RESUMO
Many fundamental cellular processes such as division, polarization, endocytosis, and motility require the assembly, maintenance, and disassembly of filamentous actin (F-actin) networks at specific locations and times within the cell. The particular function of each network is governed by F-actin organization, size, and density as well as by its dynamics. The distinct characteristics of different F-actin networks are determined through the coordinated actions of specific sets of actin-binding proteins (ABPs). Furthermore, a cell typically assembles and uses multiple F-actin networks simultaneously within a common cytoplasm, so these networks must self-organize from a common pool of shared globular actin (G-actin) monomers and overlapping sets of ABPs. Recent advances in multicolor imaging and analysis of ABPs and their associated F-actin networks in cells, as well as the development of sophisticated in vitro reconstitutions of networks with ensembles of ABPs, have allowed the field to start uncovering the underlying principles by which cells self-organize diverse F-actin networks to execute basic cellular functions.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Humanos , Proteínas dos Microfilamentos/metabolismo , Modelos Biológicos , Schizosaccharomyces/metabolismoRESUMO
The monomer-binding protein profilin 1 (PFN1) plays a crucial role in actin polymerization. However, mutations in PFN1 are also linked to hereditary amyotrophic lateral sclerosis, resulting in a broad range of cellular pathologies which cannot be explained by its primary function as a cytosolic actin assembly factor. This implies that there are important, undiscovered roles for PFN1 in cellular physiology. Here we screened knockout cells for novel phenotypes associated with PFN1 loss of function and discovered that mitophagy was significantly upregulated. Indeed, despite successful autophagosome formation, fusion with the lysosome, and activation of additional mitochondrial quality control pathways, PFN1 knockout cells accumulate depolarized, dysmorphic mitochondria with altered metabolic properties. Surprisingly, we also discovered that PFN1 is present inside mitochondria and provide evidence that mitochondrial defects associated with PFN1 loss are not caused by reduced actin polymerization in the cytosol. These findings suggest a previously unrecognized role for PFN1 in maintaining mitochondrial integrity and highlight new pathogenic mechanisms that can result from PFN1 dysregulation.
Assuntos
Actinas , Mitocôndrias , Profilinas , Profilinas/metabolismo , Profilinas/genética , Mitocôndrias/metabolismo , Mitocôndrias/genética , Humanos , Actinas/metabolismo , Mitofagia/genética , Lisossomos/metabolismo , Citosol/metabolismo , Técnicas de Inativação de Genes , Autofagossomos/metabolismo , Células HeLaRESUMO
Diseases caused by Leishmania and Trypanosoma parasites are a major health problem in tropical countries. Because of their complex life cycle involving both vertebrate and insect hosts, and >1 billion years of evolutionarily distance, the cell biology of trypanosomatid parasites exhibits pronounced differences to animal cells. For example, the actin cytoskeleton of trypanosomatids is divergent when compared with other eukaryotes. To understand how actin dynamics are regulated in trypanosomatid parasites, we focused on a central actin-binding protein profilin. Co-crystal structure of Leishmania major actin in complex with L. major profilin revealed that, although the overall folds of actin and profilin are conserved in eukaryotes, Leishmania profilin contains a unique α-helical insertion, which interacts with the target binding cleft of actin monomer. This insertion is conserved across the Trypanosomatidae family and is similar to the structure of WASP homology-2 (WH2) domain, a small actin-binding motif found in many other cytoskeletal regulators. The WH2-like motif contributes to actin monomer binding and enhances the actin nucleotide exchange activity of Leishmania profilin. Moreover, Leishmania profilin inhibited formin-catalyzed actin filament assembly in a mechanism that is dependent on the presence of the WH2-like motif. By generating profilin knockout and knockin Leishmania mexicana strains, we show that profilin is important for efficient endocytic sorting in parasites, and that the ability to bind actin monomers and proline-rich proteins, and the presence of a functional WH2-like motif, are important for the in vivo function of Leishmania profilin. Collectively, this study uncovers molecular principles by which profilin regulates actin dynamics in trypanosomatids.
