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1.
Theranostics ; 11(16): 7755-7766, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34335962

RESUMO

Background: Myocardial infarction (MI) evokes an organized remodeling process characterized by the activation and transdifferentiation of quiescent cardiac fibroblasts to generate a stable collagen rich scar. Early fibroblast activation may be amenable to targeted therapy, but is challenging to identify in vivo. We aimed to non-invasively image active fibrosis by targeting the fibroblast activation protein (FAP) expressed by activated (myo)fibroblasts, using a novel positron emission tomography (PET) radioligand [68Ga]MHLL1 after acute MI. Methods: One-step chemical synthesis and manual as well as module-based radiolabeling yielded [68Ga]MHLL1. Binding characteristics were evaluated in murine and human FAP-transfected cells, and stability tested in human serum. Biodistribution in healthy animals was interrogated by dynamic PET imaging, and metabolites were measured in blood and urine. The temporal pattern of FAP expression was determined by serial PET imaging at 7 d and 21 d after coronary artery ligation in mice as percent injected dose per gram (%ID/g). PET measurements were validated by ex vivo autoradiography and immunostaining for FAP and inflammatory macrophages. Results: [68Ga]MHLL1 displayed specific uptake in murine and human FAP-positive cells (p = 0.0208). In healthy mice the tracer exhibited favorable imaging characteristics, with low blood pool retention and dominantly renal clearance. At 7 d after coronary artery ligation, [68Ga]MHLL1 uptake was elevated in the infarct relative to the non-infarcted remote myocardium (1.3 ± 0.3 vs. 1.0 ± 0.2 %ID/g, p < 0.001) which persisted to 21 d after MI (1.3 ± 0.4 vs. 1.1 ± 0.4 %ID/g, p = 0.013). Excess unlabeled compound blocked tracer accumulation in both infarct and non-infarct remote myocardium regions (p < 0.001). Autoradiography and histology confirmed the regional uptake of [68Ga]MHLL1 in the infarct and especially border zone regions, as identified by Masson trichrome collagen staining. Immunostaining further delineated persistent FAP expression at 7 d and 21 d post-MI in the border zone, consistent with tracer distribution in vivo. Conclusion: The simplified synthesis of [68Ga]MHLL1 bears promise for non-invasive characterization of fibroblast activation protein early in remodeling after MI.


Assuntos
Endopeptidases/metabolismo , Radioisótopos de Gálio/farmacologia , Proteínas de Membrana/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Animais , Autorradiografia/métodos , Linhagem Celular Tumoral , Endopeptidases/fisiologia , Fibroblastos/metabolismo , Fibrose/diagnóstico por imagem , Radioisótopos de Gálio/metabolismo , Humanos , Masculino , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Imagem Molecular/métodos , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , Distribuição Tecidual/fisiologia , Tomografia Computadorizada por Raios X/métodos
2.
Theranostics ; 11(16): 8092-8111, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34335982

RESUMO

Active c-Src non-receptor tyrosine kinase localizes to the plasma membrane via N-terminal lipid modification. Membranous c-Src causes cancer initiation and progression. Even though transmembrane 4 L six family member 5 (TM4SF5), a tetraspan(in), can be involved in this mechanism, the molecular and structural influence of TM4SF5 on c-Src remains unknown. Methods: Here, we investigated molecular and structural details by which TM4SF5 regulated c-Src devoid of its N-terminus and how cell-penetrating peptides were able to interrupt c-Src activation via interference of c-Src-TM4SF5 interaction in hepatocellular carcinoma models. Results: The TM4SF5 C-terminus efficiently bound the c-Src SH1 kinase domain, efficiently to the inactively-closed form. The complex involved protein tyrosine phosphatase 1B able to dephosphorylate Tyr530. The c-Src SH1 domain alone, even in a closed form, bound TM4SF5 to cause c-Src Tyr419 and FAK Y861 phosphorylation. Homology modeling and molecular dynamics simulation studies predicted the directly interfacing residues, which were further validated by mutational studies. Cell penetration of TM4SF5 C-terminal peptides blocked the interaction of TM4SF5 with c-Src and prevented c-Src-dependent tumor initiation and progression in vivo. Conclusions: Collectively, these data demonstrate that binding of the TM4SF5 C-terminus to the kinase domain of inactive c-Src leads to its activation. Because this binding can be abolished by cell-penetrating peptides containing the TM4SF5 C-terminus, targeting this direct interaction may be an effective strategy for developing therapeutics that block the development and progression of hepatocellular carcinoma.


