RESUMO
BACKGROUND: MARCKS (myristoylated alanine-rich C kinase substrates) serves as a substrate for protein kinase C, residing in the plasma membrane while acts as an actin filament crosslinking protein. This investigation aims to elucidate phosphorylated MARCKS (p-MARCKS) levels and activity in allergic asthma patients and explore the therapeutic potential of peptide inhibitors targeting p-MARCKS in an acute mouse model of allergic asthma. METHODS: Immunohistochemistry and histology staining were employed on lung tissue slides to evaluate p-MARCKS expression and allergic asthma symptoms. Airway resistance was measured using invasive whole-body plethysmography. Flow cytometry detected lung dendritic cell migration, and migration/maturation assays were conducted on isolated murine bone marrow-derived dendritic cells (BM-DCs). RESULTS: Elevated p-MARCKS expression was observed in both human asthmatic tissues and animal models immunized with ovalbumin or Alternaria alternata. Remarkably, asthmatic individuals showed elevated high p-MARCKS expression in lung tissues. Intraperitoneal injection of the peptide MPS, targeting the MARCKS phosphorylation site domain, before allergen challenged, effectively suppressed MARCKS phosphorylation in murine lung tissues. MPS inhibited both in vivo and in vitro migration and maturation of dendritic cells (BM-DCs) and reduced Th2-related lymphocyte activation in bronchoalveolar lavage fluid (BALF). MPS pretreatment additionally suppressed all symptoms associated with allergic airway asthma, including a reduction in inflammatory cell influx, airway mucous cell metaplasia, and airway hyperreactivity. CONCLUSION: These findings suggest that phosphorylated MARCKS occurs in asthmatic lung tissue, and the inhibition of MARCKS phosphorylation by the MPS peptide reduces dendritic cell migration and Th2-related lymphocytes in the lungs in a murine model of acute asthma.
Assuntos
Asma , Movimento Celular , Células Dendríticas , Modelos Animais de Doenças , Substrato Quinase C Rico em Alanina Miristoilada , Animais , Asma/imunologia , Asma/tratamento farmacológico , Asma/patologia , Asma/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Movimento Celular/efeitos dos fármacos , Substrato Quinase C Rico em Alanina Miristoilada/metabolismo , Fosforilação , Camundongos , Humanos , Feminino , Pulmão/patologia , Pulmão/imunologia , Pulmão/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Doença Aguda , Peptídeos/farmacologia , MasculinoRESUMO
The Pcdhg gene cluster encodes 22 γ-Protocadherin (γ-Pcdh) cell adhesion molecules that critically regulate multiple aspects of neural development, including neuronal survival, dendritic and axonal arborization, and synapse formation and maturation. Each γ-Pcdh isoform has unique protein domains-a homophilically interacting extracellular domain and a juxtamembrane cytoplasmic domain-as well as a C-terminal cytoplasmic domain shared by all isoforms. The extent to which isoform-specific versus shared domains regulate distinct γ-Pcdh functions remains incompletely understood. Our previous in vitro studies identified protein kinase C (PKC) phosphorylation of a serine residue within a shared C-terminal motif as a mechanism through which γ-Pcdh promotion of dendrite arborization via myristoylated alanine-rich C-kinase substrate (MARCKS) is abrogated. Here, we used CRISPR/Cas9 genome editing to generate two new mouse lines expressing only non-phosphorylatable γ-Pcdhs, due either to a serine-to-alanine mutation (PcdhgS/A) or to a 15-amino acid C-terminal deletion resulting from insertion of an early stop codon (PcdhgCTD). Both lines are viable and fertile, and the density and maturation of dendritic spines remain unchanged in both PcdhgS/A and PcdhgCTD cortex. Dendrite arborization of cortical pyramidal neurons, however, is significantly increased in both lines, as are levels of active MARCKS. Intriguingly, despite having significantly reduced levels of γ-Pcdh proteins, the PcdhgCTD mutation yields the strongest phenotype, with even heterozygous mutants exhibiting increased arborization. The present study confirms that phosphorylation of a shared C-terminal motif is a key γ-Pcdh negative regulation point and contributes to a converging understanding of γ-Pcdh family function in which distinct roles are played by both individual isoforms and discrete protein domains.