Assuntos
Citoesqueleto de Actina , Actinas , Leishmania major , Parasitos , Profilinas , Animais , Humanos , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Actinas/química , Actinas/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , Sequência Conservada , Cristalização , Cristalografia por Raios X , Leishmania major/citologia , Leishmania major/metabolismo , Parasitos/citologia , Parasitos/metabolismo , Profilinas/química , Profilinas/metabolismo , Ligação Proteica , Domínios ProteicosRESUMO
Membrane polyphosphoinositides (PPIs) are lipid-signaling molecules that undergo metabolic turnover and influence a diverse range of cellular functions. PPIs regulate the activity and/or spatial localization of a number of actin-binding proteins (ABPs) through direct interactions; however, it is much less clear whether ABPs could also be an integral part in regulating PPI signaling. In this study, we show that ABP profilin1 (Pfn1) is an important molecular determinant of the cellular content of PI(4,5)P2 (the most abundant PPI in cells). In growth factor (EGF) stimulation setting, Pfn1 depletion does not impact PI(4,5)P2 hydrolysis but enhances plasma membrane (PM) enrichment of PPIs that are produced downstream of activated PI3-kinase, including PI(3,4,5)P3 and PI(3,4)P2, the latter consistent with increased PM recruitment of SH2-containing inositol 5' phosphatase (SHIP2) (a key enzyme for PI(3,4)P2 biosynthesis). Although Pfn1 binds to PPIs in vitro, our data suggest that Pfn1's affinity to PPIs and PM presence in actual cells, if at all, is negligible, suggesting that Pfn1 is unlikely to directly compete with SHIP2 for binding to PM PPIs. Additionally, we provide evidence for Pfn1's interaction with SHIP2 in cells and modulation of this interaction upon EGF stimulation, raising an alternative possibility of Pfn1 binding as a potential restrictive mechanism for PM recruitment of SHIP2. In conclusion, our findings challenge the dogma of Pfn1's binding to PM by PPI interaction, uncover a previously unrecognized role of Pfn1 in PI(4,5)P2 homeostasis and provide a new mechanistic avenue of how an ABP could potentially impact PI3K signaling byproducts in cells through lipid phosphatase control.
Assuntos
Fosfatidilinositóis , Profilinas , Fator de Crescimento Epidérmico/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Fosfatidilinositóis/metabolismo , Humanos , Células HEK293 , Profilinas/metabolismoRESUMO
Profilin 4 (Pfn4) is expressed during spermiogenesis and localizes to the acrosome-acroplaxome-manchette complex. Here, we generated PFN4-deficient mice, with sperm displaying severe impairment in manchette formation. Interestingly, HOOK1 staining suggests that the perinuclear ring is established; however, ARL3 staining is disrupted, suggesting that lack of PFN4 does not interfere with the formation of the perinuclear ring and initial localization of HOOK1, but impedes microtubular organization of the manchette. Furthermore, amorphous head shape and flagellar defects were detected, resulting in reduced sperm motility. Disrupted cis- and trans-Golgi networks and aberrant production of proacrosomal vesicles caused impaired acrosome biogenesis. Proteomic analysis showed that the proteins ARF3, SPECC1L and FKBP1, which are involved in Golgi membrane trafficking and PI3K/AKT pathway, are more abundant in Pfn4-/- testes. Levels of PI3K, AKT and mTOR were elevated, whereas AMPK level was reduced, consistent with inhibition of autophagy. This seems to result in blockage of autophagic flux, which could explain the failure in acrosome formation. In vitro fertilization demonstrated that PFN4-deficient sperm is capable of fertilizing zona-free oocytes, suggesting a potential treatment for PFN4-related human infertility.
