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Autophagy Aug 2023Macroautophagy/autophagy is a catabolic process by which cytosolic content is engulfed, degraded and recycled. It has been implicated as a critical pathway in advanced...
Macroautophagy/autophagy is a catabolic process by which cytosolic content is engulfed, degraded and recycled. It has been implicated as a critical pathway in advanced stages of cancer, as it maintains tumor cell homeostasis and continuous growth by nourishing hypoxic or nutrient-starved tumors. Autophagy also supports alternative cellular trafficking pathways, providing a mechanism of non-canonical secretion of inflammatory cytokines. This opens a significant therapeutic opportunity for using autophagy inhibitors in cancer and acute inflammatory responses. Here we developed a high throughput compound screen to identify inhibitors of protein-protein interaction (PPI) in autophagy, based on the protein-fragment complementation assay (PCA). We chose to target the ATG12-ATG3 PPI, as this interaction is indispensable for autophagosome formation, and the analyzed structure of the interaction interface predicts that it may be amenable to inhibition by small molecules. We screened 41,161 compounds yielding 17 compounds that effectively inhibit the ATG12-ATG3 interaction in the PCA platform, and which were subsequently filtered by their ability to inhibit autophagosome formation in viable cells. We describe a lead compound (#189) that inhibited GFP-fused MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta) puncta formation in cells with IC50 value corresponding to 9.3 μM. This compound displayed a selective inhibitory effect on the growth of autophagy addicted tumor cells and inhibited secretion of IL1B/IL-1β (interleukin 1 beta) by macrophage-like cells. Compound 189 has the potential to be developed into a therapeutic drug and its discovery documents the power of targeting PPIs for acquiring specific and selective compound inhibitors of autophagy. ANOVA: analysis of variance; ATG: autophagy related; CQ: chloroquine; GFP: green fluorescent protein; GLuc: Luciferase; HEK: human embryonic kidney; IL1B: interleukin 1 beta; LPS: lipopolysaccharide; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; PCA: protein-fragment complementation assay; PDAC: pancreatic ductal adenocarcinoma; PMA: phorbol 12-myristate 13-acetate; PPI: protein-protein interaction. VCL: vinculin.
Topics: Humans; Autophagy; Interleukin-1beta; Microtubule-Associated Proteins; Autophagy-Related Proteins; Green Fluorescent Proteins; Pancreatic Neoplasms; Ubiquitin-Conjugating Enzymes; Autophagy-Related Protein 12
PubMed: 37184247
DOI: 10.1080/15548627.2023.2178159 -
An ensemble of cadherin-catenin-vinculin complex employs vinculin as the major F-actin binding mode.Biophysical Journal Jun 2023The cell-cell adhesion cadherin-catenin complexes recruit vinculin to the adherens junction (AJ) to modulate the mechanical couplings between neighboring cells. However,...
The cell-cell adhesion cadherin-catenin complexes recruit vinculin to the adherens junction (AJ) to modulate the mechanical couplings between neighboring cells. However, it is unclear how vinculin influences the AJ structure and function. Here, we identified two patches of salt bridges that lock vinculin in the head-tail autoinhibited conformation and reconstituted the full-length vinculin activation mimetics bound to the cadherin-catenin complex. The cadherin-catenin-vinculin complex contains multiple disordered linkers and is highly dynamic, which poses a challenge for structural studies. We determined the ensemble conformation of this complex using small-angle x-ray and selective deuteration/contrast variation small-angle neutron scattering. In the complex, both α-catenin and vinculin adopt an ensemble of flexible conformations, but vinculin has fully open conformations with the vinculin head and actin-binding tail domains well separated from each other. F-actin binding experiments show that the cadherin-catenin-vinculin complex binds and bundles F-actin. However, when the vinculin actin-binding domain is removed from the complex, only a minor fraction of the complex binds to F-actin. The results show that the dynamic cadherin-catenin-vinculin complex employs vinculin as the primary F-actin binding mode to strengthen AJ-cytoskeleton interactions.
Topics: Cadherins; Actins; Vinculin; alpha Catenin; Protein Binding; Actin Cytoskeleton; Cell Adhesion
PubMed: 37147801
DOI: 10.1016/j.bpj.2023.04.026 -
Frontiers in Cell and Developmental... 2023The actin cytoskeleton remodels to enable diverse processes essential to immunity, such as cell adhesion, migration and phagocytosis. A panoply of actin-binding...