Assuntos
Proteínas Relacionadas a Caderinas , Caderinas , Córtex Cerebral , Dendritos , Proteína Quinase C , Animais , Córtex Cerebral/metabolismo , Córtex Cerebral/citologia , Caderinas/metabolismo , Caderinas/genética , Fosforilação/fisiologia , Dendritos/metabolismo , Camundongos , Proteína Quinase C/metabolismo , Proteína Quinase C/genética , Substrato Quinase C Rico em Alanina Miristoilada/metabolismo , Substrato Quinase C Rico em Alanina Miristoilada/genética , Motivos de Aminoácidos/fisiologia , Camundongos TransgênicosRESUMO
Peptides and proteins encoded by noncanonical open reading frames (ORFs) of circRNAs have recently been recognized to play important roles in disease progression, but the biological functions and mechanisms of these peptides and proteins are largely unknown. Here, we identified a potential coding circular RNA, circTRIM1, that was upregulated in doxorubicin-resistant TNBC cells by intersecting transcriptome and translatome RNA-seq data, and its expression was correlated with clinicopathological characteristics and poor prognosis in patients with TNBC. CircTRIM1 possesses a functional IRES element along with an 810 nt ORF that can be translated into a novel endogenously expressed protein termed TRIM1-269aa. Functionally, we demonstrated that TRIM1-269aa, which is involved in the biological functions of circTRIM1, promoted chemoresistance and metastasis in TNBC cells both in vitro and in vivo. In addition, we found that TRIM1-269aa can be packaged into exosomes and transmitted between TNBC cells. Mechanistically, TRIM1-269aa enhanced the interaction between MARCKS and calmodulin, thus promoting the calmodulin-dependent translocation of MARCKS, which further initiated the activation of the PI3K/AKT/mTOR pathway. Overall, circTRIM1, which encodes TRIM1-269aa, promoted TNBC chemoresistance and metastasis by enhancing MARCKS translocation and PI3K/AKT/mTOR activation. Our investigation has yielded novel insights into the roles of protein-coding circRNAs and supported circTRIM1/TRIM1-269aa as a novel promising prognostic and therapeutic target for patients with TNBC.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Proteínas Associadas aos Microtúbulos , Substrato Quinase C Rico em Alanina Miristoilada , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , RNA Circular , Serina-Treonina Quinases TOR , Fatores de Transcrição , Neoplasias de Mama Triplo Negativas , Animais , Feminino , Humanos , Camundongos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Metástase Neoplásica , Fosfatidilinositol 3-Quinases/metabolismo , Prognóstico , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Circular/genética , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Proteínas com Motivo Tripartido/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Calmodulina/metabolismo , Substrato Quinase C Rico em Alanina Miristoilada/metabolismoRESUMO
In this paper, we study the nonlinear dynamics of the MARCKS protein between cytosol and cytoplasmic membrane through the modulational instability phenomenon. The reaction-diffusion generic model used here is firstly transformed into a cubic complex Ginzburg-Landau equation. Then, modulational instability (MI) is carried out in order to derive the MI criteria. We find the domains of some parameter space where nonlinear patterns are expected in the model. The analytical results on the MI growth rate predict that phosphorylation and binding rates affect MARCKS dynamics in opposite way: while the phosphorylation rate tends to support highly localized structures of MARCKS, the binding rate in turn tends to slow down such features. On the other hand, self-diffusion process always amplifies the MI phenomenon. These predictions are confirmed by numerical simulations. As a result, the cyclic transport of MARCKS protein from membrane to cytosol may be done by means of multisolitons-like patterns.
Assuntos
Dinâmica não Linear , Substrato Quinase C Rico em Alanina Miristoilada , Membrana Celular , Difusão , FosforilaçãoRESUMO
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.
