Myristoylated Alanine-rich C Kinase Substrate Research Articles

PIP2 Is An Electrostatic Catalyst for Vesicle Fusion by Lowering the Hydration Energy: Arresting Vesicle Fusion by Masking PIP2.

Lipids are key factors in regulating membrane fusion. Lipids are not only structural components to form membranes but also active catalysts for vesicle fusion and neurotransmitter release, which are driven by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. SNARE proteins seem to be partially assembled before fusion, but the mechanisms that arrest vesicle fusion before Ca2+ influx are still not clear. Here, we show that phosphatidylinositol 4,5-bisphosphate (PIP2) electrostatically triggers vesicle fusion as an electrostatic catalyst by lowering the hydration energy and that a myristoylated alanine-rich C-kinase substrate (MARCKS), a PIP2-binding protein, arrests vesicle fusion in a vesicle docking state where the SNARE complex is partially assembled. Vesicle-mimicking liposomes fail to reproduce vesicle fusion arrest by masking PIP2, indicating that native vesicles are essential for the reconstitution of physiological vesicle fusion. PIP2 attracts cations to repel water molecules from membranes, thus lowering the hydration energy barrier.

Abstract 3360: Myristoylated alanine-rich C-kinase substrate (MARCKS) as a target for pathological tunneling nanotubes in glioblastoma

Abstract Background: Glioblastoma (GBM) is a lethal brain malignancy with a median survival of <2 years due to relapse in 100% of cases. Tunneling nanotubes (TNTs) facilitate the development of treatment resistance by establishing tumor-promoting networks between GBM and surrounding cells. Only having emerged in 2004, many key players remain to be identified which is why we propose that MARCKS regulates TNT directionality through phosphorylation of its effector domain (ED). Methods: We quantified TNT sinuosity, numbers, branching points, and average length in (3) PTEN-null doxycycline-inducible U87-MARCKS-mutant models in serum-free conditions. Mutants overexpress MARCKS as wild-type (U87-WT), non-phosphorylated (U87-NP), or pseudo-phosphorylated (U87-PP). TNTs were characterized using immunofluorescence, confocal imaging, ICY manual TNT plugin, and negative staining. We also investigated a cytotoxic peptide derived from MARCKS-ED, termed MED2, on PTEN-null brain tumor-initiating cell (BTIC) TNTs. Statistical analysis was conducted with one or two-way ANOVA and multiple comparisons. Results: The most striking difference between all mutants is the loss of rigidity and directionality in U87-NP TNTs. Negative staining revealed U87-NP TNTs have deformed bodies with clubbed tips compared to ordered filaments in U87-WT and U87-PP. 4h MED2 treatment in non-mutated BTICs results in cytotoxicity as well as truncated TNTs that phenotypically resemble U87-NP. Conclusions: This is the first report supporting MARCKS to be a novel regulator of TNTs through ED phosphorylation. Future directions will investigate the functionality of TNTs in U87 mutants to determine if the lack of MARCKS phosphorylation renders TNTs nonfunctional. Defining MARCKS' role in GBM TNTs not only advances the field of cellular communication but also offers a new perspective on GBM treatment. Quantitative TNT Characterization Sinuosity TNT Number Branch Points Average Length MARCKS/F-actin Correlation U87-WT 1.00 +/- 0.00 29.0 +/- 2.94 17.0 +/- 2.94 9.80 +/- 1.74 R2=0.9245 U87-NP 1.21 +/- 0.10 11.5 +/- 1.29 9.00 +/- 0.82 9.90 +/- 1.79 R2=0.5497 U87-PP 1.03 +/- 0.02 34.3 +/- 11.2 8.50 +/- 3.41 7.90 +/- 1.05 R2=0.7249 P-value WT vs. NP: P=0.0026 WT vs. PP: P=0.7804 NP vs. PP: P=0.0070 WT vs. NP: P=0.0126 WT vs. PP: P=0.5344 NP vs. PP: P=0.0025 WT vs. NP: P=0.0053 WT vs. PP: P=0.0036 NP vs. PP: P=0.9616 WT vs. NP: P=0.9975 WT vs. PP: P=0.0363 NP vs. PP: P=0.0312 U87-WT: P<0.0001 U87-NP: P<0.0001 U87-PP: P<0.0001 BTIC + No treatment 1.00 +/- 0.00 13.5 +/- 1.29 8.00 +/- 1.83 23.2 +/- 10.3 Not determined BTIC + 3μM MED2 1.48 +/- 0.33 13.3 +/- 3.5 7.50 +/- 1.92 15.0 +/- 11.7 Not determined P-value NT vs. MED2: P=0.0198 NT vs. MED2: P>0.9999 NT vs. MED2: P>0.9999 NT vs. MED2 P=0.2003 Not determined Citation Format: Lauren C. Nassour-Caswell, Nicholas J. Eustace, Shane T. Rich-New, Taylor L. Schanel, Hasan Alrefai, Andee M. Beierle, Manoj Kumar, Patricia H. Hicks, Johsua C. Anderson, Christopher D. Willey. Myristoylated alanine-rich C-kinase substrate (MARCKS) as a target for pathological tunneling nanotubes in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3360.