The actin cytoskeleton remodels to enable diverse processes essential to immunity, such as cell adhesion, migration and phagocytosis. A panoply of actin-binding proteins regulate these rapid rearrangements to induce actin-based shape changes and to generate force. L-plastin (LPL) is a leukocyte-specific, actin-bundling protein that is regulated in part by phosphorylation of the Ser-5 residue. LPL deficiency in macrophages impairs motility, but not phagocytosis; we recently found that expression of LPL in which the S5 residue is converted to a non-phosphorylatable alanine (S5A-LPL) resulted in diminished phagocytosis, but unimpaired motility. To provide mechanistic insight into these findings, we now compare the formation of podosomes (an adhesive structure) and phagosomes in alveolar macrophages derived from wild-type (WT), LPL-deficient, or S5A-LPL mice. Both podosomes and phagosomes require rapid remodeling of actin, and both are force-transmitting. Actin rearrangement, force generation, and signaling rely upon recruitment of many actin-binding proteins, including the adaptor protein vinculin and the integrin-associated kinase Pyk2. Prior work suggested that vinculin localization to podosomes was independent of LPL, while Pyk2 was displaced by LPL deficiency. We therefore chose to compare vinculin and Pyk2 co-localization with F-actin at sites of adhesion of phagocytosis in AMs derived from WT, S5A-LPL or LPL mice, using Airyscan confocal microscopy. As described previously, podosome stability was significantly disrupted by LPL deficiency. In contrast, LPL was dispensable for phagocytosis and was not recruited to phagosomes. Recruitment of vinculin to sites of phagocytosis was significantly enhanced in cells lacking LPL. Expression of S5A-LPL impeded phagocytosis, with reduced appearance of ingested bacteria-vinculin aggregates. Our systematic analysis of the regulation of LPL during podosome vs. phagosome formation illuminates essential remodeling of actin during key immune processes.
PubMed: 37138794
DOI: 10.3389/fcell.2023.1020091 -
Pharmaceuticals (Basel, Switzerland) Apr 2023Meldonium (MID) is a synthetic drug designed to decrease the availability of L-carnitine-a main player in mitochondrial energy generation-thus modulating the cell...
Meldonium (MID) is a synthetic drug designed to decrease the availability of L-carnitine-a main player in mitochondrial energy generation-thus modulating the cell pathways of energy metabolism. Its clinical effects are mostly evident in blood vessels during ischemic events, when the hyperproduction of endogenous carnitine enhances cell metabolic activities, leading to increased oxidative stress and apoptosis. MID has shown vaso-protective effects in model systems of endothelial dysfunction induced by high glucose or by hypertension. By stimulating the endothelial nitric oxide synthetase (eNOS) via PI3 and Akt kinase, it has shown beneficial effects on the microcirculation and blood perfusion. Elevated intraocular pressure (IOP) and endothelial dysfunction are major risk factors for glaucoma development and progression, and IOP remains the main target for its pharmacological treatment. IOP is maintained through the filtration efficiency of the trabecular meshwork (TM), a porous tissue derived from the neuroectoderm. Therefore, given the effects of MID on blood vessels and endothelial cells, we investigated the effects of the topical instillation of MID eye drops on the IOP of normotensive rats and on the cell metabolism and motility of human TM cells in vitro. Results show a significant dose-dependent decrease in the IOP upon topic treatment and a decrease in TM cell motility in the wound-healing assay, correlating with an enhanced expression of vinculin localized in focal adhesion plaques. Motility inhibition was also evident on scleral fibroblasts in vitro. These results may encourage a further exploration of MID eye drops in glaucoma treatment.
PubMed: 37111351
DOI: 10.3390/ph16040594 -
BMC Microbiology Apr 2023Rickettsia helvetica, a spotted fever rickettsia, is transmitted to humans via ticks in Europe, North Africa, and Asia. The central nervous system is a crucial target...
BACKGROUND
Rickettsia helvetica, a spotted fever rickettsia, is transmitted to humans via ticks in Europe, North Africa, and Asia. The central nervous system is a crucial target for rickettsial diseases, which has been reported for 12 of the 31 species, of which R. helvetica is one. This study aimed, in an experimental model, to identify characteristics of R. helvetica infection in a mouse neuronal cell line, NSC-34.
