expression via transcriptional regulation through directly binding to the 5' promoter region of its host gene SMG1, We showed that ERα-suppressed circ-SMG1.72 could sponge and inhibit the expression of the microRNA (miRNA, miR), miR-141-3p, which could then result in increasing the GSN messenger RNA translation via reduced miR binding to its 3' untranslated region (3'UTR). The preclinical study using an in vivo mouse model with orthotopic xenografts of HCC cells confirmed the in vitro data, and the human HCC clinical sample survey and tissue staining also confirmed the linkage of ERα/miR-141-3p/GSN signaling to the HCC progression. Together, our findings suggest that ERα can suppress HCC cell invasion via altering the ERα/circRNA-SMG1.72/miR-141-3p/GSN signaling, and targeting this newly identified signaling with small molecules may help in the development of novel therapies to better suppress the HCC progression.

Gelsolin superfamily proteins, consisting of multiple domains (usually six), sever actin filaments and cap the barbed ends in a Ca 2+ -dependent manner. Two types of evolutionally conserved Ca 2+ -binding sites have been identified in this family; type-1 (between gelsolin and actin) and type-2 (within the gelsolin domain). Fragmin, a member in the slime mold Physarum polycephalum, consists of three domains (F1-F3) that are highly similar to the N-terminal half of mammalian gelsolin (G1-G3). Despite their similarities, the two proteins exhibit a significant difference in the Ca 2+ dependency; F1-F3 absolutely requires Ca 2+ for the filament severing whereas G1-G3 does not. In this study, we examined the strong dependency of fragmin on Ca 2+ using biochemical and structural approaches. Our co-sedimentation assay demonstrated that Ca 2+ significantly enhanced the binding of F2-F3 to actin. We determined the crystal structure of F2-F3 in the presence of Ca 2+ . F2-F3 binds a total of three calcium ions; while two are located in type-2 sites within F2 or F3, the remaining one resides between the F2 long helix and the F3 short helix. The inter-domain Ca 2+ -coordination appears to stabilize F2-F3 in a closely packed configuration. Notably, the F3 long helix exhibits a bent conformation which is different from the straight G3 long helix in the presence of Ca 2+ . Our results provide the first structural evidence for the existence of an unconventional Ca 2+ -binding site in the gelsolin superfamily proteins.

The assembly of protein actin into double-helical filaments promotes many eukaryotic cellular processes that are regulated by actin-binding proteins (ABPs). Actin filaments can adopt multiple conformations, known as structural polymorphism, which possibly influences the interaction between filaments and ABPs. Gelsolin is a Ca 2+ -regulated ABP that severs and caps actin filaments. Gelsolin binding modulates filament structure; however, it is not known how polymorphic actin filament structures influence an interaction of gelsolin S1 with the barbed-end of filament. Herein, we investigated how polymorphic structures of actin filaments affect the interactions near interfaces between the gelsolin segment 1 (S1) domain and the filament barbed-end. Using all-atom molecular dynamics simulations, we demonstrate that different tilted states of subunits modulate gelsolin S1 interactions with the barbed-end of polymorphic filaments. Hydrogen bonding and interaction energy at the filament-gelsolin S1 interface indicate distinct conformations of filament barbed ends, resulting in different interactions of gelsolin S1. This study demonstrates that filament's structural multiplicity plays important roles in the interactions of actin with ABPs., (© 2019 Wiley Periodicals, Inc.)

Mutations in the gelsolin protein are responsible for a rare conformational disease known as AGel amyloidosis. Four of these mutations are hosted by the second domain of the protein (G2): D187N/Y, G167R and N184K. The impact of the latter has been so far evaluated only by studies on the isolated G2. Here we report the characterization of full-length gelsolin carrying the N184K mutation and compare the findings with those obtained on the wild type and the other variants. The crystallographic structure of the N184K variant in the Ca 2+ -free conformation shows remarkable similarities with the wild type protein. Only minimal local rearrangements can be observed and the mutant is as efficient as the wild type in severing filamentous actin. However, the thermal stability of the pathological variant is compromised in the Ca 2+ -free conditions. These data suggest that the N to K substitution causes a local disruption of the H-bond network in the core of the G2 domain. Such a subtle rearrangement of the connections does not lead to significant conformational changes but severely affects the stability of the protein.