MARCKS Inhibition Alters Bovine Neutrophil Responses to Salmonella Typhimurium.

Neutrophils are innate immune cells that respond quickly to sites of bacterial infection and play an essential role in host defense. Interestingly, some bacterial pathogens benefit from exuberant neutrophil inflammation. Salmonella is one such pathogen that can utilize the toxic mediators released by neutrophils to colonize the intestine and cause enterocolitis. Because neutrophils can aid gut colonization during Salmonella infection, neutrophils represent a potential host-directed therapeutic target. Myristoylated alanine-rich C-kinase substrate (MARCKS) is an actin-binding protein that plays an essential role in many neutrophil effector responses. We hypothesized that inhibition of MARCKS protein would alter bovine neutrophil responses to Salmonella Typhimurium (STm) ex vivo. We used a MARCKS inhibitor peptide to investigate the role of MARCKS in neutrophil responses to STm. This study demonstrates that MARCKS inhibition attenuated STm-induced neutrophil adhesion and chemotaxis. Interestingly, MARCKS inhibition also enhanced neutrophil phagocytosis and respiratory burst in response to STm. This is the first report describing the role of MARCKS protein in neutrophil antibacterial responses.

MARCKS and PI(4,5)P2 reciprocally regulate actin-based dendritic spine morphology.

Myristoylated, alanine-rich C-kinase substrate (MARCKS) is an F-actin and phospholipid binding protein implicated in numerous cellular activities, including the regulation of morphology in neuronal dendrites and dendritic spines. MARCKS contains a lysine-rich effector domain that mediates its binding to plasma membrane phosphatidylinositol-4,5-biphosphate (PI(4,5)P2) in a manner controlled by PKC and calcium/calmodulin. In neurons, manipulations of MARCKS concentration and membrane targeting strongly affect the numbers, shapes, and F-actin properties of dendritic spines, but the mechanisms remain unclear. Here, we tested the hypothesis that the effects of MARCKS on dendritic spine morphology are due to its capacity to regulate the availability of plasma membrane PI(4,5)P2. We observed that the concentration of free PI(4,5)P2 on the dendritic plasma membrane was inversely proportional to the concentration of MARCKS. Endogenous PI(4,5)P2 levels were increased or decreased, respectively, by acutely overexpressing either phosphatidylinositol-4-phosphate 5-kinase (PIP5K) or inositol polyphosphate 5-phosphatase (5ptase). PIP5K, like MARCKS depletion, induced severe spine shrinkage; 5ptase, like constitutively membrane-bound MARCKS, induced aberrant spine elongation. These phenotypes involved changes in actin properties driven by the F-actin severing protein cofilin. Collectively, these findings support a model in which neuronal activity regulates actin-dependent spine morphology through antagonistic interactions of MARCKS and PI(4,5)P2.