Assuntos
Acrossomo , Profilinas , Espermátides , Espermatogênese , Acrossomo/metabolismo , Animais , Masculino , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Profilinas/genética , Profilinas/metabolismo , Proteômica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sêmen , Motilidade dos Espermatozoides , Espermátides/metabolismo , Espermatogênese/genética , EspermatozoidesRESUMO
Overexpression of actin-binding protein profilin-1 (Pfn1) correlates with advanced disease features and adverse clinical outcome of patients with clear cell renal carcinoma, the most prevalent form of renal cancer. We previously reported that Pfn1 is predominantly overexpressed in tumor-associated vascular endothelial cells in human clear cell renal carcinoma. In this study, we combined in vivo strategies involving endothelial cell-specific depletion and overexpression of Pfn1 to demonstrate a role of vascular endothelial Pfn1 in promoting tumorigenicity and enabling progressive growth and metastasis of renal carcinoma cells in a syngeneic orthotopic mouse model of kidney cancer. We established an important role of endothelial Pfn1 in tumor angiogenesis and further identified endothelial Pfn1-dependent regulation of several pro- (VEGF, SERPINE1, CCL2) and anti-angiogenic factors (platelet factor 4) in vivo. Endothelial Pfn1 overexpression increases tumor infiltration by macrophages and concomitantly diminishes tumor infiltration by T cells including CD8+ T cells in vivo, correlating with the pattern of endothelial Pfn1-dependent changes in tumor abundance of several prominent immunomodulatory cytokines. These data were also corroborated by multiplexed quantitative immunohistochemistry and immune deconvolution analyses of RNA-seq data of clinical samples. Guided by Upstream Regulator Analysis of tumor transcriptome data, we further established endothelial Pfn1-induced Hif1α elevation and suppression of STAT1 activation. In conclusion, this study demonstrates for the first time a direct causal relationship between vascular endothelial Pfn1 dysregulation, immunosuppressive tumor microenvironment, and disease progression with mechanistic insights in kidney cancer. Our study also provides a conceptual basis for targeting Pfn1 for therapeutic benefit in kidney cancer.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Profilinas , Microambiente Tumoral , Animais , Humanos , Camundongos , Carcinoma de Células Renais/genética , Células Endoteliais/metabolismo , Neoplasias Renais/genética , Profilinas/genética , Profilinas/metabolismo , Progressão da DoençaRESUMO
Profilin is a small protein that controls actin polymerization in yeast and higher eukaryotes. In addition, profilin has emerged as a multifunctional protein that contributes to other processes in multicellular organisms. This study focuses on profilin (Pfy1) in the budding yeast Saccharomyces cerevisiae. The primary sequences of yeast Pfy1 and its metazoan orthologs diverge vastly. However, structural elements of profilin are conserved among different species. To date, the full spectrum of Pfy1 functions has yet to be defined. The current work explores the possible involvement of yeast profilin in nuclear protein import. To this end, a panel of well-characterized yeast profilin mutants was evaluated. The experiments demonstrate that yeast profilin (i) regulates nuclear protein import, (ii) determines the subcellular localization of essential nuclear transport factors, and (iii) controls the relative abundance of actin and tubulin. Together, these results define yeast profilin as a moonlighting protein that engages in multiple essential cellular activities.
Assuntos
Actinas , Profilinas , Animais , Actinas/genética , Actinas/metabolismo , Profilinas/genética , Profilinas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Transporte Ativo do Núcleo Celular , Proteínas Nucleares/metabolismoRESUMO
The actin cytoskeleton is a biosensor of cellular stress and a potential prognosticator of human disease. In particular, aberrant cytoskeletal structures such as stress granules formed in response to energetic and oxidative stress are closely linked to ageing, cancer, cardiovascular disease, and viral infection. Whether these cytoskeletal phenomena can be harnessed for the development of biosensors for cytoskeletal dysfunction and, by extension, disease progression, remains an open question. In this work, we describe the design and development of an optogenetic iteration of profilin, an actin monomer binding protein with critical functions in cytoskeletal dynamics. We demonstrate that this optically activated profilin ('OptoProfilin') can act as an optically triggered biosensor of applied cellular stress in select immortalized cell lines. Notably, OptoProfilin is a single component biosensor, likely increasing its utility for experimentalists. While a large body of preexisting work closely links profilin activity with cellular stress and neurodegenerative disease, this, to our knowledge, is the first example of profilin as an optogenetic biosensor of stress-induced changes in the cytoskeleton.