Assuntos
Proteína Tirosina Quinase CSK/metabolismo , Carcinoma Hepatocelular/metabolismo , Proteínas de Membrana/metabolismo , Proteína Tirosina Quinase CSK/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Genes src/genética , Genes src/fisiologia , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Peptídeos/metabolismo , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Tetraspaninas/genética , Tetraspaninas/metabolismo
3.
FASEB J ; 35(8): e21829, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34314069

RESUMO

Retinal ischemia is a leading cause of irreversible blindness worldwide. Inner retinal dysfunction including loss of retinal ganglion cells is encountered in a number of retinal ischemic disorders. We previously reported administration of two different hypoxia-inducible factor (HIF) inhibitors exerted neuroprotective effects in a murine model of retinal ischemia/reperfusion (I/R) which mimics these disorders, as inner retinal degeneration could be involved in pathological HIF induction. However, this notion needs further investigation. Therefore, in this study, we attempted to use retina-specific Hif-1α conditional knockout (cKO) mice to uncover this notion more clearly under the same condition. Hif-1α cKO mice showed inner retinal neurodegeneration to a lesser extent than control mice. Hif-1α depletion in a murine 661W retinal cell line reduced cell death under pseudohypoxic and hypoxic conditions. Among hypoxia-related genes, the expression of BCL2 19 kDa protein-interacting protein 3 (Bnip3) was substantially upregulated in the inner retinal layer after retinal I/R. In this regard, we further examined Bnip3 depletion in retinal neurons in vitro and in vivo and found the similar neuroprotective effects. Our results support the notion that the HIF-1α/BNIP3 pathway may have a critical role in inner retinal neurodegeneration, which can be linked with the development of new promising therapeutics for inner retinal ischemic disorders.


Assuntos
Hipóxia Celular , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Proteínas de Membrana/fisiologia , Proteínas Mitocondriais/fisiologia , Neuroproteção , Retina , Degeneração Retiniana/metabolismo , Animais , Linhagem Celular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Retina/metabolismo , Retina/patologia
4.
Int J Mol Sci ; 22(13)2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201472

RESUMO

The ubiquitously expressed transmembrane protein a disintegrin and metalloproteinase 10 (ADAM10) functions as a "molecular scissor", by cleaving the extracellular regions from its membrane protein substrates in a process termed ectodomain shedding. ADAM10 is known to have over 100 substrates including Notch, amyloid precursor protein, cadherins, and growth factors, and is important in health and implicated in diseases such as cancer and Alzheimer's. The tetraspanins are a superfamily of membrane proteins that interact with specific partner proteins to regulate their intracellular trafficking, lateral mobility, and clustering at the cell surface. We and others have shown that ADAM10 interacts with a subgroup of six tetraspanins, termed the TspanC8 subgroup, which are closely related by protein sequence and comprise Tspan5, Tspan10, Tspan14, Tspan15, Tspan17, and Tspan33. Recent evidence suggests that different TspanC8/ADAM10 complexes have distinct substrates and that ADAM10 should not be regarded as a single scissor, but as six different TspanC8/ADAM10 scissor complexes. This review discusses the published evidence for this "six scissor" hypothesis and the therapeutic potential this offers.


Assuntos
Proteína ADAM10/fisiologia , Tetraspaninas/fisiologia , Secretases da Proteína Precursora do Amiloide/fisiologia , Animais , Caderinas/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Terapia de Alvo Molecular/métodos , Tetraspaninas/química
5.
Int J Mol Sci ; 22(13)2021 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-34281239

RESUMO

HOPS is a ubiquitin-like protein implicated in many aspects of cellular function including the regulation of mitotic activity, proliferation, and cellular stress responses. In this study, we focused on the complex relationship between HOPS and the tumor suppressor p53, investigating both transcriptional and non-transcriptional p53 responses. Here, we demonstrated that Hops heterozygous mice and mouse embryonic fibroblasts exhibit an impaired DNA-damage response to etoposide-induced double-strand breaks when compared to wild-type genes. Specifically, alterations in HOPS levels caused significant defects in the induction of apoptosis, including a reduction in p53 protein level and percentage of apoptotic cells. We also analyzed the effect of reduced HOPS levels on the DNA-damage response by examining the transcript profiles of p53-dependent genes, showing a suggestive deregulation of the mRNA levels for a number of p53-dependent genes. Taken together, these results show an interesting haploinsufficiency effect mediated by Hops monoallelic deletion, which appears to be enough to destabilize the p53 protein and its functions. Finally, these data indicate a novel role for Hops as a tumor-suppressor gene in DNA damage repair in mammalian cells.