Assuntos
Substrato Quinase C Rico em Alanina Miristoilada , Fosfatidilinositol 4,5-Difosfato , Fosfatidilserinas , Ligação Proteica , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Substrato Quinase C Rico em Alanina Miristoilada/metabolismo , Substrato Quinase C Rico em Alanina Miristoilada/química , Peptídeos/química , Peptídeos/metabolismo , Domínios Proteicos , Água/química , Membrana Celular/metabolismo , Membrana Celular/química , Sequência de AminoácidosRESUMO
MicroRNAs (miRNAs) have emerged as pivotal regulators of gene expression, playing essential roles in diverse cellular processes, including the development and progression of cancer. Among the numerous proteins influenced by miRNAs, the MARCKS/MARCKSL1 protein, a key regulator of cellular cytoskeletal dynamics and membrane-cytosol communication, has garnered significant attention due to its multifaceted involvement in various cancer-related processes, including cell migration, invasion, metastasis, and drug resistance. Motivated by the encouraging early clinical success of peptides targeting MARCKS in several pathological conditions, this review article delves into the intricate interplay between miRNAs and the MARCKS protein in cancer. Herein, we have highlighted the latest findings on specific miRNAs that modulate MARCKS/MARCKSL1 expression, providing a comprehensive overview of their roles in different cancer types. We have underscored the need for in-depth investigations into the therapeutic feasibility of targeting the miRNA-MARCKS axis in cancer, taking cues from the successes witnessed in related fields. Unlocking the full potential of miRNA-mediated MARCKS regulation could pave the way for innovative and effective therapeutic interventions against various cancer types.
Assuntos
MicroRNAs , Neoplasias , Humanos , Substrato Quinase C Rico em Alanina Miristoilada/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Proteína Quinase C/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias/genética , Fosforilação , Proteínas de Ligação a Calmodulina/metabolismo , Proteínas dos Microfilamentos/metabolismoRESUMO
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.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , Receptor 4 Toll-Like , Humanos , Lipopolissacarídeos/farmacologia , Substrato Quinase C Rico em Alanina Miristoilada , Macrófagos , Inflamação , FosforilaçãoRESUMO
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.
Assuntos
Diabetes Mellitus , Proteínas de Membrana , Camundongos , Animais , Substrato Quinase C Rico em Alanina Miristoilada/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteólise , Isoformas de Proteínas/metabolismo , Rim/metabolismo , Fosforilação , Camundongos Endogâmicos , Catepsinas/metabolismo , Peptídeo Hidrolases/metabolismo , Glucose/metabolismo , Diabetes Mellitus/metabolismoRESUMO
INTRODUCTION: MARCKS protein, a protein kinase C (PKC) substrate, is known to be at the intersection of several intracellular signaling pathways and plays a pivotal role in cellular physiology. Unlike PKC inhibitors, MARCKS-targeting drug (BIO-11006) has shown early success in clinical trials involving lung diseases. Recent research investigations have identified two MARCKS-targeting peptides which possess multifaceted implications against asthma, cancer, inflammation, and lung diseases. AREAS COVERED: This review article provides the patent landscape and recent developments on peptides targeting MARCKS for therapeutic purposes. Online free open-access databases were used to fetch out the patent information, and research articles were fetched using PubMed. EXPERT OPINION: Research studies highlighting the intriguing role of MARCKS in human disease and physiology have dramatically increased in recent years. A similar increasing trend in the number of patents has also been observed related to the MARCKS-targeting peptides. Thus, there is a need to amalgamate and translate such a trend into therapeutic intervention. Our review article provides an overview of such recent advances, and we believe that our compilation will fetch the interest of researchers around the globe to develop MARCKS-targeting peptides in future for human diseases.
Assuntos
Pneumopatias , Proteínas de Membrana , Humanos , Proteínas de Membrana/metabolismo , Substrato Quinase C Rico em Alanina Miristoilada/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Patentes como Assunto , Peptídeos/farmacologia , Proteína Quinase C/metabolismo , Alanina , FosforilaçãoRESUMO
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 ß2-integrins. We hypothesized that MARCKS inhibition would alter neutrophil ß2-integrin activation and signaling. We utilized a MARCKS-targeting peptide to inhibit MARCKS in inside-out and outside-in ß2-integrin activation in neutrophils. MANS-mediated MARCKS inhibition had no significant effect on inside-out ß2-integrin activation. MANS treatment significantly attenuated ICAM-1/Mn2+-stimulated static adhesion, cell spreading and ß2-integrin clustering, suggesting a role for MARCKS function in outside-in ß2-integrin activation. Additional work is needed to better understand the molecular mechanisms of MARCKS role in outside-in ß2-integrin activation and signaling.