Assuntos
Técnicas Biossensoriais , Profilinas , Profilinas/metabolismo , Humanos , Optogenética/métodos , Estresse FisiológicoRESUMO
Small cell lung cancer (SCLC) is one of the most malignant tumors that has an extremely poor prognosis. RNA-binding protein (RBP) and long noncoding RNA (lncRNA) have been shown to be key regulators during tumorigenesis as well as lung tumor progression. However, the role of RBP ELAVL4 and lncRNA LYPLAL1-DT in SCLC remains unclear. In this study, we verified that lncRNA LYPLAL1-DT acts as an SCLC oncogenic lncRNA and was confirmed in vitro and in vivo. Mechanistically, LYPLAL1-DT negatively regulates the expression of miR-204-5p, leading to the upregulation of PFN2, thus, promoting SCLC cell proliferation, migration, and invasion. ELAVL4 has been shown to enhance the stability of LYPLAL1-DT and PFN2 mRNA. Our study reveals a regulatory pathway, where ELAVL4 stabilizes PFN2 and LYPLAL1-DT with the latter further increasing PFN2 expression by blocking the action of miR-204-5p. Upregulated PFN2 ultimately promotes tumorigenesis and invasion in SCLC. These findings provide novel prognostic indicators as well as promising new therapeutic targets for SCLC.
Assuntos
Neoplasias Pulmonares , MicroRNAs , RNA Longo não Codificante , Carcinoma de Pequenas Células do Pulmão , Humanos , RNA Longo não Codificante/genética , Profilinas/genética , Carcinoma de Pequenas Células do Pulmão/genética , Transformação Celular Neoplásica/genética , Carcinogênese/genética , Neoplasias Pulmonares/genética , MicroRNAs/genética , Proteína Semelhante a ELAV 4RESUMO
Among small-molecule CDK4/6 inhibitors (palbociclib, ribociclib, and abemaciclib) approved for metastatic breast cancers, abemaciclib has a more tolerable adverse effects in clinic. This is attributable to preferential inhibition of CDK4 over CDK6. In our search for a biased CDK4 inhibitor, we discovered a series of pyrimidine-indazole inhibitors. SAR studies led us to TQB3616 as a preferential CDK4 inhibitor. TQB3616 exhibited improvements in both enzymatic and cellular proliferation inhibitory potency when tested side-by-side with the FDA approved palbociclib and abemaciclib. TQB3616 also possessed favorable PK profile in multiple species. These differentiated properties, together with excellent GLP safety profile warranted TQB3616 moving to clinic. TQB3616 entered into clinical development in 2019 and currently in phase III clinical trials (NCT05375461, NCT05365178).
