Role of Clathrin Light Chains in Regulating Invadopodia Formation

One of the most fundamental processes of the cell is the uptake of molecules from the surrounding environment. Clathrin-mediated endocytosis (CME) is the best-described uptake pathway and regulates nutrient uptake, protein and lipid turnover at the plasma membrane (PM), cell signaling, cell motility and cell polarity. The main protein in CME is clathrin, which assembles as a triskelion-looking building block made of three clathrin heavy chains and three clathrin light chains. Compared to clathrin heavy chains (CHCs), the role of the two isoforms of clathrin light chains (CLCA and CLCB) is poorly understood. Here, we confirm that the simultaneous deletion of both CLCA/B causes abnormal actin structures at the ventral PM and we describe them, for the first time, as functional invadopodia rather than disorganized actin-cytoskeleton assembly sites. Their identification is based on the occurrence of common invadopodia markers as well as functional invadopodia activity characterized by an increased local proteolytic activity of the extracellular matrix proteins. We demonstrate that CLCA/B deletion impacts the intracellular trafficking and recovery of the matrix metalloproteinase 14 (MMP14) leading to its accumulation at the plasma membrane and induction of invadopodia formation. Importantly, we show that invadopodia formation can be prevented by depletion of MMP14. As such, we propose that CLCA/B regulate invadopodia formation by regulating MMP14 delivery to the plasma membrane.

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The roles of grouper clathrin light chains in regulating the infection of a novel marine DNA virus, Singapore grouper iridovirus

Scientific Reports ◽

10.1038/s41598-019-51725-5 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Liqun Wang ◽

Qiang Li ◽

Songwei Ni ◽

Youhua Huang ◽

Jingguang Wei ◽

...

Keyword(s):

Virus Infection ◽

Light Chains ◽

Dna Virus ◽

Virus Attachment ◽

Heavy Chains ◽

Fish Virus ◽

Early Endosomes ◽

Epinephelus Akaara ◽

First Time

Abstract Clathrins, composed of clathrin heavy chains (CHCs) and clathrin light chains (CLCs), are usually hijacked by viruses for infection. However, the role of CLCs, especially in regulating fish virus infection, remains poorly understood. Here, two isoforms of CLCs were cloned from the red-spotted grouper (Epinephelus akaara) (EaCLCa and EaCLCb). Both EaCLC transcripts were expressed in all examined tissues, and the expression of EaCLCa was much higher than that of EaCLCb. Over-expressing EaCLCa-W119R mutant significantly reduced Singapore grouper iridovirus (SGIV) infectivity. However, no effect of EaCLCb-W122R on SGIV infection was observed. The detailed steps were further studied, mainly including virus attachment, entry and the following transport to early endosomes. EaCLCa-W119R mutant notably inhibited internalization of SGIV particles with no effect on SGIV attachment. Furthermore, EaCLCa-W119R mutant obviously impaired the delivery of SGIV to early endosomes after virus internalization. In addition, the EaCLCa-W119R mutant markedly reduced the colocalization of SGIV and actin. However, EaCLCb is not required for such events during SGIV infection. Taken together, these results demonstrate for the first time that EaCLCa and EaCLCb exerted different impacts on iridovirus infection, providing a better understanding of the mechanisms of SGIV infection and opportunities for the design of new antiviral strategies.

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A Conserved Tryptophan in the Ebola Virus Matrix Protein C-Terminal Domain Is Required for Efficient Virus-Like Particle Formation

Pathogens ◽

10.3390/pathogens9050402 ◽

2020 ◽

Vol 9 (5) ◽

pp. 402 ◽

Cited By ~ 1

Author(s):

Kristen A. Johnson ◽

Rudramani Pokhrel ◽

Melissa R. Budicini ◽

Bernard S. Gerstman ◽

Prem P. Chapagain ◽

...