Assuntos
Antígenos CD18 , Substrato Quinase C Rico em Alanina Miristoilada , Neutrófilos , Alanina , Antígenos CD18/metabolismo , Peptídeos/farmacologia , Transdução de Sinais , Substrato Quinase C Rico em Alanina Miristoilada/antagonistas & inibidoresRESUMO
Inflammatory breast cancer (IBC) is the most pro-metastatic form of breast cancer (BC). We previously demonstrated that protein overexpression of Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) protein was associated with shorter survival in IBC patients. MARCKS has been associated with the PI3K/AKT pathway. MARCKS inhibitors are in development. Our objective was to investigate MARCKS, expressed preferentially in IBC that non-IBC (nIBC), as a novel potential therapeutic target for IBC. The biologic activity of MPS, a MARCKS peptide inhibitor, on cell proliferation, migration, invasion, and mammosphere formation was evaluated in IBC (SUM149 and SUM190) and nIBC (MDA-MB-231 and MCF7) cell lines, as well as its effects on protein expression in the PTEN/AKT and MAPK pathways. The prognostic relevance of MARCKS and phosphatase and tensin homolog (PTEN) protein expression as a surrogate marker of metastasis-free survival (MFS) was evaluated by immunohistochemistry (IHC) in a retrospective series of archival tumor samples derived from 180 IBC patients and 355 nIBC patients. In vitro MPS impaired cell proliferation, migration and invasion, and mammosphere formation in IBC cells. MARCKS inhibition upregulated PTEN and downregulated pAKT and pMAPK expression in IBC cells, but not in nIBC cells. By IHC, MARCKS expression and PTEN expression were negatively correlated in IBC samples and were associated with shorter MFS and longer MFS, respectively, in multivariate analysis. The combination of MARCKS-/PTEN+ protein status was associated with longer MFS in IBC patient only (p = 8.7 × 10-3), and mirrored the molecular profile (MARCKS-downregulated/PTEN-upregulated) of MPS-treated IBC cell lines. In conclusion, our results uncover a functional role of MARCKS implicated in IBC aggressiveness. Associated with the good-prognosis value of the MARCKS-/PTEN+ protein status that mirrors the molecular profile of MPS-treated IBC cell lines, our results suggest that MARCKS could be a potential therapeutic target in patients with MARCKS-positive IBC. Future preclinical studies using a larger panel of IBC cell lines, animal models and analysis of a larger series of clinical samples are warranted in order to validate our results.
Assuntos
Produtos Biológicos , Neoplasias Inflamatórias Mamárias , Substrato Quinase C Rico em Alanina Miristoilada , Produtos Biológicos/uso terapêutico , Humanos , Neoplasias Inflamatórias Mamárias/tratamento farmacológico , Neoplasias Inflamatórias Mamárias/patologia , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Estudos Retrospectivos , TensinasRESUMO
Epithelial sodium channel (ENaC) is a pivotal regulator of alveolar fluid clearance in the airway epithelium and plays a key role in the treatment of acute lung injury (ALI), which is mainly composed of the three homologous subunits (α, ß and γ). The mechanisms of microRNAs in small extracellular vesicles (sEVs) derived from mesenchymal stem cell (MSC-sEVs) on the regulation of lung ion transport are seldom reported. In this study, we aimed at investigating whether miR-34c had an effect on ENaC dysfunction induced by lipopolysaccharide and explored the underlying mechanism in this process. Primarily, the effect of miR-34c on lung edema and histopathology changes in an ALI mouse model was investigated. Then the uptake of PKH26-labeled sEVs was observed in recipient cells, and we observed that the overexpression of miR-34c in MSC-sEVs could upregulate the LPS-inhibited γ-ENaC expression. The dual luciferase reporter gene assay demonstrated that myristoylated alanine-rich C kinase substrate (MARCKS) was one of target genes of miR-34c, the protein expression of which was negatively correlated with miR-34c. Subsequently, either upregulating miR-34c or knocking down MARCKS could increase the protein expression of phospho-phosphatidylinositol 3-kinase (p-PI3K) and phospho-protein kinase B (p-AKT), implying a downstream regulation pathway was involved. All of the above suggest that miR-34c in MSC-sEVs can attenuate edematous lung injury via enhancing γ-ENaC expression, at least partially, through targeting MARCKS and activating the PI3K/AKT signaling pathway subsequently.