Binding-induced lipid domains: Peptide-membrane interactions with PIP2 and PS

Cell signaling is an important process involving complex interactions between lipids and proteins. The myristoylated alanine-rich C-kinase substrate (MARCKS) has been established as a key signaling regulator, serving a range of biological roles. Its effector domain (ED), which anchors the protein to the plasma membrane, induces domain formation in membranes containing phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylserine (PS). The mechanisms governing the MARCKS-ED binding to membranes remain elusive. Here, we investigate the composition-dependent affinity and MARCKS-ED-binding-induced changes in interfacial environments using two-dimensional infrared spectroscopy and fluorescence anisotropy. Both negatively charged lipids facilitate the MARCKS-ED binding to lipid vesicles. Although the hydrogen-bonding structure at the lipid-water interface remains comparable across vesicles with varied lipid compositions, the dynamics of interfacial water show divergent patterns due to specific interactions between lipids and peptides. Our findings also reveal that PIP2 becomes sequestered by bound peptides, while the distribution of PS exhibits no discernible change upon peptide binding. Interestingly, PIP2 and PS become colocalized into domains both in the presence and absence of MARCKS-ED. More broadly, this work offers molecular insights into the effects of membrane composition on binding.

Myristoylated, alanine-rich C-kinase substrate (MARCKS) regulates toll-like receptor 4 signaling in macrophages

MARCKS (myristoylated alanine-rich C-kinase substrate) is a membrane-associated protein expressed in many cell types, including macrophages. MARCKS is functionally implicated in cell adhesion, phagocytosis, and inflammation. LPS (lipopolysaccharide) triggers inflammation via TLR4 (toll-like receptor 4).The presence of MARCKS and the formation of phospho-MARCKS in various cell types have been described, but the role(s) of MARCKS in regulating macrophage functions remain unclear. We investigated the role of MARCKS in inflammation. Confocal microscopy revealed that MARCKS and phospho-MARCKS increased localization to endosomes and the Golgi apparatus upon LPS stimulation.CRISPR-CAS9 mediated knockout of MARCKS in macrophages downregulated the production of TNF and IL6, suggesting a role for MARCKS in inflammatory responses. Our comprehensive proteomics analysis together with real-time metabolic assays comparing LPS-stimulation of WT and MARCKS knock-out macrophages provided insights into the involvement of MARCKS in specific biological processes including innate immune response, inflammatory response, cytokine production, and molecular functions such as extracellularly ATP-gated cation channel activity, electron transfer activity and oxidoreductase activity, uncovering specific proteins involved in regulating MARCKS activity upon LPS stimulation. MARCKS appears to be a key regulator of inflammation whose inhibition might be beneficial for therapeutic intervention in inflammatory diseases.

Far-Infrared Ameliorates Pb-Induced Renal Toxicity via Voltage-Gated Calcium Channel-Mediated Calcium Influx.

Far-infrared (FIR), characterized by its specific electromagnetic wavelengths, has emerged as an adjunctive therapeutic strategy for various diseases, particularly in ameliorating manifestations associated with renal disorders. Although FIR was confirmed to possess antioxidative and anti-inflammatory attributes, the intricate cellular mechanisms through which FIR mitigates lead (Pb)-induced nephrotoxicity remain enigmatic. In this study, we investigated the effects of FIR on Pb-induced renal damage using in vitro and in vivo approaches. NRK52E rat renal cells exposed to Pb were subsequently treated with ceramic-generated FIR within the 9~14 μm range. Inductively coupled plasma mass spectrometry (ICP-MS) enabled quantitative Pb concentration assessment, while proteomic profiling unraveled intricate cellular responses. In vivo investigations used Wistar rats chronically exposed to lead acetate (PbAc) at 6 g/L in their drinking water for 15 weeks, with or without a concurrent FIR intervention. Our findings showed that FIR upregulated the voltage-gated calcium channel, voltage-dependent L type, alpha 1D subunit (CaV1.3), and myristoylated alanine-rich C kinase substrate (MARCKS) (p < 0.05), resulting in increased calcium influx (p < 0.01), the promotion of mitochondrial activity, and heightened ATP production. Furthermore, the FIR intervention effectively suppressed ROS production, concurrently mitigating Pb-induced cellular death. Notably, rats subjected to FIR exhibited significantly reduced blood Pb levels (30 vs. 71 μg/mL; p < 0.01), attenuated Pb-induced glomerulosclerosis, and enhanced Pb excretion compared to the controls. Our findings suggest that FIR has the capacity to counteract Pb-induced nephrotoxicity by modulating calcium influx and optimizing mitochondrial function. Overall, our data support FIR as a novel therapeutic avenue for Pb toxicity in the kidneys.