The second domain of gelsolin (G2) hosts mutations responsible for a hereditary form of amyloidosis. The active form of gelsolin is Ca 2+ -bound; it is also a dynamic protein, hence structural biologists often rely on the study of the isolated G2. However, the wild type G2 structure that have been used so far in comparative studies is bound to a crystallographic Cd 2+ , in lieu of the physiological calcium. Here, we report the wild type structure of G2 in complex with Ca 2+ highlighting subtle ion-dependent differences. Previous findings on different G2 mutations are also briefly revised in light of these results., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Adseverin (Ads) is a Ca 2+ -dependent actin-capping and severing protein that is highly expressed in gastric, prostate and bladder cancer cells. Currently it is unknown whether Ads contributes to the subcortical actin remodeling associated with the formation of cell extensions and matrix invasion in cancer. We compared cell extension formation and matrix degradation in Ads wildtype and Ads-null MCF7 breast cancer cells generated by CRISPR/Cas9. Compared with wildtype, Ads-null cells plated on fibronectin or collagen exhibited a more circular morphology with shorter cell extensions (37% reduction on fibronectin; p < 0.001). Reconstitution of Ads in Ads-null cells restored the formation of cell extensions (p < 0.05). While cell migration on two-dimensional matrices was unchanged by Ads deletion, the formation of cell extensions across Transwell membranes was reduced (~40% reduction, p < 0.05). When plated on fibrillar collagen, compared with wildtype, Ads-null cells showed reduced expression of MT1-MMP, collagen degradation (p < 0.05) and phagocytosis of collagen-coated beads (25% reduction; p = 0.001). We conclude that Ads is involved in the formation of cell extensions and collagen degradation in MCF7 cells, which may in turn affect matrix invasion and metastasis., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: Previous clinical studies have shown frequent cardiac symptoms in patients with hereditary gelsolin (AGel) amyloidosis, possibly related to amyloid deposition in the heart and other internal organs. Previous studies on internal organ amyloid deposition in AGel amyloidosis have been based on small patient series. Methods: Paraffin-embedded tissue sections from 25 autopsied individuals (age at death 44.4-88.6 years) with AGel amyloidosis were stained with HE, Congo red and Herovici stains and immunohistochemistry against the low molecular weight gelsolin fraction was performed. The amount of amyloid was estimated semi-quantitatively. Results: AGel-based amyloid deposits were found in the myocardium and cardiac blood vessels in every patient. The deposits were mainly small and co-localized with regions with excess fibrosis in the myocardium. The lungs were positive for amyloid in 79%, renal parenchyma in 54% and renal blood vessels in 71% of the cases. The amount of myocardial, renal and hepatic amyloid correlated with age at death of the patients. Conclusions: We show the constant presence of AGel amyloid in the hearts of patients with AGel amyloidosis. Although the deposits were mainly small, the co-localization of amyloid with fibrosis may amplify the effect of pure amyloid deposition, possibly leading to clinical signs and symptoms.

Synaptic plasticity underlies central brain functions, such as learning. Ca2+ signaling is involved in both strengthening and weakening of synapses, but it is still unclear how one signal molecule can induce two opposite outcomes. By identifying molecules, which can distinguish between signaling leading to weakening or strengthening, we can improve our understanding of how synaptic plasticity is regulated. Here, we tested gelsolin's response to the induction of chemical long-term potentiation (cLTP) or long-term depression (cLTD) in cultured rat hippocampal neurons. We show that gelsolin relocates from the dendritic shaft to dendritic spines upon cLTD induction while it did not show any relocalization upon cLTP induction. Dendritic spines are small actin-rich protrusions on dendrites, where LTD/LTP-responsive excitatory synapses are located. We propose that the LTD-induced modest - but relatively long-lasting - elevation of Ca2+ concentration increases the affinity of gelsolin to F-actin. As F-actin is enriched in dendritic spines, it is probable that increased affinity to F-actin induces the relocalization of gelsolin.