RESULTS
NSC-34, a fusion cell line of mouse motor spinal cord neurons and neuroblastoma cells, was used as a model. Propagation of R. helvetica in neurons was confirmed. Short actin tails were shown at the polar end of the bacteria, which makes it likely that they can move intracellularly, and even spread between cells. Another protein, Sca4, which with the cell adhesion protein vinculin enables the passage of the cell membrane, was expressed during infection. No significant increase in TNFα levels was seen in the infected neurons, which is of interest because TNFα protects the host cell from infection-induced apoptotic death which is crucial for host cell survival. The bacteria were also shown to invade and grow in the cell nucleus of the neuron.
CONCLUSIONS
The findings suggest that a R. helvetica infection may be harmful to NSC-34 neurons under these in vitro conditions, but the full effects of the infection on the cell need to be studied further, also on human neurons, to also understand the possible significance of this infection in relation to pathogenetic mechanisms.
Topics: Animals; Mice; Humans; Tumor Necrosis Factor-alpha; Rickettsia; Cell Nucleus; Neurons; Ixodes
PubMed: 37085774
DOI: 10.1186/s12866-023-02859-0 -
Journal of Cell Science Apr 2023Talin (herein referring to the talin-1 form), is a cytoskeletal adapter protein that binds integrin receptors and F-actin, and is a key factor in the formation and...
Talin (herein referring to the talin-1 form), is a cytoskeletal adapter protein that binds integrin receptors and F-actin, and is a key factor in the formation and regulation of integrin-dependent cell-matrix adhesions. Talin forms the mechanical link between the cytoplasmic domain of integrins and the actin cytoskeleton. Through this linkage, talin is at the origin of mechanosignaling occurring at the plasma membrane-cytoskeleton interface. Despite its central position, talin is not able to fulfill its tasks alone, but requires help from kindlin and paxillin to detect and transform the mechanical tension along the integrin-talin-F-actin axis into intracellular signaling. The talin head forms a classical FERM domain, which is required to bind and regulate the conformation of the integrin receptor, as well as to induce intracellular force sensing. The FERM domain allows the strategic positioning of protein-protein and protein-lipid interfaces, including the membrane-binding and integrin affinity-regulating F1 loop, as well as the interaction with lipid-anchored Rap1 (Rap1a and Rap1b in mammals) GTPase. Here, we summarize the structural and regulatory features of talin and explain how it regulates cell adhesion and force transmission, as well as intracellular signaling at integrin-containing cell-matrix attachment sites.
Topics: Animals; Talin; Actins; Integrins; Cell Adhesion; Cytoskeletal Proteins; Lipids; Mammals
PubMed: 37078342
DOI: 10.1242/jcs.260576 -
Shigella IpaA mediates actin bundling through diffusible vinculin oligomers with activation imprint.Cell Reports Apr 2023Upon activation, vinculin reinforces cytoskeletal anchorage during cell adhesion. Activating ligands classically disrupt intramolecular interactions between the vinculin...
Upon activation, vinculin reinforces cytoskeletal anchorage during cell adhesion. Activating ligands classically disrupt intramolecular interactions between the vinculin head and tail domains that bind to actin filaments. Here, we show that Shigella IpaA triggers major allosteric changes in the head domain, leading to vinculin homo-oligomerization. Through the cooperative binding of its three vinculin-binding sites (VBSs), IpaA induces a striking reorientation of the D1 and D2 head subdomains associated with vinculin oligomerization. IpaA thus acts as a catalyst producing vinculin clusters that bundle actin at a distance from the activation site and trigger the formation of highly stable adhesions resisting the action of actin relaxing drugs. Unlike canonical activation, vinculin homo-oligomers induced by IpaA appear to keep a persistent imprint of the activated state in addition to their bundling activity, accounting for stable cell adhesion independent of force transduction and relevant to bacterial invasion.
Topics: Bacterial Proteins; Antigens, Bacterial; Actins; Vinculin; Shigella; Protein Binding
PubMed: 37071535
DOI: 10.1016/j.celrep.2023.112405 -
ACS Applied Materials & Interfaces Apr 2023The substratum topography of both natural and synthetic materials is a prominent regulator of cell behaviors including adhesion, migration, matrix fibrillogenesis, and...
Contact Guidance of Connective Tissue Fibroblasts on Submicrometer Anisotropic Topographical Cues Is Dependent on Tissue of Origin, β1 Integrins, and Tensin-1 Recruitment.