Assuntos
Apoptose , Reparo do DNA , Haploinsuficiência , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas de Membrana/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Dano ao DNA , Feminino , Heterozigoto , Masculino , Camundongos
6.
Neuron ; 109(13): 2131-2149.e15, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34089643

RESUMO

CIB2 is a Ca2+- and Mg2+-binding protein essential for mechanoelectrical transduction (MET) by cochlear hair cells, but not by vestibular hair cells that co-express CIB2 and CIB3. Here, we show that in cochlear hair cells, CIB3 can functionally substitute for CIB2. Using X-ray crystallography, we demonstrate that CIB2 and CIB3 are structurally similar to KChIP proteins, auxiliary subunits of voltage-gated Kv4 channels. CIB2 and CIB3 bind to TMC1/2 through a domain in TMC1/2 flanked by transmembrane domains 2 and 3. The co-crystal structure of the CIB-binding domain in TMC1 with CIB3 reveals that interactions are mediated through a conserved CIB hydrophobic groove, similar to KChIP1 binding of Kv4. Functional studies in mice show that CIB2 regulates TMC1/2 localization and function in hair cells, processes that are affected by deafness-causing CIB2 mutations. We conclude that CIB2 and CIB3 are MET channel auxiliary subunits with striking similarity to Kv4 channel auxiliary subunits.


Assuntos
Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/fisiologia , Células Ciliadas Auditivas/fisiologia , Mecanotransdução Celular/fisiologia , Animais , Cristalografia por Raios X , Células HEK293 , Humanos , Proteínas Interatuantes com Canais de Kv/química , Proteínas Interatuantes com Canais de Kv/fisiologia , Proteínas de Membrana/química , Proteínas de Membrana/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
7.
Commun Biol ; 4(1): 820, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34188173

RESUMO

Host lipid metabolism and viral responses are intimately connected. However, the process by which the acquired immune systems adapts lipid metabolism to meet demands, and whether or not the metabolic rewiring confers a selective advantage to host immunity, remains unclear. Here we show that viral infection attenuates the expression of genes related to lipid metabolism in murine CD4+ T cells, which in turn increases the expression of antiviral genes. Inhibition of the fatty acid synthesis pathway substantially increases the basal expression of antiviral genes via the spontaneous production of type I interferon (IFN). Using a combination of CRISPR/Cas9-mediated genome editing technology and a global lipidomics analysis, we found that the decrease in monounsaturated fatty acid caused by genetic deletion of Scd2 in mice was crucial for the induction of an antiviral response through activation of the cGAS-STING pathway. These findings demonstrate the important relationship between fatty acid biosynthesis and type I IFN responses that enhances the antiviral response.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Ácidos Graxos Monoinsaturados/metabolismo , Interferon Tipo I/farmacologia , Proteínas de Membrana/fisiologia , Nucleotidiltransferases/fisiologia , Estearoil-CoA Dessaturase/fisiologia , Viroses/imunologia , Animais , Interações Hospedeiro-Patógeno , Metabolismo dos Lipídeos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transdução de Sinais , Viroses/metabolismo
8.
Mol Cell ; 81(12): 2596-2610.e7, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-33961796

RESUMO

p53-binding protein 1 (53BP1) regulates both the DNA damage response and p53 signaling. Although 53BP1's function is well established in DNA double-strand break repair, how its role in p53 signaling is modulated remains poorly understood. Here, we identify the scaffolding protein AHNAK as a G1 phase-enriched interactor of 53BP1. We demonstrate that AHNAK binds to the 53BP1 oligomerization domain and controls its multimerization potential. Loss of AHNAK results in hyper-accumulation of 53BP1 on chromatin and enhanced phase separation, culminating in an elevated p53 response, compromising cell survival in cancer cells but leading to senescence in non-transformed cells. Cancer transcriptome analyses indicate that AHNAK-53BP1 cooperation contributes to the suppression of p53 target gene networks in tumors and that loss of AHNAK sensitizes cells to combinatorial cancer treatments. These findings highlight AHNAK as a rheostat of 53BP1 function, which surveys cell proliferation by preventing an excessive p53 response.