Chondrocytes dedifferentiate as a result of monolayer culture for cell number expansion. This is associated with the development of an elongated shape, increased actin polymerization, development of stress fibres, and expression of contractile molecules. Given the changes in actin status with dedifferentiation, the hypothesis of this study was that adseverin, an actin severing and capping protein, plays a role in regulating chondrocyte phenotype and function. This study reports that serial passaging of articular chondrocytes in monolayer culture resulted in loss of adseverin protein expression as early as Day 14 of culture and remained repressed in Passage 2 (P2) cells. Knockdown of adseverin by siRNA in primary chondrocytes promoted an increase in cell size and an elongated shape, actin stress fibres, decreased G-/F-actin ratio, and increased number of actin-free barbed ends. The cells also showed increased expression of the contractile genes and proteins, vinculin and α-smooth muscle actin, and increased ability to contract collagen gels. These are all features of dedifferentiation. These effects were due to adseverin as adseverin overexpression following transfection of the green fluorescent protein-adseverin plasmid partially reversed all of these changes in P2 chondrocytes. Furthermore, sox9 and aggrecan chondrogenic gene expression was upregulated, and collagen type I genes expression was downregulated with adseverin overexpression. The change in aggrecan mRNA expression had functional consequence as these cells exhibited increased total proteoglycan synthesis. These findings demonstrate that adseverin regulates features indicative of redifferentiation in passaged articular chondrocytes through modulation of the actin cytoskeleton status and potentially may regulate the maintenance of phenotype in primary chondrocytes., (© 2019 John Wiley & Sons, Ltd.)

Actin binding compounds are widely used tools in cell biology. We compare the biological and biochemical effects of miuraenamide A and jasplakinolide, a structurally related prototypic actin stabilizer. Though both compounds have similar effects on cytoskeletal morphology and proliferation, they affect migration and transcription in a distinctive manner, as shown by a transcriptome approach in endothelial cells. In vitro, miuraenamide A acts as an actin nucleating, F-actin polymerizing and stabilizing compound, just like described for jasplakinolide. However, in contrast to jasplakinolide, miuraenamide A competes with cofilin, but not gelsolin or Arp2/3 for binding to F-actin. We propose a binding mode of miuraenamide A, explaining both its similarities and its differences to jasplakinolide. Molecular dynamics simulations suggest that the bromophenol group of miurenamide A interacts with residues Tyr133, Tyr143, and Phe352 of actin. This shifts the D-loop of the neighboring actin, creating tighter packing of the monomers, and occluding the binding site of cofilin. Since relatively small changes in the molecular structure give rise to this selectivity, actin binding compounds surprisingly are promising scaffolds for creating actin binders with specific functionality instead of just "stabilizers".

AGel amyloidosis, formerly known as familial amyloidosis of the Finnish-type, is caused by pathological aggregation of proteolytic fragments of plasma gelsolin. So far, four mutations in the gelsolin gene have been reported as responsible for the disease. Although D187N is the first identified variant and the best characterized, its structure has been hitherto elusive. Exploiting a recently-developed nanobody targeting gelsolin, we were able to stabilize the G2 domain of the D187N protein and obtained, for the first time, its high-resolution crystal structure. In the nanobody-stabilized conformation, the main effect of the D187N substitution is the impairment of the calcium binding capability, leading to a destabilization of the C-terminal tail of G2. However, molecular dynamics simulations show that in the absence of the nanobody, D187N-mutated G2 further misfolds, ultimately exposing its hydrophobic core and the furin cleavage site. The nanobody's protective effect is based on the enhancement of the thermodynamic stability of different G2 mutants (D187N, G167R and N184K). In particular, the nanobody reduces the flexibility of dynamic stretches, and most notably decreases the conformational entropy of the C-terminal tail, otherwise stabilized by the presence of the Ca 2+ ion. A Caenorhabditis elegans-based assay was also applied to quantify the proteotoxic potential of the mutants and determine whether nanobody stabilization translates into a biologically relevant effect. Successful protection from G2 toxicity in vivo points to the use of C. elegans as a tool for investigating the mechanisms underlying AGel amyloidosis and rapidly screen new therapeutics., (Copyright © 2019 Elsevier B.V. All rights reserved.)