Assuntos
Lesão Pulmonar Aguda , Vesículas Extracelulares , Células-Tronco Mesenquimais , MicroRNAs , Edema Pulmonar , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/terapia , Animais , Canais Epiteliais de Sódio/genética , Canais Epiteliais de Sódio/metabolismo , Vesículas Extracelulares/metabolismo , Transporte de Íons , Lipopolissacarídeos/farmacologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , MicroRNAs/metabolismo , Substrato Quinase C Rico em Alanina Miristoilada/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Edema Pulmonar/metabolismo , Transdução de SinaisRESUMO
Purpose: To determine if inhibition of Myristoylated Alanine Rich C Kinase Substrate (MARCKS) protein, using novel MARCKS inhibitor peptides, will reduce the severity of endotoxin-induced uveitis (EIU) in rats. Methods: EIU was induced in Lewis rats using subcutaneous administration of lipopolysaccharide. In the first phase of the study, 3 different novel MARCKS inhibitor peptides that mimic the N-terminal region of MARCKS (BIO-11006, or lower molecular weight analogs BIO-91201 or BIO-91202; Biomarck Pharmaceuticals, Ltd., Newtown, PA) were administered intravitreally (IVT) at 50 and 100 µM. In the second phase, BIO-91201 was administered IVT at 10, 50, and 100 µM and topically at the 100 µM concentration. The efficacy of MARCKS inhibitor peptides was assessed by clinical examination using slit lamp biomicroscopy, optical coherence tomography (OCT) anterior chamber cell counts, histopathology, and aqueous humor cytokine analysis. Results: Clinical scores were significantly reduced 24 h following uveitis induction in the first phase of the study in the following treatment groups: BIO-11006 50 µM IVT and 100 µM IVT, BIO-91201 50 µM IVT, and BIO-91202 100 µM IVT (P < 0.05). OCT anterior chamber cell counts were significantly reduced in the first phase of the study in all treatment groups (P < 0.001). OCT anterior chamber cell counts and histopathology scores were significantly reduced in the second phase of the study in the BIO-91201 50 µM IVT group (P < 0.05). No effect was seen with topical administration. Conclusion: MARCKS inhibitor peptides were effective in reducing the severity of ocular inflammation and cellular influx in EIU.
Assuntos
Endotoxinas , Uveíte , Animais , Humor Aquoso/metabolismo , Endotoxinas/toxicidade , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Substrato Quinase C Rico em Alanina Miristoilada/metabolismo , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Ratos , Ratos Endogâmicos Lew , Uveíte/induzido quimicamente , Uveíte/tratamento farmacológico , Uveíte/patologiaRESUMO
Ependymal cells (ECs) line the ventricular surfaces of the mammalian central nervous system (CNS) and their development is indispensable to structural integrity and functions of the CNS. We previously reported that EC-specific genetic deletion of the myristoylated alanine-rich protein kinase C substrate (Marcks) disrupts barrier functions and elevates oxidative stress and lipid droplet accumulation in ECs causing precocious cellular aging. However, little is known regarding the mechanisms that mediate these changes in ECs. To gain insight into Marcks-mediated mechanisms, we performed mass spectrometric analyses on Marcks-associated proteins in young and aged ECs in the mouse forebrain using an integrated approach. Network analysis on annotated proteins revealed that the identified Marcks-associated complexes are in part involved in protein transport mechanisms in young ECs. In fact, we found perturbed intracellular vesicular trafficking in cultured ECs with selective deletion of Marcks (Marcks-cKO mice), or upon pharmacological alteration to phosphorylation status of Marcks. In comparison, Marcks-associated protein complexes in aged ECs appear to be involved in regulation of lipid metabolism and responses to oxidative stress. Confirming this, we found elevated signatures of inflammation in the cerebral cortices and the hippocampi of young Marcks-cKO mice. Interestingly, behavioral testing using a water maze task indicated that spatial learning and memory is diminished in young Marcks-cKO mice similar to aged wildtype mice. Taken together, our study provides first line of evidence for potential mechanisms that may mediate differential Marcks functions in young and old ECs, and their effect on forebrain homeostasis during aging.