Assuntos
Proliferação de Células , Quinase 4 Dependente de Ciclina , Inibidores de Proteínas Quinases , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/metabolismo , Humanos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/síntese química , Relação Estrutura-Atividade , Estrutura Molecular , Proliferação de Células/efeitos dos fármacos , Animais , Descoberta de Drogas , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Relação Dose-Resposta a Droga , Pirimidinas/química , Pirimidinas/farmacologia , Pirimidinas/síntese química , Ratos , Ensaios de Seleção de Medicamentos Antitumorais , Avaliação Pré-Clínica de MedicamentosRESUMO
BACKGROUND: Circular RNAs (circRNAs) are single-stranded RNAs with covalently closed structures that have been implicated in cancer progression. However, the regulatory mechanisms remain largely unclear. So, the aim of this study was to reveal the role and regulatory mechanisms of circ-SLC16A1. METHODS: In this study, next-generation sequencing was used to identify abnormally expressed circRNAs between cancerous and para-carcinoma tissues. Fluorescence in situ hybridization and quantitative reverse transcription polymerase chain reaction were performed to assess the expression patterns of circ-solute carrier family 16 member 1 (SLC16A1) in non-small cell lung cancer (NSCLC) cells and tissue specimens. The dual-luciferase reporter assay was utilized to identify downstream targets of circ-SLC16A1. Transwell migration, wound healing, 5-ethynyl-2'-deoxyuridine incorporation, cell counting, and colony formation assays were conducted to assess the proliferation and migration of NSCLC cells. A mouse tumor xenograft model was employed to determine the roles of circ-SLC16A1 in NSCLC progression and metastasis in vivo. RESULTS: The results found that circ-SLC16A1 was upregulated in NSCLC cells and tissues. Downregulation of circ-SLC16A1 inhibited tumor growth by reducing proliferation, lung metastasis, and lymphatic metastasis of NSCLC cells, and arrested the cell cycle in the G1 phase. Also, silencing of circ-SLC16A1 promoted apoptosis of NSCLC cells. The results of bioinformatics analysis and the dual-luciferase reporter assay confirmed that microRNA (miR)-1287-5p and profilin 2 (PFN2) are downstream targets of circ-SLC16A1. PFN2 overexpression or circ-SLC16A1 inhibition restored proliferation and migration of NSCLC cells after silencing of circ-SLC16A1. PFN2 overexpression restored migration and proliferation of NSCLC cells post miR-1287-5p overexpression. CONCLUSIONS: Collectively, these findings show that miR-1287-5p/PFN2 signaling was associated with downregulation of circ-SLC16A1 and reduced invasion and proliferation of NSCLC cells. So, circ-SLC16A1 is identified as a mediator of multiple pro-oncogenic signaling pathways in NSCLC and can be targeted to suppress tumor progression.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , MicroRNAs , Animais , Humanos , Camundongos , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Modelos Animais de Doenças , Hibridização in Situ Fluorescente , Luciferases , Neoplasias Pulmonares/genética , MicroRNAs/genética , Profilinas , RNA Circular/genéticaRESUMO
Profilin-1 (PFN1) plays important roles in modulating actin dynamics through binding both monomeric actin and proteins enriched with polyproline motifs. Mutations in PFN1 have been linked to the neurodegenerative disease amyotrophic lateral sclerosis (ALS). However, whether ALS-linked mutations affect PFN1 function has remained unclear. To address this question, we employed an unbiased proteomics analysis in mammalian cells to identify proteins that differentially interact with mutant and wild-type (WT) PFN1. These studies uncovered differential binding between two ALS-linked PFN1 variants, G118V and M114T, and select formin proteins. Furthermore, both variants augmented formin-mediated actin assembly relative to PFN1 WT. Molecular dynamics simulations revealed mutation-induced changes in the internal dynamic couplings within an alpha helix of PFN1 that directly contacts both actin and polyproline, as well as structural fluctuations within the actin- and polyproline-binding regions of PFN1. These data indicate that ALS-PFN1 variants have the potential for heightened flexibility in the context of the ternary actin-PFN1-polyproline complex during actin assembly. Conversely, PFN1 C71G was more severely destabilized than the other PFN1 variants, resulting in reduced protein expression in both transfected and ALS patient lymphoblast cell lines. Moreover, this variant exhibited loss-of-function phenotypes in the context of actin assembly. Perturbations in actin dynamics and assembly can therefore result from ALS-linked mutations in PFN1. However, ALS-PFN1 variants may dysregulate actin polymerization through different mechanisms that depend upon the solubility and stability of the mutant protein.