BACKGROUND Gelsolin (GSN) is an actin-binding and PIP₂/Ca²⁺-regulated protein found in the cytoplasm and blood plasma. Hypogelsolinemia occurs in a wide range of traumatic injuries and inflammatory reactions. We hypothesize that blood GSN levels will be altered in patients diagnosed with acute myeloid leukemia (AML) that develop sepsis, and assessment of GSN concentration will be a useful marker to determine their clinical outcome. To achieve this task, we evaluated the plasma gelsolin concentration in blood samples collected from patients diagnosed with acute myeloid leukemia (AML) at initial stages of sepsis. MATERIAL AND METHODS To assess if AML patients might be at risk of sepsis, a SOFA score was determined. Plasma gelsolin concentration was evaluated using an immunoblotting technique. RESULTS We found that GSN concentration in the blood of the AML group with developing sepsis was significantly lower (32±41 µg/ml; p<0.05) compared to the AML group (65±35 µg/ml) and control group (176±37 µg/ml; p<0.001). Additionally, low gelsolin concentration in the blood of AML patients developing sepsis was associated with a high SOFA score. A decrease of GSN concentration in the blood of AML subjects with developing sepsis suggests that GSN level in blood reflects not only chronic inflammation stage associated with leukemia, but that GSN depletion also manifests the inflammation associated with sepsis development. CONCLUSIONS The results presented here suggest the possible utility of GSN evaluation for diagnostic purposes. Overall, these data support the that reversing plasma GSN deficiency might be a possible new strategy in sepsis treatment.

Gelsolin (GSN), one of the most abundant actin-binding proteins, is involved in cell motility, shape and metabolism. As a member of the GSN superfamily, GSN is a highly structured protein in eukaryotic cells that can be regulated by calcium concentration, intracellular pH, temperature and phosphatidylinositol-4,5-bisphosphate. GSN plays an important role in cellular mechanisms as well as in different cellular interactions. Because of its participation in immunologic processes and its interaction with different cells of the immune system, GSN is a potential candidate for various therapeutic applications. In this review, we summarise the structure of GSN as well as its regulating and functional roles, focusing on distinct diseases such as Alzheimer's disease, rheumatoid arthritis and cancer. A short overview of GSN as a therapeutic target in today's medicine is also provided.

Consumption of high fat diet (HFD) is associated with increased cardiovascular risk factors among elderly people. Aging and obesity induced-cardiac remodeling includes hypertrophy and fibrosis. Gelsolin (GSN) induces cardiac hypertrophy and TGF-β, a key cytokine, which induces fibrosis. The relationship between TGF-β and GSN in aging induced cardiac remodeling is still unknown. We evaluated the expressions of TGF-β and GSN in HFD fed 22 months old aging SD rats, followed by the administration of either probucol or alcalase potato protein hydrolysate (APPH). Western blotting and Masson trichrome staining showed that APPH (45 and 75 mg/kg/day) and probucol (500 mg/kg/day) treatments significantly reduced the aging and HFD-induced hypertrophy and fibrosis. Echocardiograph showed that the performance of the hearts was improved in APPH, and probucol treated HFD aging rats. Serum from all rats was collected and H9c2 cells were cultured with collected serums separately. The GSN dependent hypertrophy was inhibited with an exogenous TGF-β in H9c2 cells cultured in HFD+ APPH treated serum. Thus, we propose that along with its role in cardiac fibrosis, TGF-β also acts as an upstream activator of GSN dependent hypertrophy. Hence, TGF-β in serum could be a promising therapeutic target for cardiac remodeling in aging and/or obese subjects., (© 2018 Wiley Periodicals, Inc.)