Keyword(s):

Plasma Membrane ◽

Matrix Protein ◽

Ebola Virus ◽

Membrane Localization ◽

Virus Like Particles ◽

Plasma Membrane Localization ◽

The Matrix ◽

Dynamics Simulations

The Ebola virus (EBOV) harbors seven genes, one of which is the matrix protein eVP40, a peripheral protein that is sufficient to induce the formation of virus-like particles from the host cell plasma membrane. eVP40 can form different structures to fulfil different functions during the viral life cycle, although the structural dynamics of eVP40 that warrant dimer, hexamer, and octamer formation are still poorly understood. eVP40 has two conserved Trp residues at positions 95 and 191. The role of Trp95 has been characterized in depth as it serves as an important residue in eVP40 oligomer formation. To gain insight into the functional role of Trp191 in eVP40, we prepared mutations of Trp191 (W191A or W191F) to determine the effects of mutation on eVP40 plasma membrane localization and budding as well as eVP40 oligomerization. These in vitro and cellular experiments were complemented by molecular dynamics simulations of the wild-type (WT) eVP40 structure versus that of W191A. Taken together, Trp is shown to be a critical amino acid at position 191 as mutation to Ala reduces the ability of VP40 to localize to the plasma membrane inner leaflet and form new virus-like particles. Further, mutation of Trp191 to Ala or Phe shifted the in vitro equilibrium to the octamer form by destabilizing Trp191 interactions with nearby residues. This study has shed new light on the importance of interdomain interactions in stability of the eVP40 structure and the critical nature of timing of eVP40 oligomerization for plasma membrane localization and viral budding.

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Role of matrix metalloproteinase in the aneurismatic aortic disease

Monaldi Archives for Chest Disease ◽

10.4081/monaldi.2004.663 ◽

2004 ◽

Vol 62 (3) ◽

Author(s):

Nicola Troisi ◽

Alfredo Mazza ◽

Felice Mazza ◽

Gabriele Iannelli

Keyword(s):

Matrix Metalloproteinases ◽

Aortic Disease ◽

Endothelial Damage ◽

Matrix Proteins ◽

Tissue Destruction ◽

The Matrix ◽

Drug Inhibition ◽

Atherosclerotic Aneurysm ◽

Aneurysm Diameter

The aorta is involved in a large variety of diseases and the atherosclerotic aneurysms represent the most common type of these. Recent reports have attempted to clarify the mechanisms, that cause the formation and the progression of the atherosclerotic aneurysms, caused not only by the atherosclerosis. One of the features of this disease is the extensive proteolytic destruction of structural matrix proteins in the aortic wall realized by the matrix metalloproteinases. The atherosclerotic aneurysm can be considered a disease caused by an imbalance between connective tissue destruction and its repair. Knowledge of the role played by matrix metalloproteinases in the formation process of the aneurysms has made the inhibition of these proteins a logical therapeutic strategy. Once completed the aneurysm treatment, surgical or endovascular, the endothelial damage must disappear; the persistence of this damage, after endovascular procedure, is the cause of the formation of the endoleaks. The preoperative matrix metalloproteinases plasmatic levels are related to the aneurysm diameter and after endovascular treatment these values come back normal, except in the case of presence of an endoleak, that don’t make possible the reduction of these values. In spite of that, obscure points still remain, above all about the dosage of these proteins and their inhibition through drugs with clear metalloproteinases- inhibiting properties. The aim of this study is to clarify further on the mechanisms of the formation of the aneurysms with particular care to the matrix metalloproteinases, their dosage and their drug inhibition.

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Catestatin Induces Glucose Uptake and GLUT4 Trafficking in Adult Rat Cardiomyocytes

BioMed Research International ◽

10.1155/2018/2086109 ◽

2018 ◽

Vol 2018 ◽

pp. 1-7 ◽

Cited By ~ 5

Author(s):

Maria Pia Gallo ◽

Saveria Femminò ◽

Susanna Antoniotti ◽

Giulia Querio ◽

Giuseppe Alloatti ◽

...

Keyword(s):