Small Organic Compounds Mimicking the Effector Domain of Myristoylated Alanine-Rich C-Kinase Substrate Stimulate Female-Specific Neurite Outgrowth.

Myristoylated alanine-rich C-kinase substrate (MARCKS) is a critical member of a signaling cascade that influences disease-relevant neural functions such as neural growth and plasticity. The effector domain (ED) of MARCKS interacts with the extracellular glycan polysialic acid (PSA) through the cell membrane to stimulate neurite outgrowth in cell culture. We have shown that a synthetic ED peptide improves functional recovery after spinal cord injury in female but not male mice. However, peptides themselves are unstable in therapeutic applications, so we investigated more pharmacologically relevant small organic compounds that mimic the ED peptide to maximize therapeutic potential. Using competition ELISAs, we screened small organic compound libraries to identify molecules that structurally and functionally mimic the ED peptide of MARCKS. Since we had shown sex-specific effects of MARCKS on spinal cord injury recovery, we assayed neuronal viability as well as neurite outgrowth from cultured cerebellar granule cells of female and male mice separately. We found that epigallocatechin, amiodarone, sertraline, tegaserod, and nonyloxytryptamine bind to a monoclonal antibody against the ED peptide, and compounds stimulate neurite outgrowth in cultured cerebellar granule cells of female mice only. Therefore, a search for compounds that act in males appears warranted.

Circ-AMOTL1 enhances cardiac fibrosis through binding with EIF4A3 and stabilizing MARCKS expression in diabetic cardiomyopathy

ObjectiveTo evaluate the effects and possible mechanisms of circular RNAs (circRNAs) on diabetic myocardial fibrosis (DMF). MethodsWe used an in vivo mice model of streptozotocin (STZ)-induced diabetes and conducted in vitro studies using cultured mouse cardiac fibroblast cells (CFs). ResultsWe found that the expression of circ-AMOTL1 was significantly upregulated in the myocardial tissue of diabetic mice compared to that in normal tissues. Inhibition of circ-AMOTL1 improved cardiac function in mice with type I diabetes and significantly repressed STZ-induced myocardial mesenchymal and perivascular fibrosis. In addition, silencing circ-AMOTL1 inhibited cell proliferation, decreased the expression levels of TGF-β1, collagen 1, collagen III, and α-SMA, and reduced the levels of ROS and NO in HG-treated CFs. Our data also indicated that silencing circ-AMOTL1 significantly reduced the expression of myristoylated alanine-rich C-kinase substrate (MARCKS). Finally, circ-AMOTL1 combined with the RNA-binding protein EIF4A3 to improve MARCKS stability. Moreover, co-transfection with si-circ-AMOTL1 and MARCKS reversed the effects of si-circ-AMOTL1 on cell proliferation, fibrotic marker proteins, and ROS and NO levels in vitro. ConclusionOur data suggest that circ-AMOTL1 plays a key role in STZ-induced DMF by modulating MARCKS, and that targeting circ-AMOTL1 may be a potential strategy to treat DMF.

Targeting carcinoma-associated mesothelial cells with antibody-drug conjugates in ovarian carcinomatosis.