Galectin-3 (Gal-3) is a chimeric protein structurally composed of unusual tandem repeats of proline and short glycine-rich segments fused onto a carbohydrate recognition domain. Our studies have previously demonstrated that Gal-3 drives oligodendrocyte (OLG) differentiation to control myelin integrity and function. The cytoskeleton plays a key role in OLG maturation: the initial stage of OLG process extension requires dynamic actin filament assembly, while subsequent myelin wrapping coincides with the upregulation of actin disassembly proteins which are dependent on myelin basic protein (MPB) expression. In this context, the aim of the present work was to elucidate the mechanism by which recombinant Gal-3 (rGal-3) induces OLG maturation, giving special attention to the actin cytoskeleton. Our results show that rGal-3 induced early actin filament assembly accompanied by Erk signaling deactivation, which led to a decrease in the number of platelet-derived growth factor receptor α (PDGFRα)+ cells concomitantly with an increase in the number of 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase)+ cells at 1 day of treatment (TD1), and Akt signaling activation at TD1 and TD3. Strikingly, rGal-3 induced an accelerated shift from polymerized to depolymerized actin between TD3 and TD5, accompanied by a significant increase in MBP, gelsolin, Rac1, Rac1-GTP, and β-catenin expression at TD5. These results were strongly supported by assays using Erk 1/2 and Akt inhibitors, indicating that both pathways are key to rGal-3-mediated effects. Erk 1/2 inhibition in control-treated cells resembled an rGal-3 like state characterized by an increase in MBP, β-catenin, and gelsolin expression. In contrast, Akt inhibition in rGal-3-treated cells reduced MBP, β-catenin, and gelsolin expression, indicating a blockade of rGal-3 effects. Taken together, these results indicate that rGal-3 accelerates OLG maturation by modulating signaling pathways and protein expression which lead to changes in actin cytoskeleton dynamics.

Biomechanical stress and cytoskeletal remodeling are key determinants of cellular homeostasis and tissue responses to mechanical stimuli and injury. Here we document the increased activity of gelsolin, an actin filament severing and capping protein, in failing human hearts. Deletion of gelsolin prevents biomechanical stress-induced adverse cytoskeletal remodeling and heart failure in mice. We show that phosphatidylinositol (3,4,5)-triphosphate (PIP3) lipid suppresses gelsolin actin-severing and capping activities. Accordingly, loss of PI3Kα, the key PIP3-producing enzyme in the heart, increases gelsolin-mediated actin-severing activities in the myocardium in vivo, resulting in dilated cardiomyopathy in response to pressure-overload. Mechanical stretching of adult PI3Kα-deficient cardiomyocytes disrupts the actin cytoskeleton, which is prevented by reconstituting cells with PIP3. The actin severing and capping activities of recombinant gelsolin are effectively suppressed by PIP3. Our data identify the role of gelsolin-driven cytoskeletal remodeling in heart failure in which PI3Kα/PIP3 act as negative regulators of gelsolin activity.

Background Cardiac hypertrophy is an adaptive remodeling event that may improve or diminish contractile performance of the heart. Physiologic and pathologic hypertrophy yield distinct outcomes, yet both are dependent on caspase-directed proteolysis. This suggests that each form of myocardial growth may derive from a specific caspase cleavage event(s). We examined whether caspase 3 cleavage of the actin capping/severing protein gelsolin is essential for the development of pathologic hypertrophy. Methods and Results Caspase targeting of gelsolin was established through protein analysis of hypertrophic cardiomyocytes and mass spectrometry mapping of cleavage sites. Pathologic agonists induced late-stage caspase-mediated cleavage of gelsolin. The requirement of caspase-mediated gelsolin cleavage for hypertrophy induction was evaluated in primary cardiomyocytes by cell size analysis, monitoring of prohypertrophy markers, and measurement of hypertrophy-related transcription activity. The in vivo impact of caspase-mediated cleavage was investigated by echo-guided intramyocardial injection of adenoviral-expressed gelsolin. Expression of the N-terminal gelsolin caspase cleavage fragment was necessary and sufficient to cause pathologic remodeling in isolated cardiomyocytes and the intact heart, whereas expression of a noncleavable form prevents cardiac remodeling. Alterations in myocardium structure and function were determined by echocardiography and end-stage cardiomyocyte cell size analysis. Gelsolin secretion was also monitored for its impact on naïve cells using competitive antibody trapping, demonstrating that hypertrophic agonist stimulation of cardiomyocytes leads to gelsolin secretion, which induces hypertrophy in naïve cells. Conclusions These results suggest that cell autonomous caspase cleavage of gelsolin is essential for pathologic hypertrophy and that cardiomyocyte secretion of gelsolin may accelerate this negative remodeling response.