Plasma Membrane ◽

Glucose Uptake ◽

Cardiac Metabolism ◽

Glut4 Translocation ◽

Rat Cardiomyocytes ◽

Adult Rat ◽

Hydrophobic Peptide ◽

First Time ◽

Intracellular Pathway

Catestatin is a cationic and hydrophobic peptide derived from the enzymatic cleavage of the prohormone Chromogranin A. Initially identified as a potent endogenous nicotinic–cholinergic antagonist, Catestatin has recently been shown to act as a novel regulator of cardiac function and blood pressure and as a cardioprotective agent in both pre- and postconditioning through AKT-dependent mechanisms. The aim of this study is to investigate the potential role of Catestatin also on cardiac metabolism modulation, particularly on cardiomyocytes glucose uptake. Experiments were performed on isolated adult rat cardiomyocytes. Glucose uptake was assessed by fluorescent glucose incubation and confocal microscope analysis. Glut4 plasma membrane translocation was studied by immunofluorescence experiments and evaluation of the ratio peripheral vs internal Glut4 staining. Furthermore, we performed immunoblot experiments to investigate the involvement of the intracellular pathway AKT/AS160 in the Catestatin dependent Glut4 trafficking. Our results show that 10 nM Catestatin induces a significant increase in the fluorescent glucose uptake, comparable to that exerted by 100 nM Insulin. Moreover, Catestatin stimulates Glut4 translocation to plasma membrane and both AKT and AS160 phosphorylation. All these effects were inhibited by Wortmannin. On the whole, we show for the first time that Catestatin is able to modulate cardiac glucose metabolism, by inducing an increase in glucose uptake through Glut4 translocation to the plasma membrane and that this mechanism is mediated by the AKT/AS160 intracellular pathway.

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An Electrostatic Net Model for the Role of Extracellular DNA in Biofilm Formation by Staphylococcus aureus

Journal of Bacteriology ◽

10.1128/jb.00726-15 ◽

2015 ◽

Vol 197 (24) ◽

pp. 3779-3787 ◽

Cited By ~ 48

Author(s):

Vanina Dengler ◽

Lucy Foulston ◽

Alicia S. DeFrancesco ◽

Richard Losick

Keyword(s):

Staphylococcus Aureus ◽

Biofilm Formation ◽

Extracellular Dna ◽

Matrix Proteins ◽

Exogenous Dna ◽

Content Type ◽

Cytoplasmic Proteins ◽

The Matrix ◽

Biofilm Matrix

ABSTRACTStaphylococcus aureusis an important human pathogen that can form biofilms on various surfaces. These cell communities are protected from the environment by a self-produced extracellular matrix composed of proteins, DNA, and polysaccharide. The exact compositions and roles of the different components are not fully understood. In this study, we investigated the role of extracellular DNA (eDNA) and its interaction with the recently identified cytoplasmic proteins that have a moonlighting role in the biofilm matrix. These matrix proteins associate with the cell surface upon the drop in pH that naturally occurs during biofilm formation, and we found here that this association is independent of eDNA. Conversely, the association of eDNA with the matrix was dependent on matrix proteins. Both proteinase and DNase treatments severely reduced clumping of resuspended biofilms; highlighting the importance of both proteins and eDNA in connecting cells together. By adding an excess of exogenous DNA to DNase-treated biofilm, clumping was partially restored, confirming the crucial role of eDNA in the interconnection of cells. On the basis of our results, we propose that eDNA acts as an electrostatic net, interconnecting cells surrounded by positively charged matrix proteins at a low pH.IMPORTANCEExtracellular DNA (eDNA) is an important component of the biofilm matrix of diverse bacteria, but its role in biofilm formation is not well understood. Here we report that inStaphylococcus aureus, eDNA associates with cells in a manner that depends on matrix proteins and that eDNA is required to link cells together in the biofilm. These results confirm previous studies that showed that eDNA is an important component of theS. aureusbiofilm matrix and also suggest that eDNA acts as an electrostatic net that tethers cells together via the proteinaceous layer of the biofilm matrix.

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A Complete Absence of Missense Mutation in Myosin Regulatory and Essential Light Chain Genes of South Indian Hypertrophic and Dilated Cardiomyopathies

Cardiology ◽

10.1159/000495027 ◽

2018 ◽

Vol 141 (3) ◽

pp. 156-166

Author(s):

Deepa Selvi Rani ◽

Pratibha Nallari ◽

Jhansi Rani ◽

Sheikh Nizamuddin ◽

Thulasamma Seelamneni ◽

...

Keyword(s):

South India ◽

Myosin Head ◽

Light Chains ◽

Healthy Controls ◽

Missense Mutations ◽

Regulatory Light Chains ◽

Heavy Chains ◽

Essential Light Chain ◽

South Indian ◽

First Time

Background: Myosin is a hexameric contractile protein composed of 2 heavy chains associated with 4 light chains of 2 distinct classes – 2 regulatory light chains (MYL2) and 2 essential light chains (MYL3). The myosin light chains stabilize the long alpha helical neck of the myosin head and regulate the myosin ATPase activities. Objectives: Mutations in MYL2 and MYL3 are reported to be associated with cardiomyopathies. However, there is no study available on these genes in Indian cardiomyopathies, and therefore we planned to study them. Method: For the first time we sequenced MYL2 and MYL3 genes in a total of 248 clinically well-characterized cardiomyopathies consisting of 101 hypertrophic and 147 dilated cases along with 207 healthy controls from south India. Results: Our study revealed a total of 10 variations – 7 in MYL2 and 3 in MYL3, of which 3 are novel variations observed exclusively in cases. However, the 15 causative missense mutations previously reported are totally absent in our study, which showed that the sequences of MYL2 and MYL3 are highly conserved in Indian cases/controls. Conclusions: MYL2 and MYL3 mutations are rare and the least cause of cardiomyopathies in Indians.