Assuntos
Actinas/metabolismo , Esclerose Lateral Amiotrófica/genética , Forminas/efeitos adversos , Polimerização , Profilinas/genética , Profilinas/metabolismo , Animais , Células HeLa , Humanos , Proteínas Mutantes/química , Mutação , Doenças Neurodegenerativas , Fenótipo , Profilinas/química , Conformação Proteica em alfa-Hélice , Deficiências na ProteostaseRESUMO
Summary: Background. Gibberellin Regulated Proteins (GRPs) are small glycoproteins that induce allergy to various types of fruit. This study aimed to evaluate co-sensitization to cypress pollen and other molecules responsible for fruit allergy, such as nsLTP (Pru p 3), PR-10 (Bet v1), and Profilin (Bet v2). Methods. Sixty subjects sensitized to peach GRP (Pru p 7) were consecutively recruited from four Italian centers: 28 males and 32 females (mean age 37.9 years; range 11-79). Specific IgE for Pru p 7, Pru p 3, Bet v 1, Bet v 2, cypress pollen extract (Cup s), and Cup a 1 were determined in all subjects. Results. Sensitization rates to Cup s, Cup a 1, Pru p 3, Bet v 1, and Bet v 2 in the entire studied population were 90.0%, 83.3%, 45.8%, 40.0%, and 30.0%, respectively. In subjects residing in Northern Italy, the respective sensitization rates were 96.4%, 80.0%, 50.0%, 73.3%, and 40.0%, while in those residing in Southern Italy, they were 83.3%, 86.7%, 40.0%, 6.7%, and 20.0%. The only significant difference was observed for PR-10 (p less than 0.0001) Co-sensitization to PR-10 was found to be associated with a reduced risk of anaphylaxis (OR: 0.125). Allergic reactions were most commonly triggered by peach (26/40), followed by orange (12/40), with other foods being less frequently implicated. Conclusions. This study confirms a high association between sensitization to Pru p 7 and cypress pollen and highlights a high percentage of co-sensitization to nsLTP, PR-10, and profilin. PR-10 emerged as a protective factor against anaphylaxis.
RESUMO
Dynamic remodeling of the actin cytoskeleton is an essential feature for virtually all actin-dependent cellular processes, including cell migration, cell cycle progression, chromatin remodeling and gene expression, and even the DNA damage response. An altered actin cytoskeleton is a structural hallmark associated with numerous pathologies ranging from cardiovascular diseases to immune disorders, neurological diseases and cancer. The actin cytoskeleton in cells is regulated through the orchestrated actions of a myriad of actin-binding proteins. In this Review, we provide a brief overview of the structure and functions of the actin-monomer-binding protein profilin-1 (Pfn1) and then discuss how dysregulated expression of Pfn1 contributes to diseases associated with the cardiovascular system.
Assuntos
Doenças Cardiovasculares , Profilinas , Citoesqueleto de Actina/genética , Actinas/genética , Doenças Cardiovasculares/genética , Humanos , Proteínas dos Microfilamentos , Profilinas/genéticaRESUMO
hPFN1 mutations including C71G cause ALS by gain of toxicity but the mechanism still remains unknown. Stress granules (SGs) are formed by phase separation of the prion-like domain (PLD) of RNA-binding proteins including FUS, whose inclusion was also associated with ALS. C71G-hPFN1 triggers seed-dependent co-aggregation with FUS/TDP-43 to manifest the prion-like propagandation but its biophysical basis remains unexplored. Here by DIC imaging we first showed that three hPFN1 mutants have differential capacity in disrupting the dynamics of liquid droplets formed by phase separation of FUS prion-like domain (PLD). C71G-hPFN1 co-exists with the folded and unfolded states, thus allowing to simultaneously characterize conformations, hydrodynamics and dynamics of the interactions of both states with the phase separated FUS PLD by NMR. The results reveal that the folded state is not significantly affected while by contrast, the unfolded state has extensive interactions with FUS PLD. As a consequence, the dynamics of FUS liquid droplets become significantly reduced. Such interactions might act to recruit C71G-hPFN1 into the droplets, thus leading to the increase of the local concentrations and subsequent co-aggregation of C71G-hPFN1 with FUS. Our study sheds the first light on the biophysical basis by which hPFN1 mutants gain toxicity to cause ALS. As other aggregation-prone proteins have no fundamental difference from hPFN1 mutants, aggregation-prone proteins might share a common capacity in disrupting phase separation responsible for organizing various membrane-less organelles. As such, the mechanism for C71G-hPFN1 might also be utilized by other aggregation-prone proteins for gain of toxicity to trigger diseases and aging.