We have recently performed the characterization of alternative splicing events (ASEs) in head and neck squamous cell carcinoma, which allows dysregulation of protein expression common for cancer cells. Such analysis demonstrated a high ASE prevalence among tumor samples, including tumor-specific alternative splicing in the GSN gene.In vitro studies confirmed that overall expression of either ASE-GSN or wild-type GSN (WT-GSN) isoform inversely correlated with cell proliferation, whereas the high ratio of ASE-GSN to WT-GSN correlated with increased cellular invasion. Additionally, a change in expression of either isoform caused compensatory changes in expression of the other isoform. Our results suggest that the overall expression and the balance between GSN isoforms are mediating factors in proliferation, while increased overall expression of ASE-GSN is specific to cancer tissues. As a result, we propose ASE-GSN can serve not only as a biomarker of disease and disease progression, but also as a neoantigen for head and neck squamous cell carcinoma treatment, for which only a limited number of disease-specific targeted therapies currently exist., (Copyright © 2018. Published by Elsevier Inc.)

For the acquisition of the ability to fertilize the egg, mammalian spermatozoa should undergo a series of biochemical transformations in the female reproductive tract, collectively called capacitation. The capacitated sperm can undergo the acrosomal exocytosis process near or on the oocyte, which allows the spermatozoon to penetrate and fertilize it. One of the main processes in capacitation involves dynamic cytoskeletal remodeling particularly of actin. Actin polymerization occurs during sperm capacitation and the produced F-actin should be depolymerized prior to the acrosomal exocytosis. In the present review, we describe the mechanisms that regulate F-actin formation during sperm capacitation and the F-actin dispersion prior to the acrosomal exocytosis. During sperm capacitation, the actin severing proteins gelsolin and cofilin are inactive and they undergo activation prior to the acrosomal exocytosis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Recently there has been a growing interest in three-dimensional (3D) cell culture systems for drug discovery and development. These 3D culture systems better represent the in vivo cellular environment compared to two-dimensional (2D) cell culture, thereby providing more physiologically reliable information on drug screening and testing. Here we present the quantitative profiling of a drug-induced proteome in 2D- and 3D-cultured colorectal cancer SW480 cells using 2D nanoflow liquid chromatography-tandem mass spectrometry (2D-nLC-MS/MS) integrated with isobaric tags for relative and absolute quantitation (iTRAQ). We identified a total of 4854 shared proteins between 2D- and 3D-cultured SW480 cells and 136/247 differentially expressed proteins (up/down-regulated in 3D compared to 2D). These up/down-regulated proteins were mainly involved in energy metabolism, cell growth, and cell-cell interactions. We also investigated the XAV939 (tankyrase inhibitor)-induced proteome to reveal factors involved in the 3D culture-selective growth inhibitory effect of XAV939 on SW480 cells. We identified novel XAV939-induced proteins, including gelsolin (a possible tumor suppressor) and lactate dehydrogenase A (a key enzyme of glycolysis), which were differentially expressed between 2D- and 3D-cultured SW480 cells. These results provide a promising informative protein dataset to determine the effect of XAV939 on the expression levels of proteins involved in SW480 cell growth.

Background: Diabetes is a leading cause of mortality and morbidity across the world. Over 50% of deaths among diabetic patients are caused by cardiovascular diseases. Cardiac diastolic dysfunction is one of the key early signs of diabetic cardiomyopathy, which often occurs before systolic dysfunction. However, no drug is currently licensed for its treatment., Methods: Type 9 adeno-associated virus combined with cardiac Troponin T promoter were employed to manipulate miR-21 expression in the leptin receptor-deficient (db/db) mice. Cardiac structure and functions were measured by echocardiography and hemodynamic examinations. Primary cardiomyocytes and cardiomyocyte cell lines were used to perform gain/loss-of-function assays in vitro., Results: We observed a significant reduction of miR-21 in the diastolic dysfunctional heart of db/db mice. Remarkably, delivery of miR-21 efficiently protected against the early impairment in cardiac diastolic dysfunction, represented by decreased ROS production, increased bioavailable NO and relieved diabetes-induced cardiomyocyte hypertrophy in db/db mice. Through bioinformatic analysis and Ago2 co-immunoprecipitation, we identified that miR-21 directly targeted gelsolin, a member of the actin-binding proteins, which acted as a transcriptional cofactor in signal transduction. Moreover, down-regulation of gelsolin by siRNA also attenuated the early phase of diabetic cardiomyopathy., Conclusion: Our findings reveal a new role of miR-21 in attenuating diabetic cardiomyopathy by targeting gelsolin, and provide a molecular basis for developing a miRNA-based therapy against diabetic cardiomyopathy.