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Trial Proteomic Qualitative and Quantitative Analysis of the Protein Matrix of Submandibular Sialoliths

Molecules ◽

10.3390/molecules26216725 ◽

2021 ◽

Vol 26 (21) ◽

pp. 6725

Author(s):

Paulina Czaplewska ◽

Aleksandra E. Bogucka ◽

Natalia Musiał ◽

Dmitry Tretiakow ◽

Andrzej Skorek ◽

...

Keyword(s):

Quantitative Analysis ◽

Protein Extraction ◽

Sodium Dodecyl ◽

Extraction Process ◽

The Matrix ◽

Human Proteins ◽

Protein Components ◽

First Time

Our studies aimed to explore the protein components of the matrix of human submandibular gland sialoliths. A qualitative analysis was carried out based on the filter aided sample preparation (FASP) methodology. In the protein extraction process, we evaluated the applicability of the standard demineralization step and the use of a lysis buffer containing sodium dodecyl sulfate (SDS) and dithiothreitol (DTT). The analysis of fragmentation spectra based on the human database allowed for the identification of 254 human proteins present in the deposits. In addition, the use of multi-round search in the PEAKS Studio program against the bacterial base allowed for the identification of 393 proteins of bacterial origin present in the extract obtained from sialolith, which so far has not been carried out for this biological material. Furthermore, we successfully applied the SWATH methodology, allowing for a relative quantitative analysis of human proteins present in deposits. The obtained results correlate with the classification of sialoliths proposed by Tretiakow. The performed functional analysis allowed for the first time the selection of proteins, the levels of which differ between the tested samples, which may suggest the role of these proteins in the calcification process in different types of sialoliths. These are preliminary studies, and drawing specific conclusions requires research on a larger group, but it provides us the basis for the continuation of the work that has already begun.

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Annexin 1 localisation in tissue eosinophils as detected by electron microscopy

Mediators of Inflammation ◽

10.1080/09629350210000015683 ◽

2002 ◽

Vol 11 (5) ◽

pp. 287-292 ◽

Cited By ~ 18

Author(s):

Sonia M. Oliani ◽

Amilcar S. Damazo ◽

Mauro Perretti

Keyword(s):

Electron Microscopy ◽

Plasma Membrane ◽

Ultrastructural Analysis ◽

Annexin 1 ◽

Cellular Localisation ◽

N Terminus ◽

The Matrix ◽

Inducible Protein ◽

First Time ◽

Carrageenin Injection

Background: Human and rodent leukocytes express high levels of the glucocorticoid-inducible protein annexin 1 (ANXA1) (previously referred to as lipocortin 1). Neutrophils and monocytes have abundant ANXA1 levels.Aim: We have investigated, for the first time, ANXA1 ultrastructural expression in rat eosinophils and compared it with that of extravasated neutrophils. The effect of inflammation (carrageenin peritonitis) was also monitored.Methods: Electron microscopy was used to define the sub-cellular localisation of ANXA1 in rat eosinophils and neutrophils extravasated in the mesenteric tissue. A pair of antibodies raised against the ANXA1 N-terminus (i.e. able to recognise intact ANXA1, termed LCPS1) or the whole protein (termed LCS3) was used to perform the ultrastructural analysis.Results: The majority of ANXA1 was localised in the eosinophil cytosol (~60%) and nucleus (30-40%), whereas a small percentage was found on the plasma membrane (< 10%). Within the cytosol, the protein was equally distributed in the matrix and in the granules, including those containing the typical crystalloid. The two anti-ANXA1 antibodies gave similar results, with the exception that LCPS1 gave a lower degree of immunoreactivity in the plasma membrane. Inflammation (i.e. carrageenin injection) produced a modest increase in eosinophil-associated ANXA1 reactivity (significant only in the cytoplasm compartment). Extravasated neutrophils, used for comparative purposes, displayed a much higher degree of immunoreactivity for the protein.Conclusion: We describe for the first time ANXA1 distribution in rat eosinophil by ultrastructural analysis, and report a different protein mobilisation from extravasated neutrophils, at least in this acute model of peritonitis.