Ovarian carcinomatosis is characterized by the accumulation of carcinoma-associated mesothelial cells (CAMs) in the peritoneal stroma and mainly originates through a mesothelial-to-mesenchymal transition (MMT) process. MMT has been proposed as a therapeutic target for peritoneal metastasis. Most ovarian cancer (OC) patients present at diagnosis with peritoneal seeding, which makes tumor progression control difficult by MMT modulation. An alternative approach is to use antibody-drug conjugates (ADCs) targeted directly to attack CAMs. This strategy could represent the cornerstone of precision-based medicine for peritoneal carcinomatosis. Here, we performed complete transcriptome analyses of ascitic fluid-isolated CAMs in advanced OC patients with primary-, high-, and low-grade, serous subtypes and following neoadjuvant chemotherapy. Our findings suggest that both cancer biological aggressiveness and chemotherapy-induced tumor mass reduction reflect the MMT-associated changes that take place in the tumor surrounding microenvironment. Accordingly, MMT-related genes, including fibroblast activation protein (FAP), mannose receptor C type 2 (MRC2), interleukin-11 receptor alpha (IL11RA), myristoylated alanine-rich C-kinase substrate (MARCKS), and sulfatase-1 (SULF1), were identified as specific actionable targets in CAMs of OC patients, which is a crucial step in the de novo design of ADCs. These cell surface target receptors were also validated in peritoneal CAMs of colorectal cancer peritoneal implants, indicating that ADC-based treatment could extend to other abdominal tumors that show peritoneal colonization. As proof of concept, a FAP-targeted ADC reduced tumor growth in an OC xenograft mouse model with peritoneal metastasis-associated fibroblasts. In summary, we propose MMT as a potential source of ADC-based therapeutic targets for peritoneal carcinomatosis. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Augmentation of Cathepsin Isoforms in Diabetic db/db Mouse Kidneys Is Associated with an Increase in Renal MARCKS Expression and Proteolysis.

The expression of the myristoylated alanine-rich C-kinase substrate (MARCKS) family of proteins in the kidneys plays an important role in the regulation of the renal epithelial sodium channel (ENaC) and hence overall blood pressure regulation. The function of MARCKS is regulated by post-translational modifications including myristoylation, phosphorylation, and proteolysis. Proteases known to cleave both ENaC and MARCKS have been shown to contribute to the development of high blood pressure, or hypertension. Here, we investigated protein expression and proteolysis of MARCKS, protein expression of multiple protein kinase C (PKC) isoforms, and protein expression and activity of several different proteases in the kidneys of diabetic db/db mice compared to wild-type littermate mice. In addition, MARCKS protein expression was assessed in cultured mouse cortical collecting duct (mpkCCD) cells treated with normal glucose and high glucose concentrations. Western blot and densitometric analysis showed less abundance of the unprocessed form of MARCKS and increased expression of a proteolytically cleaved form of MARCKS in the kidneys of diabetic db/db mice compared to wild-type mice. The protein expression levels of PKC delta and PKC epsilon were increased, while cathepsin B, cathepsin S, and cathepsin D were augmented in diabetic db/db kidneys compared to those of wild-type mice. An increase in the cleaved form of MARCKS was observed in mpkCCD cells cultured in high glucose compared to normal glucose concentrations. Taken together, these results suggest that high glucose may contribute to an increase in the proteolysis of renal MARCKS, while the upregulation of the cathepsin proteolytic pathway positively correlates with increased proteolysis of MARCKS in diabetic kidneys, where PKC expression is augmented.

A myristoylated alanine-rich C-kinase substrate (MARCKS) inhibitor peptide attenuates neutrophil outside-in β2-integrin activation and signaling

ABSTRACT MARCKS is an actin and PIP2-binding protein that plays an essential role in neutrophil migration and adhesion; however, the molecular details regarding MARCKS function in these processes remains unclear. Neutrophil adhesion and migration also require the cell surface receptors β2-integrins. We hypothesized that MARCKS inhibition would alter neutrophil β2-integrin activation and signaling. We utilized a MARCKS-targeting peptide to inhibit MARCKS in inside-out and outside-in β2-integrin activation in neutrophils. MANS-mediated MARCKS inhibition had no significant effect on inside-out β2-integrin activation. MANS treatment significantly attenuated ICAM-1/Mn2+-stimulated static adhesion, cell spreading and β2-integrin clustering, suggesting a role for MARCKS function in outside-in β2-integrin activation. Additional work is needed to better understand the molecular mechanisms of MARCKS role in outside-in β2-integrin activation and signaling.

Decreased MARCKS Protein Expression in Kidney Cortex Membrane Fractions of Cathepsin B Knockout Mice Is Associated with Reduced Lysophosphatidylcholine and Protein Kinase C Activity.