The present study aimed to identify molecules associated with lymphovascular invasion (LVI) and perineural invasion (PNI) and to examine their biological behavior in colorectal cancer (CRC). LVI- and PNI-associated molecules were identified and verified using sequential processes including (1) identification of 117 recurrence-associated genes differentially expressed on RNA-seq analysis using primary cancer tissues from 130 CRC patients with and without systemic recurrence; (2) analysis of molecules associated with LVI and PNI; (3) assessment of biological properties by measuring proliferation, anoikis, invasion/migration, epithelial-mesenchymal transition and autophagy flux; and (4) verification of disease-free survival using public datasets. Gelsolin (GSN) and 2'-5'-oligoadenylate synthetase 2 (OAS2) were associated with PNI and LVI, respectively. Invasion potential was >2-fold greater in GSN-overexpressing LoVo cells than in control cells (p<0.001-0.005), whereas OAS2-overexpressing RKO cells showed reduced invasion (p<0.001-0.005). GSN downregulated E-cadherin, β-catenin, claudin-1 and snail, and upregulated N-cadherin and ZEB1, whereas OAS2 overexpression had the opposite effects. Several autophagy-related proteins including ATG5-12, ATG6/BECN1, ATG7 and ATG101 were downregulated in GSN-overexpressing LoVo cells, whereas the opposite pattern was observed in OAS2-overexpressing RKO cells. Patients with low GSN expression had significantly higher 5-year recurrence-free survival (RFS) rates than those with GSN overexpression (73.6% vs. 64.7%, p = 0.038), whereas RFS was longer in patients with OAS2 overexpression than in those with underexpression (73.4% vs. 63.7%, p = 0.01). In conclusion, GSN and OAS2 were positively and negatively associated with recurrence, respectively, suggesting their potential value as predictors of recurrence or therapeutic targets in CRC patients., Competing Interests: The authors have declared that no competing interests exist.

Gelsolin, an actin-depolymerizing protein expressed both in extracellular fluids and in the cytoplasm of a majority of human cells, has been recently implicated in a variety of both physiological and pathological processes. Its extracellular isoform, called plasma gelsolin (pGSN), is present in blood, cerebrospinal fluid, milk, urine, and other extracellular fluids. This isoform has been recognized as a potential biomarker of inflammatory-associated medical conditions, allowing for the prediction of illness severity, recovery, efficacy of treatment, and clinical outcome. A compelling number of animal studies also demonstrate a broad spectrum of beneficial effects mediated by gelsolin, suggesting therapeutic utility for extracellular recombinant gelsolin. In the review, we summarize the current data related to the potential of pGSN as an inflammatory predictor and therapeutic target, discuss gelsolin-mediated mechanisms of action, and highlight recent progress in the clinical use of pGSN.

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 Ca 2+ -free conditions, 28-161 associates into monomer to dimer and tetramer, which later forms β-amyloids, but in presence of Ca 2+ , 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 Ca 2+ 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 Ca 2+ 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.