Assuntos
Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Cromatina/metabolismo , DNA/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Fase G1/fisiologia , Histonas/metabolismo , Humanos , Células MCF-7 , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Transdução de Sinais/fisiologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/fisiologia
9.
Commun Biol ; 4(1): 517, 2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-33941818

RESUMO

Erythropoiesis involves complex interrelated molecular signals influencing cell survival, differentiation, and enucleation. Diseases associated with ineffective erythropoiesis, such as ß-thalassemias, exhibit erythroid expansion and defective enucleation. Clear mechanistic determinants of what make erythropoiesis effective are lacking. We previously demonstrated that exogenous transferrin ameliorates ineffective erythropoiesis in ß-thalassemic mice. In the current work, we utilize transferrin treatment to elucidate a molecular signature of ineffective erythropoiesis in ß-thalassemia. We hypothesize that compensatory mechanisms are required in ß-thalassemic erythropoiesis to prevent apoptosis and enhance enucleation. We identify pleckstrin-2-a STAT5-dependent lipid binding protein downstream of erythropoietin-as an important regulatory node. We demonstrate that partial loss of pleckstrin-2 leads to worsening ineffective erythropoiesis and pleckstrin-2 knockout leads to embryonic lethality in ß-thalassemic mice. In addition, the membrane-associated active form of pleckstrin-2 occurs at an earlier stage during ß-thalassemic erythropoiesis. Furthermore, membrane-associated activated pleckstrin-2 decreases cofilin mitochondrial localization in ß-thalassemic erythroblasts and pleckstrin-2 knockdown in vitro induces cofilin-mediated apoptosis in ß-thalassemic erythroblasts. Lastly, pleckstrin-2 enhances enucleation by interacting with and activating RacGTPases in ß-thalassemic erythroblasts. This data elucidates the important compensatory role of pleckstrin-2 in ß-thalassemia and provides support for the development of targeted therapeutics in diseases of ineffective erythropoiesis.


Assuntos
Apoptose , Núcleo Celular/patologia , Eritroblastos/patologia , Eritropoese , Proteínas de Membrana/fisiologia , Talassemia beta/patologia , Animais , Núcleo Celular/metabolismo , Eritroblastos/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Talassemia beta/etiologia , Talassemia beta/metabolismo
10.
Theranostics ; 11(13): 6120-6137, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33995649

RESUMO

Rationale: Clinical interest in combining targeted radionuclide therapies (TRT) with immunotherapies is growing. External beam radiation therapy (EBRT) activates a type 1 interferon (IFN1) response mediated via stimulator of interferon genes (STING), and this is critical to its therapeutic interaction with immune checkpoint blockade. However, little is known about the time course of IFN1 activation after EBRT or whether this may be induced by decay of a TRT source. Methods: We examined the IFN1 response and expression of immune susceptibility markers in B78 and B16 melanomas and MOC2 head and neck cancer murine models using qPCR and western blot. For TRT, we used 90Y chelated to NM600, an alkylphosphocholine analog that exhibits selective uptake and retention in tumor cells including B78 and MOC2. Results: We observed significant IFN1 activation in all cell lines, with peak activation in B78, B16, and MOC2 cell lines occurring 7, 7, and 1 days, respectively, following RT for all doses. This effect was STING-dependent. Select IFN response genes remained upregulated at 14 days following RT. IFN1 activation following STING agonist treatment in vitro was identical to RT suggesting time course differences between cell lines were mediated by STING pathway kinetics and not DNA damage susceptibility. In vivo delivery of EBRT and TRT to B78 and MOC2 tumors resulted in a comparable time course and magnitude of IFN1 activation. In the MOC2 model, the combination of 90Y-NM600 and dual checkpoint blockade therapy reduced tumor growth and prolonged survival compared to single agent therapy and cumulative dose equivalent combination EBRT and dual checkpoint blockade therapy. Conclusions: We report the time course of the STING-dependent IFN1 response following radiation in multiple murine tumor models. We show the potential of TRT to stimulate IFN1 activation that is comparable to that observed with EBRT and this may be critical to the therapeutic integration of TRT with immunotherapies.


Assuntos
Carcinoma de Células Escamosas/radioterapia , Interferon Tipo I/fisiologia , Melanoma Experimental/radioterapia , Animais , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/fisiopatologia , Linhagem Celular Tumoral , Terapia Combinada , Relação Dose-Resposta à Radiação , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Técnicas de Inativação de Genes , Neoplasias de Cabeça e Pescoço/patologia , Inibidores de Checkpoint Imunológico , Interferon Tipo I/biossíntese , Interferon Tipo I/genética , Linfócitos/efeitos dos fármacos , Linfócitos/efeitos da radiação , Melanoma Experimental/imunologia , Melanoma Experimental/fisiopatologia , Proteínas de Membrana/agonistas , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/fisiologia , Compostos Radiofarmacêuticos/farmacocinética , Compostos Radiofarmacêuticos/uso terapêutico , Fatores de Tempo , Ensaio Tumoral de Célula-Tronco , Regulação para Cima , Radioisótopos de Ítrio/farmacocinética , Radioisótopos de Ítrio/uso terapêutico
11.
PLoS Pathog ; 17(5): e1009599, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34043740