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Association of 4F2hc with light chains LAT1, LAT2 or y+LAT2 requires different domains

Biochemical Journal ◽

10.1042/bj3550725 ◽

2001 ◽

Vol 355 (3) ◽

pp. 725-731 ◽

Cited By ~ 30

Author(s):

Angelika BRÖER ◽

Björn FRIEDRICH ◽

Carsten A. WAGNER ◽

Sophie FILLON ◽

Vadivel GANAPATHY ◽

...

Keyword(s):

Plasma Membrane ◽

Amino Acid ◽

Heavy Chain ◽

Membrane Surface ◽

Transmembrane Helix ◽

Surface Expression ◽

Light Chains ◽

Amino Acid Transporters ◽

Slight Reduction ◽

Heavy Chains

Heterodimeric amino acid transporters are comprised of a type-II membrane protein named the heavy chain (4F2hc or rBAT) that may associate with a number of different polytopic membrane proteins, called light chains. It is thought that the heavy chain is mainly involved in the trafficking of the complex to the plasma membrane, whereas the transport process itself is catalysed by the light chain. The 4F2heavy chain (4F2hc) associates with at least six different light chains to induce distinct amino acid-transport activites. To test if the light chains are specifically recognized and to identify domains involved in the recognition of light chains, C-terminally truncated mutants of 4F2hc were constructed and co-expressed with the light chains LAT1, LAT2 and y+LAT2. The truncated isoform T1, comprised of only 133 amino acids that form the cytosolic N-terminus and the transmembrane helix, displayed only a slight reduction in its ability to promote LAT1 expression at the membrane surface compared with the 529 amino acid wild-type 4F2hc protein. Co-expression of increasingly larger 4F2hc mutants caused a delayed translocation of LAT1. In contrast to the weak effects of 4F2hc truncations on LAT1 expression, surface expression of LAT2 and y+LAT2 was almost completely lost with all truncated heavy chains. Co-expression of LAT1 together with the other light chains did not result in displacement of LAT2 and y+LAT2. The results suggest that extracellular domains of the heavy chain are responsible mainly for recognition of light chains other than LAT1 and that the extracellular domain ensures proper translocation to the plasma membrane.

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Role of galactosylation in the renal pathogenicity of murine immunoglobulin G3 monoclonal cryoglobulins

Blood ◽

10.1182/blood.v97.11.3537 ◽

2001 ◽

Vol 97 (11) ◽

pp. 3537-3543 ◽

Cited By ~ 20

Author(s):

Tsuguo Mizuochi ◽

Yves Pastore ◽

Kohdoh Shikata ◽

Aki Kuroki ◽

Shuichi Kikuchi ◽

...

Keyword(s):

Gel Filtration ◽

Amino Acid Sequences ◽

Light Chains ◽

Remarkable Difference ◽

Pathogenic Potential ◽

Myeloma Cells ◽

Heavy Chains ◽

Direct Demonstration ◽

Direct Localization

Cryoglobulin activity associated with murine immunoglobulin G3 (IgG3) has been shown to play a significant role in the development of murine lupuslike glomerulonephritis. A fraction, but not all, IgG3 monoclonal antibodies are capable of inducing a severe acute lupuslike glomerulonephritis as a result of direct localization of IgG3 cryoglobulins, suggesting the importance of qualitative features of cryoglobulins in their nephritogenic activities. Here a remarkable difference is shown in the renal pathogenicity of 2 murine IgG3 monoclonal cryoglobulins, identical in the amino acid sequences of their heavy and light chains but different in galactosylation patterns of oligosaccharide side chains because of their synthesis in different myeloma cells. The antibody lacking the capacity to induce severe glomerulonephritis displayed an increased proportion of galactosylated heavy chains. Changes in conformation, as revealed by gel filtration analysis, reduced cryoglobulin activity, and accelerated clearance could account for the lack of the renal pathogenicity of the more galactosylated variant. This observation provides a direct demonstration for the role of IgG galactosylation in the pathogenic potential of cryoglobulins.

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