The substratum topography of both natural and synthetic materials is a prominent regulator of cell behaviors including adhesion, migration, matrix fibrillogenesis, and cell phenotype. Connective tissue fibroblasts are known to respond to repeating groove topographical modifications by aligning and exhibiting directed migration, a phenomenon termed contact guidance. Although both reside in collagen rich connective tissues, dermal and gingival fibroblasts are known to exhibit differences in phenotype during wound healing, with gingival tissue showing a fetal-like scarless response. Differences in adhesion formation and maturation are known to underlie both a scarring phenotype and cell response to topographical features. Utilizing repeating groove substrates with periodicities of 600, 900, and 1200 nm (depth, 100 nm), we investigated the roles of integrins αvβ3 and β1 associated adhesions on contact guidance of human gingival (HGFs) and dermal fibroblasts (HDFs). HGFs showed a higher degree of orientation with the groove long axis than HDFs, with alignment of both vinculin and tensin-1 evident on 600 and 900 nm periodicities in both cell types. Orientation with grooves of any periodicity in HGFs and HDFs did not alter the adhesion number or area compared to smooth control surfaces. Growth of both cell types on all periodicities reduced fibronectin fibrillogenesis compared to control surfaces. Independent inhibition of integrin αvβ3 and β1 in both cell types induced changes in spreading up to 6 h and reduced alignment with the groove long axis. At 24 h post-seeding with blocking antibodies, HGFs recovered orientation, but in HDFs, blocking of β1, but not αvβ3 integrins, inhibited alignment. Blocking of β1 and αvβ3 in HDFs, but not HGFs, inhibited tensin-1-associated fibrillar adhesion formation. Furthermore, inhibition of β1 integrins in HDFs, but not HGFs, resulted in recruitment of tensin-1 to αvβ3 focal adhesions, preventing HDFs from aligning with the groove long axis. Our work demonstrates that tensin-1 localization with specific integrins in adhesion sites is an important determinant of contact guidance. This work emphasizes further the need for tissue-specific biomaterials, when integration into host tissues is required.
Topics: Humans; Integrin beta1; Tensins; Cues; Fibroblasts; Integrin alphaVbeta3; Connective Tissue
PubMed: 37067372
DOI: 10.1021/acsami.2c22381 -
Nature Communications Apr 2023To enter mitosis, most adherent animal cells reduce adhesion, which is followed by cell rounding. How mitotic cells regulate adhesion to neighboring cells and...
To enter mitosis, most adherent animal cells reduce adhesion, which is followed by cell rounding. How mitotic cells regulate adhesion to neighboring cells and extracellular matrix (ECM) proteins is poorly understood. Here we report that, similar to interphase, mitotic cells can employ integrins to initiate adhesion to the ECM in a kindlin- and talin-dependent manner. However, unlike interphase cells, we find that mitotic cells cannot engage newly bound integrins to actomyosin via talin or vinculin to reinforce adhesion. We show that the missing actin connection of newly bound integrins leads to transient ECM-binding and prevents cell spreading during mitosis. Furthermore, β1 integrins strengthen the adhesion of mitotic cells to adjacent cells, which is supported by vinculin, kindlin, and talin1. We conclude that this dual role of integrins in mitosis weakens the cell-ECM adhesion and strengthens the cell-cell adhesion to prevent delamination of the rounding and dividing cell.
Topics: Animals; Integrins; Vinculin; Talin; Cell Adhesion; Extracellular Matrix; Mitosis
PubMed: 37059721
DOI: 10.1038/s41467-023-37760-x -
Dental Materials Journal Aug 2023The present study was designed to clarify the activity of human gingival fibroblasts (HGFs) on the fibronectin (FN)-coated silanized microgroove titanium surface. The...
The present study was designed to clarify the activity of human gingival fibroblasts (HGFs) on the fibronectin (FN)-coated silanized microgroove titanium surface. The surface elemental composition of titanium discs was detected using XPS. HGFs' adhesion to the titanium discs was detected by immunofluorescence staining of vinculin. HGFs' number on the titanium discs was detected using the CCK8 assay. HGFs' secretion of type 1 collagen after five days of culturing was detected using ELISA and qPCR. HGFs could proliferate and spread well on the surface. The viability of HGFs in the experimental group was significantly more than in the control group. The HGFs in the experimental group significantly secreted more type 1 collagen than in the control group. Therefore, FN-coated can improve the morphology, viability, and type 1 collagen secretion of HGFs silanized microgroove titanium surface, which might ameliorate the efficacy of implants.
Topics: Humans; Titanium; Surface Properties; Fibronectins; Collagen Type I; Cell Adhesion; Fibroblasts; Gingiva; Cells, Cultured
PubMed: 37045777
DOI: 10.4012/dmj.2022-137