RESUMO

Antiviral therapeutics are a front-line defense against virally induced diseases. Because viruses frequently mutate to escape direct inhibition of viral proteins, there is interest in targeting the host proteins that the virus must co-opt to complete its replication cycle. However, a detailed understanding of the interactions between the virus and the host cell is necessary in order to facilitate development of host-directed therapeutics. As a first step, we performed a genome-wide loss of function screen using the alphacoronavirus HCoV-229E to better define the interactions between coronaviruses and host factors. We report the identification and validation of an ER-resident host protein, TMEM41B, as an essential host factor for not only HCoV-229E but also genetically distinct coronaviruses including the pandemic betacoronavirus SARS-CoV-2. We show that the protein is required at an early, but post-receptor engagement, stage of the viral lifecycle. Further, mechanistic studies revealed that although the protein was not enriched at replication complexes, it likely contributes to viral replication complex formation via mobilization of cholesterol and other lipids to facilitate host membrane expansion and curvature. Continued study of TMEM41B and the development of approaches to prevent its function may lead to broad spectrum anti-coronavirus therapeutics.


Assuntos
Coronavirus Humano 229E/efeitos dos fármacos , Interações entre Hospedeiro e Microrganismos/fisiologia , Proteínas de Membrana/metabolismo , Animais , Antivirais/farmacologia , COVID-19/metabolismo , Linhagem Celular , Chlorocebus aethiops , Coronavirus Humano 229E/fisiologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Interações entre Hospedeiro e Microrganismos/genética , Humanos , Proteínas de Membrana/fisiologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidade , Células Vero , Replicação Viral/efeitos dos fármacos
12.
J Physiol Biochem ; 77(3): 355-363, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33835423

RESUMO

PKM2, pyruvate kinase type M2, has been shown to play a key role in aerobic glycolysis and to regulate the malignant behaviors of cancer cells. Recently, PKM2 has been revealed to hold dual metabolic and nonmetabolic roles. Working as both a pyruvate kinase with catalytic activity and a protein kinase that phosphorylates its substrates, PKM2 stands at the crossroads of glycolysis and tumor growth. Recently, it was revealed that the catalytic activity of PKM2 can be regulated by its posttranslational modification (PTM). Several PTM types, including phosphorylation, methylation, acetylation, oxidation, hydroxylation, succinylation, and glycylation, have been gradually identified on different amino acid residues of the PKM2 coding sequence. In this review, we highlight the recent advancements in understanding PKM2 PTMs and the regulatory roles conferred by PTMs during anaerobic glycolysis in tumors.


Assuntos
Proteínas de Transporte/fisiologia , Proteínas de Membrana/fisiologia , Neoplasias/metabolismo , Processamento de Proteína Pós-Traducional , Hormônios Tireóideos/fisiologia , Acetilação , Linhagem Celular Tumoral , Glicólise , Humanos , Metilação , Efeito Warburg em Oncologia
13.
Int J Mol Sci ; 22(5)2021 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-33800732

RESUMO

Matriptase-2, a serine protease expressed in hepatocytes, is a negative regulator of hepcidin expression. The purpose of the study was to investigate the interaction of matriptase-2 with hemojuvelin protein in vivo. Mice lacking the matriptase-2 proteolytic activity (mask mice) display decreased content of hemojuvelin protein. Vice versa, the absence of hemojuvelin results in decreased liver content of matriptase-2, indicating that the two proteins interact. To further characterize the role of matriptase-2, we investigated iron metabolism in mask mice fed experimental diets. Administration of iron-enriched diet increased liver iron stores as well as hepcidin expression. Treatment of iron-overloaded mask mice with erythropoietin increased hemoglobin and hematocrit, indicating that the response to erythropoietin is intact in mask mice. Feeding of an iron-deficient diet to mask mice significantly increased spleen weight as well as the splenic content of erythroferrone and transferrin receptor proteins, indicating stress erythropoiesis. Liver hepcidin expression was decreased; expression of Id1 was not changed. Overall, the results suggest a complex interaction between matriptase-2 and hemojuvelin, and demonstrate that hepcidin can to some extent be regulated even in the absence of matriptase-2 proteolytic activity.