Gelsolin is a severing and capping protein that targets filamentous actin and regulates filament lengths near plasma membranes, contributing to cell movement and plasma membrane morphology. Gelsolin binds to the plasma membrane via phosphatidylinositol 4,5-bisphosphate (PIP2) in a state that cannot cap F-actin, and gelsolin-capped actin filaments are uncapped by PIP2 leading to filament elongation. The process by which gelsolin is removed from PIP2 at the plasma membrane is currently unknown. Gelsolin also binds ATP with unknown function. Here we characterize the role of ATP on PIP2-gelsolin complex dynamics. Fluorophore-labeled PIP2 and ATP were used to study their interactions with gelsolin using steady-state fluorescence anisotropy, and Alexa488-labeled gelsolin was utilized to reconstitute the regulation of gelsolin binding to PIP2-containing phospholipid vesicles by ATP. Under physiological salt conditions ATP competes with PIP2 for binding to gelsolin, while calcium causes the release of ATP from gelsolin. These data suggest a cycle for gelsolin activity. Firstly, calcium activates ATP-bound gelsolin allowing it to sever and cap F-actin. Secondly, PIP2-binding removes the gelsolin cap from F-actin at low calcium levels, leading to filament elongation. Finally, ATP competes with PIP2 to release the calcium-free ATP-bound gelsolin, allowing it to undergo a further round of severing., Competing Interests: The authors have declared that no competing interests exist.

Aims: Post-operative atrial fibrillation (POAF) is a predictor of morbidity and mortality after cardiac surgery. Latent predisposing factors may reside in the epicardial adipose tissue (EAT) due to its anatomical position and high protein production rate. In order to explore a possible mechanistic link, we characterized proteins secreted by the EAT preceding the onset of POAF., Methods and Results: Epicardial adipose tissue samples were collected from 76 consecutive patients with no history of AF undergoing coronary artery bypass surgery, 50 samples for proteomic analysis and 26 for gene expression studies, further divided according to development of POAF. Ten vs. 10 matched samples representing EAT secretome were analysed by two-dimensional difference in-gel electrophoresis (2D-DIGE) and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to identify differentially expressed proteins (P < 0.05, expression change >1.2 fold). Findings were validated by Western blotting on EAT protein extracts and by gene expression studies via quantitative polymerase chain reaction (qPCR). Proteomics returned 35 differentially expressed proteins. Amongst those, gelsolin was down regulated in POAF. Western blot analysis confirmed a significant reduction in gelsolin in the AF group. Gene expression for gelsolin was significantly reduced in the AF group confirming the proteomics findings., Conclusion: For the first time we describe EAT secretome as a possible substrate for POAF. It contains various proteins differentially expressed in patients who later develop POAF. Amongst those gelsolin, involved in inflammation and ion channel regulation, was associated with maintenance of sinus rhythm. Understanding the role of EAT may offer novel insights into prevention and treatment of AF.

Renal amyloidosis is characterized by acellular Congo red positive deposits in the glomeruli, interstitium and/or arteries. Light chain restriction on immunofluorescence studies is present in AL-amyloidosis, the most common type of amyloidosis involving the kidney. The detection of Congo red positive deposits coupled with negative immunofluorescence studies is highly suggestive of non-AL amyloidosis. Some of the non-AL amyloidosis are common while others are relatively rare. The clinical features, laboratory and renal pathology findings are helpful in the diagnosis and typing of non-AL amyloidosis. Thus, ALECT2 amyloidosis is characterized by diffuse cortical interstitial amyloid deposits, AA amyloidosis shows vascular deposits in addition to the glomerular deposits, AFib amyloidosis is characterized by massive amyloid accumulation limited to the glomeruli resulting in the obliteration of glomerular architecture, AApoA1 and AApoAIV are characterized by large amyloid deposits restricted to the medulla, and AGel shows swirling patterns of amyloid fibrils on electron microscopy. While light microscopy is very helpful, accurate typing of non-AL amyloidosis then requires immunohistochemical or laser microdissection/mass spectrometry studies of the Congo red positive deposits. Immunohistochemical studies are available for some of the non-AL amyloidosis. On the other hand, mass spectrometry analysis is a one stop methodology for confirmation and typing of amyloidosis. The diagnosis and typing of amyloidosis by mass spectrometry is based on finding the signature amyloid peptides, apolipoprotein E and serum amyloid-P component, followed by detection of precursor amyloidogenic protein such as LECT2, fibrinogen-α, gelsolin, etc. To, summarize, non-AL amyloidosis is a group of amyloidosis with distinctive clinical, laboratory and renal pathology findings. Typing of the amyloidosis is best performed using mass spectrometry methodology. Accurate typing of non-AL amyloidosis is imperative for correct management, prognosis, and genetic counseling.