Cathpesin B is a multi-functional protease that plays numerous roles in physiology and pathophysiology. We hypothesized that actin cytoskeleton proteins that are substrates of cathepsin B, various lipids, and kinases that are regulated by lipids would be down-regulated in the kidney of cathepsin B knockout mice. Here, we show by Western blot and densitometric analysis that the expression and proteolysis of the actin cytoskeleton proteins myristoylated alanine-rich C-kinase substrate (MARCKS) and spectrin are significantly reduced in kidney cortex membrane fractions of cathepsin B knockout mice compared to C57B6 wild-type control mice. Lipidomic results show that specific lipids are increased while other lipids, including lysophosphatidylcholine (LPC) species LPC (16:0), LPC (18:0), LPC (18:1), and LPC (18:2), are significantly decreased in membrane fractions of the kidney cortex from Cathepsin B null mice. Protein Kinase C (PKC) activity is significantly lower in the kidney cortex of cathepsin B knockout mice compared to wild-type mice, while calcium/calmodulin-dependent protein kinase II (CaMKII) activity and phospholipase D (PLD) activity are comparable between the two groups. Together, these results provide the first evidence of altered actin cytoskeleton organization, membrane lipid composition, and PKC activity in the kidneys of mice lacking cathepsin B.

Integrated analysis identifies microRNA-188-5p as a suppressor of AKT/mTOR pathway in renal cancer.

Many current microRNA (miRNA) expression datasets for renal cell carcinoma (RCC) often show inconsistent analysis results, so a shift to comprehensive analysis of multiple datasets can effectively accelerate molecular screening for precision medicine and translational medicine research. MicroRNA (miR)-188-5p is a clinically noteworthy miRNA whose aberrant expression was previously observed in a variety of cancers, but its role in RCC is unclear. In this study, we undertook a comprehensive analysis of four RCC miRNA expression datasets and validated the results using The Cancer Genome Atlas (TCGA) dataset and a cohort of collected clinical samples. Fifteen miRNAs were identified as potential diagnostic markers by the analysis of four RCC miRNAs datasets. Analysis of the TCGA kidney renal clear cell carcinoma dataset showed significantly shorter survival in RCC patients with reduced miR-188-5p expression levels, and our collection of RCC clinical samples showed low miR-188-5p expression in the tumors. Overexpression of miR-188-5p in Caki-1 and 786-O cells inhibited cell growth, colony formation, invasion, and migration. In contrast, miR-188-5p inhibitors reversed these cell phenotypes. We identified a binding site for miR-188-5p in the 3'-UTR region of myristoylated alanine-rich C-kinase substrate (MARCKS) mRNA and demonstrated an interaction between these two molecules. Quantitative RT-PCR and western blot analysis revealed that miR-188-5p could regulate the AKT/mTOR signaling pathway through MARCKS. Mouse transplantation tumor assay indicated that miR-188-5p reduced the tumorigenicity of RCC in vivo. MicroRNA-188-5p could be a valuable new molecule for RCC diagnosis and prognosis.

Abstract 648: Divergent Roles For Endothelial Cell Marcks In Endothelial Cell Regeneration And Signaling After Arterial Injury And Ischemia

Cardiovascular interventions cause endothelial injury and trigger re-establishment of intact endothelium. After ischemia, endothelial signaling is essential for capillary angiogenesis as well as flow-mediated collateral artery enlargement to restore perfusion. We studied the role of the signaling protein MARCKS (myristoylated alanine-rich C kinase substrate) in the endothelial response to injury and ischemia-induced angiogenesis. Methods: Targeted deletion of MARCKS in endothelial cells was done with Cadherin 5-driven Cre/Lox recombination and confirmed with immunohistochemistry. All controls were Cre(-) littermates. Re-endothelialization after femoral artery wire injury was measured by arterial area quantitation after Evan’s Blue exclusion. After femoral artery ligation, limb blood flow was determined by serial laser Doppler scanning, and angiogenesis by counting CD31-positive cells in the gastrocnemius muscle on day 28. Results: Endothelial cell-specific deletion of MARCKS was confirmed in Cre (+) animals. Deletion of MARCKS did not affect endothelial regeneration on post operative day 6 after denuding femoral wire injury (68.2% versus 68.7% recovery, n=5-6, p&gt;.05). Ischemia-induced capillary angiogenesis was not affected by deletion of MARCKS (Capillary Count, Cre(+): 602 versus Cre(-): 511, n=6, p&gt;.05), however animals lacking endothelial MARCKS had worsened limb perfusion on days 1, 3, 7 and 21 after ischemia (ABI Range Cre(+) versus Cre(-): 0.04-0.22 versus 0.07-0.41, respectively, n=6-9, p&lt;.05). Conclusions: These studies demonstrate distinct and divergent roles for MARCKS in the endothelial cell response to injury and ischemia. MARCKS is not essential for endothelial regeneration after denuding large vessel injury, or for capillary angiogenesis induced by ischemia. Surprisingly, endothelial MARCKS appears to be a novel endogenous promotor of ischemia-induced limb revascularization. This suggests that endothelial cell MARCKS promotes flow-mediated endothelial cell signaling essential for ischemia-induced arterial enlargement. MARCKS activity could be a possible therapeutic target to enhance collateral artery formation in limb ischemia.