Assuntos
Epêndima , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Animais , Fosforilação , Epêndima/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Substrato Quinase C Rico em Alanina Miristoilada/metabolismo , Proteoma/metabolismo , Proteínas de Membrana/metabolismo , Proteína Quinase C/metabolismo , Homeostase , Prosencéfalo/metabolismo , Mamíferos/metabolismoRESUMO
BACKGROUND: The dysregulation of circular RNAs (circRNAs) has been shown to be correlated with the aggressiveness of renal cell carcinoma (RCC). Hence, this study investigated the role and mechanism of circ_0039569 in RCC progression. METHODS: The levels of circ_0039569, miR-133b, and MARCKS (myristoylated alanine-rich protein kinase C substrate) were detected using quantitative real-time polymerase chain reaction and Western blot. In vitro experiments were conducted by using 5-ethynyl-2'-deoxyuridine assay, colony-formation assay, Transwell assay, flow cytometry, and Western blot. The direct interactions between miR-133b and circ_0039569 or MARCKS were verified by using dual-luciferase reporter and pull-down assays. Xenograft mice models were established to conduct in vivo analysis. RESULTS: Circ_0039569 was highly expressed in RCC tissues and cells. Functionally, silencing of circ_0039569 suppressed cell proliferation, migration, and invasion, but induced cell apoptosis in RCC cells in vitro. Moreover, mice subcutaneous xenograft assay suggested that circ_0039569 knockdown impeded tumor growth in vivo. Mechanistically, circ_0039569 acted as a sponge for miR-133b to regulate the expression of its target MARCKS. Importantly, miR-133b inhibition or MARCKS knockdown attenuated the anticancer effects of circ_0039569 knockdown on RCC growth. CONCLUSION: Diminished circ_0039569 restrains the growth and propagation of RCC cells via miR-133b/MARCKS axis, indicating an underlying effective therapeutic target for RCC patients.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , MicroRNAs , Animais , Sítios de Ligação , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Movimento Celular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Renais/genética , Neoplasias Renais/patologia , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Substrato Quinase C Rico em Alanina Miristoilada/metabolismoRESUMO
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.
Assuntos
Diabetes Mellitus Experimental , Nefropatias Diabéticas , Hipertensão , Animais , Humanos , Camundongos , Pressão Sanguínea , Diabetes Mellitus Experimental/tratamento farmacológico , Nefropatias Diabéticas/tratamento farmacológico , Hipertensão/tratamento farmacológico , Camundongos Endogâmicos , Substrato Quinase C Rico em Alanina Miristoilada , Canais Epiteliais de Sódio/metabolismo , Receptores Adrenérgicos alfa 1/metabolismoRESUMO
The myristoylated alanine-rich C-kinase substrate (MARCKS) and the MARCKS-related protein (MARCKSL1) are ubiquitous, highly conserved membrane-associated proteins involved in the structural modulation of the actin cytoskeleton, chemotaxis, motility, cell adhesion, phagocytosis, and exocytosis. MARCKS includes an N-terminal myristoylated domain for membrane binding, a highly conserved MARCKS Homology 2 (MH2) domain, and an effector domain (which is the phosphorylation site). MARCKS can sequester phosphatidylinositol-4, 5-diphosphate (PIP2) at lipid rafts in the plasma membrane of quiescent cells, an action reversed by protein kinase C (PKC), ultimately modulating the immune function. Being expressed mostly in innate immune cells, MARCKS promotes the inflammation-driven migration and adhesion of cells and the secretion of cytokines such as tumor necrosis factor (TNF). From a clinical point of view, MARCKS is overexpressed in patients with schizophrenia and bipolar disorders, while the brain level of MARCKS phosphorylation is associated with Alzheimer's disease. Furthermore, MARCKS is associated with the development and progression of numerous types of cancers. Data in autoimmune diseases are limited to rheumatoid arthritis models in which a connection between MARCKS and the JAK-STAT pathway is mediated by miRNAs. We provide a comprehensive overview of the structure of MARCKS, its molecular characteristics and functions from a biological and pathogenetic standpoint, and will discuss the clinical implications of this pathway.