Assuntos
Proteínas Ligadas por GPI/fisiologia , Proteína da Hemocromatose/fisiologia , Sobrecarga de Ferro/metabolismo , Proteínas de Membrana/fisiologia , Serina Endopeptidases/fisiologia , Animais , Proteína Morfogenética Óssea 6/biossíntese , Proteína Morfogenética Óssea 6/genética , Eritropoetina/farmacologia , Feminino , Proteínas Ligadas por GPI/biossíntese , Proteínas Ligadas por GPI/deficiência , Proteínas Ligadas por GPI/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína da Hemocromatose/biossíntese , Proteína da Hemocromatose/deficiência , Proteína da Hemocromatose/genética , Hepcidinas/biossíntese , Hepcidinas/genética , Proteína 1 Inibidora de Diferenciação/biossíntese , Proteína 1 Inibidora de Diferenciação/genética , Ferro/deficiência , Ferro na Dieta/farmacologia , Fígado/metabolismo , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Especificidade de Órgãos , Regiões Promotoras Genéticas/genética , Domínios Proteicos , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/deficiência , Serina Endopeptidases/genética , Baço/metabolismo
14.
Gene ; 787: 145640, 2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-33845135

RESUMO

Obtaining detectable knockout phenotypes in the G0 generation is essential for gene function studies. Although CRISPR/Cas9-mediated gene editing has been employed to knock out molluscan genes, detectable phenotypes in the G0 generation have not been reported in these animals. In this study, we determined the knockout phenotype of a cilium-related gene, calaxin, using CRISPR/Cas9 technology in the gastropod mollusk Lottia goshimai. Injections with the Cas9-sgRNA complex caused approximately 30-80% of the injected larvae to exhibit a short-cilia phenotype characteristic of shortened cilia and decreased motility in the larvae. This phenotype was detectable in the G0 generation and was consistent for two independent sgRNAs. Genotyping of the injected larvae revealed various types of deletions and insertions in the target gene, which occurred in all sequences from the short-cilia larvae. This result indicated that the short-cilia phenotype was indeed caused by calaxin knockout. This possibility was supported by an RNAi assay targeting calaxin, which produced a highly similar short-cilia phenotype. We observed that a single SNP in the target sequences of the sgRNAs could show varied effects on the efficiency of mutagenesis. These results help to establish a foundation for future studies on molluscan gene editing using the CRISPR/Cas9 technique and contribute to the body of knowledge on molluscan ciliary functions.


Assuntos
Sistemas CRISPR-Cas , Cílios/fisiologia , Gastrópodes/fisiologia , Proteínas de Membrana/fisiologia , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/fisiologia , Cílios/genética , Gastrópodes/genética , Técnicas de Inativação de Genes , Proteínas de Membrana/genética , Mutagênese , Polimorfismo de Nucleotídeo Único
15.
Int J Mol Med ; 47(4)2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33649798

RESUMO

Severe acute respiratory syndrome (SARS) coronavirus­2 (SARS­CoV­2), the causative viral agent for the ongoing COVID­19 pandemic, enters its host cells primarily via the binding of the SARS­CoV­2 spike (S) proteins to the angiotensin­converting enzyme 2 (ACE2). A number of other cell entry mediators have also been identified, including neuropilin­1 (NRP1) and transmembrane protease serine 2 (TMPRSS2). More recently, it has been demonstrated that transmembrane protease serine 4 (TMPRSS4) along with TMPRSS2 activate the SARS­CoV­2 S proteins, and enhance the viral infection of human small intestinal enterocytes. To date, a systematic analysis of TMPRSS4 in health and disease is lacking. In the present study, using in silico tools, the gene expression and genetic alteration of TMPRSS4 were analysed across numerous tumours and compared to controls. The observations were also expanded to the level of the central nervous system (CNS). The findings revealed that TMPRSS4 was overexpressed in 11 types of cancer, including lung adenocarcinoma, lung squamous cell carcinoma, cervical squamous cell carcinoma, thyroid carcinoma, ovarian cancer, cancer of the rectum, pancreatic cancer, colon and stomach adenocarcinoma, uterine carcinosarcoma and uterine corpus endometrial carcinoma, whilst it was significantly downregulated in kidney carcinomas, acute myeloid leukaemia, skin cutaneous melanoma and testicular germ cell tumours. Finally, a high TMPRSS4 expression was documented in the olfactory tubercle, paraolfactory gyrus and frontal operculum, all brain regions which are associated with the sense of smell and taste. Collectively, these data suggest that TMPRSS4 may play a role in COVID­19 symptomatology as another SARS­CoV­2 host cell entry mediator responsible for the tropism of this coronavirus both in the periphery and the CNS.