MARCKS is a New Prognostic Biomarker in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common type of cancers, but there is still a lack of known biomarkers for the effective diagnosis or prognosis of HCC. Myristoylated alanine-rich C-kinase substrate (MARCKS) is a substrate of protein kinase C, which was located in the cell plasma membrane. The purpose of our study was to evaluate the role of MARCKS in HCC. The role of MARCKS in HCC was explored by bioinformatics and experiment. We demonstrated that MARCKS expression was significantly elevated in HCC datasets of TCGA. MARCKS was up-regulated in tumor sample in HCC. Functional enrichment indicated that MARCKS-related differentially expressed genes (DEGs) were mainly enriched in cell junction tissue, response to growth factors and cell population proliferation. Tumor and ECM-receptor interactions related pathways were enriched by the KEGG. MARCKS expression in HCC patients was higher in females, younger individuals, and those at worse clinical stages. Cox regression analysis showed that MARCKS expression was a risk factor for overall survival and disease-specific survival of patients. MARCKS was up-regulated in HCC, may play a crucial role in HCCs, and has prognostic value for clinical outcomes.

Mammalian D-Cysteine: A new addition to the growing family of biologically relevant D-amino acids.

Mammalian D-Cysteine is racemized from L-cysteine by serine racemase, a pyridoxal phosphate (PLP)-dependent enzyme. Endogenous D-Cysteine plays a role in neural development by inhibiting proliferation of neural progenitor cells (NPCs) via protein kinase B (AKT) signaling mediated by the FoxO family of transcription factors. D-Cysteine binds to Myristoylated Alanine Rich C Kinase Substrate (MARCKS) and alters phosphorylation at Ser 159/163 and its translocation from the membrane. By racemizing serine and cysteine, mammalian serine racemase may play important roles in neural development highlighting its importance in psychiatric disorders.

Abstract P6-10-15: Targeting MARCKS in inflammatory breast cancer increased paclitaxel sensitivity

Abstract Background. Because of its high metastatic potential, inflammatory breast cancer (IBC) is the most lethal and aggressive form of breast cancer. We previously demonstrated that Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) protein overexpression was associated with shorter metastasis-free survival (MFS) in IBC patients, but not in non-IBC (nIBC) patients. However, the mechanism of action of MARCKS and its particular association to poorer outcome in IBC compared to nIBC are poorly understood. Methods. We evaluated in vitro the inhibitory effect of MPS (MARCKS phosphorylation site domain), a peptide targeting MARCKS phosphorylation site domain (PSD) in single and in combination with paclitaxel treatment, on cell proliferation and cell motility in two cell lines of different phenotype (SUM149 for IBC and MDA-MB-231 for nIBC), as well as its distinct molecular mechanisms of action. We also assessed the clinical relevance of the protein expression of MARCKS and phosphatase and tensin homolog (PTEN) in a large series of IBC vs. nIBC patients. Results. In vitro, the treatment with MPS peptide impaired cell proliferation, migration, and invasion in SUM149 compared to MDA-MB-231 cells. More important, MARCKS inhibition increased paclitaxel sensitivity when using combination therapy in SUM149 cells compared to MDA-MB-231 cells. Interestingly, we could partially explain this specific inhibitory effect in IBC cells using western blot: MARCKS inhibition in single and in combination induced up and downregulation of the PTEN/AKT signaling pathway respectively in IBC compared to nIBC cells. Importantly, a negative correlation of MARCKS and PTEN was only found in the clinical IBC samples (180 patients) compared to nIBC samples (355 patients). More importantly, the group of patients with negative MARCKS and positive PTEN protein expression was associated to better 5-year MFS only in IBC patients. Conclusion. These results indicate two major findings: first, the important prognostic value of the negative correlation of MARCKS and PTEN expression in IBC patients, and second the specific role of MARCKS in regulating different downstream pathways and increasing the paclitaxel response in combination treatment in IBC compared to non-IBC. They suggest a potential IBC-specific targetable biomarker, the inhibition of which might impair disease aggressiveness and perhaps enhance patients’ survival. Citation Format: Maroua Manai, Ines Bini, Pascal Finetti, Haifa Bichiou, Carolina Reduzzi, Naziha Ben Hamida, Marc Lopez, Khaled Rahal, Karima Mrad, Mohamed Manai, Massimo Cristofanilli, Hamouda Boussen, Raoudha Doghri, Maher Kharrat, François Bertucci. Targeting MARCKS in inflammatory breast cancer increased paclitaxel sensitivity [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-15.