Assuntos
MicroRNAs , Substrato Quinase C Rico em Alanina Miristoilada , Alanina , Humanos , FosforilaçãoRESUMO
Viral vectors for vaccine delivery are challenged by recently reported safety issues like immunogenicity and risk for cancer development, and thus there is a growing need for the development of non-viral vectors. Cell penetrating peptides (CPPs) are non-viral vectors that can enter plasma membranes efficiently and deliver a broad range of cargoes. Our bioinformatic prediction and wet-lab validation data suggested that peptide P1 derived from MARCKS protein phosphorylation site domain is a new potential CPP candidate. We found that peptide P1 can efficiently internalize into various cell lines in a concentration-dependent manner. Receptor-mediated endocytosis pathway is the major mechanism of P1 penetration, although P1 also directly penetrates the plasma membrane. We also found that peptide P1 has low cytotoxicity in cultured cell lines as well as mouse red blood cells. Furthermore, peptide P1 not only can enter into cultured cells itself, but it also can interact with plasmid DNA and mediate the functional delivery of plasmid DNA into cultured cells, even in hard-to-transfect cells. Combined, these findings indicate that P1 may be a promising vector for efficient intracellular delivery of bioactive cargos.
Assuntos
Peptídeos Penetradores de Células/farmacocinética , Substrato Quinase C Rico em Alanina Miristoilada/farmacocinética , Animais , Transporte Biológico , Linhagem Celular , Membrana Celular/metabolismo , Sobrevivência Celular , Peptídeos Penetradores de Células/imunologia , Relação Dose-Resposta a Droga , Técnicas de Transferência de Genes , Meia-Vida , Hemólise/efeitos dos fármacos , Camundongos , Substrato Quinase C Rico em Alanina Miristoilada/imunologia , Plasmídeos , Transporte Proteico , TemperaturaRESUMO
BACKGROUND: Hepatocellular carcinoma is the fourth most common cause of cancer-related death. However, the cross-talk between tumor immune microenvironment and hepatocellular carcinoma (HCC) remains unclear. MATERIAL AND METHODS: We analyzed the expression of miR-143-3p in exosomes from different HCC cell lines. Differentially expressed genes (DEGs) in Tumor-associated macrophages (TAMs) co-cultured with HCC cell lines were overlapped with miR-143-3p target genes. We used the Oncomine, Kaplan-Meier plotter, and The Cancer Genome Atlas (TCGA) databases to assess Myristoylated alanine-rich C-kinase substrate (MARCKS) expression in various types of cancers. The relationship between patient clinicopathological characteristics and MARCKS expression level was identified using the Kaplan-Meier plotter database. Last, we analyzed how MARCKS expression correlated with immune infiltration makers using the TCGA database, Tumor IMmune Estimation Resource (TIMER), and Gene Expression Profiling Interactive Analysis (GEPIA). RESULTS: Exosomal miR-143-3p was elevated after IL-6 treatment in the HCC cell line. MARCKS, a target gene of miR-143-3p, was up-regulated in Tumor-associated macrophages co-cultured with high-metastatic-potential HCC cell line. MARCKS expression was identified as significantly correlated with outcome in multiple types of cancer, especially in HCC. High MARCKS expression level was associated with poorer overall survival (OS), Progress-free survival (PFS), and also with patient gender, race, hepatitis virus background, stage, grade, AJCC_T, and vascular invasion. MARCKS was positively associated with levels of T follicular helper cells (TFH) (R = .48, p < .001), T helper type 2 (Th2) cells (R = .47, p < .001), macrophages (R = .41, p ≤ .001), T helper cells (R = .40, p < .001), T helper type 1 (Th1) cells (R = .38, p < .001), T cells (R = .34, p < .001), NK CD56bright cells (R = .34, p < .001) and immature DC (iDC) (R = .33, p < .001), and negatively associated with levels of T helper 17 (Th17) cells. Also, MARCKS may influence the M2 polarization and immune escape. CONCLUSION: The present study suggests that MARCKS on TAMs is associated with poor prognosis and immune cell infiltration in HCC.