Assuntos
COVID-19/enzimologia , COVID-19/genética , Proteínas de Membrana/genética , SARS-CoV-2/fisiologia , SARS-CoV-2/patogenicidade , Serina Endopeptidases/genética , Internalização do Vírus , Encéfalo/enzimologia , COVID-19/virologia , Sistema Nervoso Central/enzimologia , Simulação por Computador , Bases de Dados Genéticas , Feminino , Trato Gastrointestinal/enzimologia , Perfilação da Expressão Gênica , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Masculino , Proteínas de Membrana/fisiologia , Neoplasias/enzimologia , Neoplasias/genética , Pandemias , Serina Endopeptidases/fisiologia
16.
J Biol Chem ; 296: 100481, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33647313

RESUMO

The extracellular matrix (ECM) plays an important role in maintaining tissue homeostasis and poses a significant physical barrier to in vivo cell migration. Accordingly, as a means of enhancing tissue invasion, tumor cells use matrix metalloproteinases to degrade ECM proteins. However, the in vivo ECM is comprised not only of proteins but also of a variety of nonprotein components. Hyaluronan (HA), one of the most abundant nonprotein components of the interstitial ECM, forms a gel-like antiadhesive barrier that is impenetrable to particulate matter and cells. Mechanisms by which tumor cells penetrate the HA barrier have not been addressed. Here, we demonstrate that transmembrane protein 2 (TMEM2), the only known transmembrane hyaluronidase, is the predominant mediator of contact-dependent HA degradation and subsequent integrin-mediated cell-substrate adhesion. We show that a variety of tumor cells are able to eliminate substrate-bound HA in a tightly localized pattern corresponding to the distribution of focal adhesions (FAs) and stress fibers. This FA-targeted HA degradation is mediated by TMEM2, which itself is localized at site of FAs. TMEM2 depletion inhibits the ability of tumor cells to attach and migrate in an HA-rich environment. Importantly, TMEM2 directly binds at least two integrins via interaction between extracellular domains. Our findings demonstrate a critical role for TMEM2-mediated HA degradation in the adhesion and migration of cells on HA-rich ECM substrates and provide novel insight into the early phase of FA formation.


Assuntos
Ácido Hialurônico/metabolismo , Proteínas de Membrana/metabolismo , Animais , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Movimento Celular/fisiologia , Matriz Extracelular/metabolismo , Adesões Focais/metabolismo , Adesões Focais/fisiologia , Humanos , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/fisiologia , Hialuronoglucosaminidase/metabolismo , Integrinas/metabolismo , Proteínas de Membrana/fisiologia , Camundongos
17.
Science ; 371(6533): 1059-1063, 2021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33674494

RESUMO

Color detection is used by animals of diverse phyla to navigate colorful natural environments and is thought to require evolutionarily conserved opsin photoreceptor genes. We report that Caenorhabditis elegans roundworms can discriminate between colors despite the fact that they lack eyes and opsins. Specifically, we found that white light guides C. elegans foraging decisions away from a blue-pigment toxin secreted by harmful bacteria. These foraging decisions are guided by specific blue-to-amber ratios of light. The color specificity of color-dependent foraging varies notably among wild C. elegans strains, which indicates that color discrimination is ecologically important. We identified two evolutionarily conserved cellular stress response genes required for opsin-independent, color-dependent foraging by C. elegans, and we speculate that cellular stress response pathways can mediate spectral discrimination by photosensitive cells and organisms-even by those lacking opsins.


Assuntos
Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/efeitos da radiação , Visão de Cores , Comportamento Alimentar , Animais , Aprendizagem da Esquiva , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/fisiologia , Sequência Conservada , Escherichia coli , Luz , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Proteínas Quinases/genética , Proteínas Quinases/fisiologia , Pseudomonas aeruginosa/metabolismo , Piocianina/metabolismo , Piocianina/toxicidade
18.
PLoS Biol ; 19(3): e3001139, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33657094

RESUMO

Mutations in mitochondrial replicative polymerase PolγA lead to progressive external ophthalmoplegia (PEO). While PolγA is the known central player in mitochondrial DNA (mtDNA) replication, it is unknown whether a regulatory process exists on the mitochondrial outer membrane which controlled its entry into the mitochondria. We now demonstrate that PolγA is ubiquitylated by mitochondrial E3 ligase, MITOL (or MARCH5, RNF153). Ubiquitylation in wild-type (WT) PolγA occurs at Lysine 1060 residue via K6 linkage. Ubiquitylation of PolγA negatively regulates its binding to Tom20 and thereby its mitochondrial entry. While screening different PEO patients for mitochondrial entry, we found that a subset of the PolγA mutants is hyperubiquitylated by MITOL and interact less with Tom20. These PolγA variants cannot enter into mitochondria, instead becomes enriched in the insoluble fraction and undergo enhanced degradation. Hence, mtDNA replication, as observed via BrdU incorporation into the mtDNA, was compromised in these PEO mutants. However, by manipulating their ubiquitylation status by 2 independent techniques, these PEO mutants were reactivated, which allowed the incorporation of BrdU into mtDNA. Thus, regulated entry of non-ubiquitylated PolγA may have beneficial consequences for certain PEO patients.