Phosphoinositides regulation of hyperpolarization-activated cyclic-nucleotide gated (HCN) channels.

Several ion channels are regulated by protein-lipid interactions. Hyperpolarization-activated cyclic-nucleotide gated (HCN) channels specifically, have been shown to be regulated by phosphoinositides (PIPs). PIPs enhance HCN activation via a rightward shift in voltage-dependency and has importance applications in neuronal and cardiac function. Using computational and electrophysiological approaches, we aim to examine putative sites for HCN-PIP interactions. Computational docking and coarse-grained simulations indicate that PIP binding to HCN1 and HCN4 channels are not well coordinated, but rather occurs over a broad surface of charged residues primarily in the HCN-domain, S2 and S3 helices that can be loosely organized in 2 or 3 overlapping clusters. Thus, HCN-PIP interactions are more resembling of electrostatic interactions that occur in myristoylated alanine-rich C kinase substrate (MARCKS) proteins, than the specifically coordinated interactions that occur in pleckstrin homology domains (PH domains) or ion channels such as inward rectifier potassium (Kir) channels. Our results also indicate that phosphatidylinositol (PI) interactions with HCN1 are even lower affinity, explaining why unphosphorylated PI have no effect on HCN1 activation unlike phosphorylated PIPs. Experimental validation of putative binding sites are addressed by electrophysiological approaches.

Human Alpha-1 Antitrypsin Attenuates ENaC and MARCKS and Lowers Blood Pressure in Hypertensive Diabetic db/db Mice.

Hypertension may develop before or after the onset of diabetes and it is known to increase the risk of developing diabetic nephropathy. Alpha-1 antitrypsin (AAT) is a multi-functional protein with beneficial effects in various diseases but its role in reducing blood pressure in the diabetic kidney has not been thoroughly studied. Like blood pressure, epithelial sodium channels (ENaC) and its adaptor protein myristoylated alanine-rich C-kinase substrate (MARCKS) are regulated by circadian rhythms. Our hypothesis is that administration of human AAT (hAAT) reduces blood pressure in hypertensive diabetic mice by attenuating membrane expression of ENaC and its association with the actin cytoskeleton. First, we show hAAT administration results in reduced blood pressure in diabetic db/db mice compared to vehicle treatment in both the inactive and active cycles. Western blotting and immunohistochemistry analyses showed a reduction of ENaC and the actin cytoskeleton protein, MARCKS in the kidneys of diabetic db/db mice treated with hAAT compared to vehicle. hAAT treatment resulted in elevated amounts of extracellular vesicles present in the urine of diabetic db/db mice compared to vehicle treatment both in the inactive and active cycles. Multiple hexosylceramides, among other lipid classes increased in urinary EVs released from hAAT treated hypertensive diabetic mice compared to vehicle treated mice. Taken together, these data suggest hAAT treatment could normalize blood pressure in the diabetic kidney in a mechanism involving attenuation of renal ENaC and MARCKS protein expression and possibly ceramide metabolism to hexosylceramide in kidney cells.