Assuntos
Esclerose Lateral Amiotrófica , Príons , Humanos , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Mutação , Espectroscopia de Ressonância Magnética , Imageamento por Ressonância Magnética , Príons/metabolismo , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismoRESUMO
Neocortex development during embryonic stages requires the precise control of mRNA metabolism. Human antigen R (HuR) is a well-studied mRNA-binding protein that regulates mRNA metabolism, and it is highly expressed in the neocortex during developmental stages. Deletion of HuR does not impair neural progenitor cell proliferation or differentiation, but it disturbs the laminar structure of the neocortex. We report that HuR is expressed in postmitotic projection neurons during mouse brain development. Specifically, depletion of HuR in these neurons led to a mislocalization of CDP+ neurons in deeper layers of the cortex. Time-lapse microscopy showed that HuR was required for the promotion of cell motility in migrating neurons. PCR array identified profilin 1 (Pfn1) mRNA as a major binding partner of HuR in neurons. HuR positively mediated the stability of Pfn1 mRNA and influenced actin polymerization. Overexpression of Pfn1 successfully rescued the migration defects of HuR-deleted neurons. Our data reveal a post-transcriptional mechanism that maintains actin dynamics during neuronal migration.
Assuntos
Movimento Celular , Proteína Semelhante a ELAV 1/fisiologia , Neurônios/fisiologia , RNA Mensageiro/metabolismo , Animais , Padronização Corporal/genética , Movimento Celular/genética , Células Cultivadas , Embrião de Mamíferos , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Neurais/fisiologia , Neurogênese/genética , Gravidez , Profilinas/fisiologia , Processamento Pós-Transcricional do RNA/genéticaRESUMO
PLP-dependent enzymes represent an important class of highly "druggable" enzymes that perform a wide array of critical reactions to support all organisms. Inhibition of individual members of this family of enzymes has been validated as a therapeutic target for pathologies ranging from infection with Mycobacterium tuberculosis to epilepsy. Given the broad nature of the activities within this family of enzymes, we envisioned a universally acting probe to characterize existing and putative members of the family that also includes the necessary chemical moieties to enable activity-based protein profiling experiments. Hence, we developed a probe that contains an N-hydroxyalanine warhead that acts as a covalent inhibitor of PLP-dependent enzymes, a linear diazirine for UV crosslinking, and an alkyne moiety to enable enrichment of crosslinked proteins. Our molecule was used to study PLP-dependent enzymes inâ vitro as well as look at whole-cell lysates of M. tuberculosis and assess inhibitory activity. The probe was able to enrich and identify LysA, a PLP-dependent enzyme crucial for lysine biosynthesis, through mass spectrometry. Overall, our study shows the utility of this trifunctional first-generation probe. We anticipate further optimization of probes for PLP-dependent enzymes will enable the characterization of rationally designed covalent inhibitors of PLP-dependent enzymes, which will expedite the preclinical characterization of these important therapeutic targets.
Assuntos
Fosfato de Piridoxal , Fosfato de Piridoxal/química , Modelos Moleculares , Espectrometria de MassasRESUMO
Previous reports from this laboratory have demonstrated the involvement of histone deacetylase 6 (HDAC6) in sperm motility. As the presence of HDAC6 has also been reported in the earlier stage germ cells, studies were undertaken to explore its role during these stages of spermatogenesis. HDAC6 was overexpressed in GC-1spg cells, which represent the stage between type B spermatogonia and primary spermatocyte, and its effect on germ cell transcriptome was investigated by microarray. Among the many transcripts that were differentially regulated, Profilin 2, reported previously as a neuronal specific isoform, was observed as one of the genes highly upregulated at the transcript level, which was further confirmed by real-time PCR, and the protein confirmed by indirect immunofluorescence (IIF). Profilin 2 colocalized with HDAC6, as seen both in GC-1 cells and sperm. On the sperm, the presence of Profilin 2 was detected throughout the flagella with its colocalization with HDAC6 seen conspicuously in the mid-piece region of the flagella. Co-immunoprecipitation studies confirmed Profilin 2 interaction with HDAC6. Docking studies using Z dock suggested the interaction of 8 residues of HDAC6 with 6 residues of Profilin 2. The novel observation of Profilin 2 in spermatogonial cells, its significant upregulation on HDAC6 overexpression and its interaction with HDAC6 suggests that HDAC6 in collaboration with Profilin 2 may play a role in regulating the movement of germ cells from one stage/compartment to the next.