Assuntos
DNA Polimerase gama/metabolismo , Proteínas de Membrana/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , DNA Polimerase gama/fisiologia , Replicação do DNA , DNA Mitocondrial/genética , Células HEK293 , Humanos , Proteínas de Membrana/fisiologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Mutação , Ubiquitina-Proteína Ligases/fisiologia , Ubiquitinação
19.
Cell Immunol ; 362: 104298, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33592541

RESUMO

Myeloid derived suppressor cells (MDSCs) are a highly heterogeneous population of immature immune cells with immunosuppressive functions that are recruited to the tumor microenvironment (TME). MDSCs promote tumor growth and progression by inhibiting immune effector cell proliferation and function. MDSCs are affected by both novel anti-cancer therapies targeting the immune system to promote anti-tumor immunity, as well as by conventional treatments such as radiotherapy. Following radiotherapy, cytoplasmic double stranded DNA stimulates the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway, resulting in type I interferon production. Effectiveness of radiotherapy and cGAS/STING signaling are closely intertwined: activation of cGAS and STING is key to generate systemic anti-tumor immunity after irradiation. This review focuses on how radiotherapy and cGAS/STING signaling in MDSCs and/or tumor cells impact MDSC recruitment, expansion and function. The influence of conventional and ablative radiotherapy treatment schedules, inflammatory response following radiotherapy, and hypoxia are discussed as MDSC modulators.


Assuntos
Proteínas de Membrana/metabolismo , Células Supressoras Mieloides/imunologia , Nucleotidiltransferases/metabolismo , Humanos , Imunidade Inata , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Proteínas de Membrana/fisiologia , Células Supressoras Mieloides/fisiologia , Neoplasias/patologia , Nucleotidiltransferases/genética , Nucleotidiltransferases/fisiologia , Radioterapia/métodos , Transdução de Sinais/imunologia , Microambiente Tumoral/imunologia , Microambiente Tumoral/fisiologia
20.
Theranostics ; 11(7): 3089-3108, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33537076

RESUMO

Rationale: Siglec15 is an emerging target for normalization cancer immunotherapy. However, pan-cancer anti-Siglec15 treatment is not yet validated and the potential role of Siglec15 in bladder cancer (BLCA) remains elusive. Methods: We comprehensively evaluated the expression pattern and immunological role of Siglec15 using pan-cancer analysis based on RNA sequencing data obtained from The Cancer Genome Atlas. We then systematically correlated Siglec15 with immunological characteristics in the BLCA tumor microenvironment (TME), including immunomodulators, cancer immunity cycles, tumor-infiltrating immune cells (TIICs), immune checkpoints, and T cell inflamed score. We also analyzed the role of Siglec15 in predicting the molecular subtype and the response to several treatment options in BLCA. Our results were validated in several public cohorts as well as our BLCA tumor microarray cohort, the Xiangya cohort. We developed an immune risk score (IRS), validated it, and tested its ability to predict the prognosis and response to cancer immunotherapy. Results: We found that Siglec15 was specifically overexpressed in the TME of various cancers. We hypothesize that Siglec15 designs a non-inflamed TME in BLCA based on the evidence that Siglec15 negatively correlated with immunomodulators, TIICs, cancer immunity cycles, immune checkpoints, and T cell inflamed score. Bladder cancer with high Siglec15 expression was not sensitive to cancer immunotherapy, but exhibited a higher incidence of hyperprogression. High Siglec15 levels indicated a luminal subtype of BLCA characterized by lower immune infiltration, lower response to cancer immunotherapy and neoadjuvant chemotherapy, but higher response to anti-angiogenic therapy and targeted therapies such as blocking Siglec15, ß-catenin, PPAR-γ, and FGFR3 pathways. Notably, a combination of anti-Siglec15 and cancer immunotherapy may be a more effective strategy than monotherapy. IRS can accurately predict the prognosis and response to cancer immunotherapy. Conclusions: Anti-Siglec15 immunotherapy might be suitable for BLCA treatment as Siglec15 correlates with a non-inflamed TME in BLCA. Siglec15 could also predict the molecular subtype and the response to several treatment options.


Assuntos
Imunoglobulinas/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Biomarcadores Tumorais/genética , China , Biologia Computacional/métodos , Bases de Dados Genéticas , Progressão da Doença , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Imunoglobulinas/fisiologia , Imunoterapia , Proteínas de Membrana/fisiologia , Prognóstico , Análise de Sequência de RNA/métodos , Microambiente Tumoral/imunologia , Neoplasias da Bexiga Urinária/patologia
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