Assuntos
Profilinas , Testículo , Masculino , Camundongos , Animais , Testículo/metabolismo , Desacetilase 6 de Histona/metabolismo , Profilinas/genética , Profilinas/metabolismo , Regulação para Cima , Motilidade dos Espermatozoides , Sêmen/metabolismoRESUMO
Toxoplasma gondii is a highly prevalent protozoan that infects a broad spectrum of warm-blooded animals. Profilin is a critical protein that plays a role in the movement and invasion of T. gondii. In the current study, we assessed how profilin stimulates inflammasomes and how it induces transcription and secretion of IL-1ß. For this purpose, we assessed the level of TLR 2, 4, 5, and 9 expressions in a THP-1 cell line treated with profilin from T. gondii (TgP). In addition, we analyzed the expression levels of various inflammasomes, as well as IL-1ß, and IL-18 in THP-1 cells treated with the NLRP3 inhibitor MCC950. TgP significantly increased the expression of TLR5 but the expression of TLR2, 4, and 9 was not significantly increased. In addition, TgP did not significantly increase the level of inflammasomes after 5 h. Treatment with MCC950 significantly reduced NLRP3 and IL-1ß on both transcription and protein levels. Although the transcription level of NLRP3 was reduced 5 h after treatment with TgP, western blot analysis showed an increase in NLRP3. The western blot and ELISA analysis also showed that TgP increased both pro- and mature IL-1ß. In summary, our study showed that NLRP3 most probably plays a pivotal role in the expression and production levels of IL-1ß during the interaction between TgP and macrophages.
Assuntos
Toxoplasma , Animais , Humanos , Toxoplasma/metabolismo , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Células THP-1 , Profilinas , Interleucina-1beta/metabolismoRESUMO
BACKGROUND: Circular RNA (circRNA) has been proven to play a critical role in breast cancer progression. Therefore, this study was designed to clarify the role and underlying molecular mechanisms of circ-disintegrin and metalloproteinase 9 (circ-ADAM9) in breast cancer. METHODS: A quantitative real-time polymerase chain reaction (RT-qPCR) was conducted to assess the expression levels of circ-ADAM9, microRNA-383-5p (miR-383-5p), and profilin 2 (PFN2). Cellular growth curves of breast cancer cells were determined by colony-forming assay. Cell viability and apoptosis were measured by MTT and flow cytometry, respectively. The protein expression level was analyzed by western blot. Cell migration and invasion were evaluated by wound healing and Transwell assays. A xenograft experiment was established to clarify the functional role of circ-ADAM9 inhibition in vivo. The interactions among circ-ADAM9, miR-383-5p, and PFN2 were analyzed by dual-luciferase reporter, RNA immunoprecipitation (RIP), and RNA pull-down assays. RESULTS: We found that circ-ADAM9 was upregulated in breast cancer tissues and cells compared to controls. Inhibition of circ-ADAM9 expression impaired proliferation, migration, and invasion, but increased radiosensitivity and apoptosis in breast cancer cells; besides, radiotherapy combined with circ-ADAM9 inhibition showed significant inhibitory effects on tumor growth. The functional effects of circ-ADAM9 were related to miR-383-5p, a target of circ-ADAM9. Overexpression of miR-383-5p-mediated malignant behaviors and radiosensitivity of breast cancer cells were dependent on PFN2. CONCLUSION: Circ-ADAM9 was found to participate in breast cancer progression through targeting the miR-383-5p/PFN2 axis.