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1.
Front Cell Infect Microbiol ; 14: 1391288, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38919703

RESUMEN

The coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, is a highly contagious respiratory disease with widespread societal impact. The symptoms range from cough, fever, and pneumonia to complications affecting various organs, including the heart, kidneys, and nervous system. Despite various ongoing efforts, no effective drug has been developed to stop the spread of the virus. Although various types of medications used to treat bacterial and viral diseases have previously been employed to treat COVID-19 patients, their side effects have also been observed. The way SARS-CoV-2 infects the human body is very specific, as its spike protein plays an important role. The S subunit of virus spike protein cleaved by human proteases, such as furin protein, is an initial and important step for its internalization into a human host. Keeping this context, we attempted to inhibit the furin using phytochemicals that could produce minimal side effects. For this, we screened 408 natural phytochemicals from various plants having antiviral properties, against furin protein, and molecular docking and dynamics simulations were performed. Based on the binding score, the top three compounds (robustaflavone, withanolide, and amentoflavone) were selected for further validation. MM/GBSA energy calculations revealed that withanolide has the lowest binding energy of -57.2 kcal/mol followed by robustaflavone and amentoflavone with a binding energy of -45.2 kcal/mol and -39.68 kcal/mol, respectively. Additionally, ADME analysis showed drug-like properties for these three lead compounds. Hence, these natural compounds robustaflavone, withanolide, and amentoflavone, may have therapeutic potential for the management of SARS-CoV-2 by targeting furin.


Asunto(s)
Antivirales , Tratamiento Farmacológico de COVID-19 , Furina , Simulación del Acoplamiento Molecular , Fitoquímicos , SARS-CoV-2 , Furina/antagonistas & inhibidores , Furina/metabolismo , Fitoquímicos/farmacología , Fitoquímicos/química , Humanos , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/enzimología , Antivirales/farmacología , Antivirales/química , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/antagonistas & inhibidores , Glicoproteína de la Espiga del Coronavirus/química , COVID-19/virología , Unión Proteica
2.
EBioMedicine ; 104: 105181, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38838469

RESUMEN

BACKGROUND: Although several SARS-CoV-2-related coronaviruses (SC2r-CoVs) were discovered in bats and pangolins, the differences in virological characteristics between SARS-CoV-2 and SC2r-CoVs remain poorly understood. Recently, BANAL-20-236 (B236) was isolated from a rectal swab of Malayan horseshoe bat and was found to lack a furin cleavage site (FCS) in the spike (S) protein. The comparison of its virological characteristics with FCS-deleted SARS-CoV-2 (SC2ΔFCS) has not been conducted yet. METHODS: We prepared human induced pluripotent stem cell (iPSC)-derived airway and lung epithelial cells and colon organoids as human organ-relevant models. B236, SARS-CoV-2, and artificially generated SC2ΔFCS were used for viral experiments. To investigate the pathogenicity of B236 in vivo, we conducted intranasal infection experiments in hamsters. FINDINGS: In human iPSC-derived airway epithelial cells, the growth of B236 was significantly lower than that of the SC2ΔFCS. A fusion assay showed that the B236 and SC2ΔFCS S proteins were less fusogenic than the SARS-CoV-2 S protein. The infection experiment in hamsters showed that B236 was less pathogenic than SARS-CoV-2 and even SC2ΔFCS. Interestingly, in human colon organoids, the growth of B236 was significantly greater than that of SARS-CoV-2. INTERPRETATION: Compared to SARS-CoV-2, we demonstrated that B236 exhibited a tropism toward intestinal cells rather than respiratory cells. Our results are consistent with a previous report showing that B236 is enterotropic in macaques. Altogether, our report strengthens the assumption that SC2r-CoVs in horseshoe bats replicate primarily in the intestinal tissues rather than respiratory tissues. FUNDING: This study was supported in part by AMED ASPIRE (JP23jf0126002, to Keita Matsuno, Kazuo Takayama, and Kei Sato); AMED SCARDA Japan Initiative for World-leading Vaccine Research and Development Centers "UTOPIA" (JP223fa627001, to Kei Sato), AMED SCARDA Program on R&D of new generation vaccine including new modality application (JP223fa727002, to Kei Sato); AMED SCARDA Hokkaido University Institute for Vaccine Research and Development (HU-IVReD) (JP223fa627005h0001, to Takasuke Fukuhara, and Keita Matsuno); AMED Research Program on Emerging and Re-emerging Infectious Diseases (JP21fk0108574, to Hesham Nasser; JP21fk0108493, to Takasuke Fukuhara; JP22fk0108617 to Takasuke Fukuhara; JP22fk0108146, to Kei Sato; JP21fk0108494 to G2P-Japan Consortium, Keita Matsuno, Shinya Tanaka, Terumasa Ikeda, Takasuke Fukuhara, and Kei Sato; JP21fk0108425, to Kazuo Takayama and Kei Sato; JP21fk0108432, to Kazuo Takayama, Takasuke Fukuhara and Kei Sato; JP22fk0108534, Terumasa Ikeda, and Kei Sato; JP22fk0108511, to Yuki Yamamoto, Terumasa Ikeda, Keita Matsuno, Shinya Tanaka, Kazuo Takayama, Takasuke Fukuhara, and Kei Sato; JP22fk0108506, to Kazuo Takayama and Kei Sato); AMED Research Program on HIV/AIDS (JP22fk0410055, to Terumasa Ikeda; and JP22fk0410039, to Kei Sato); AMED Japan Program for Infectious Diseases Research and Infrastructure (JP22wm0125008 to Keita Matsuno); AMED CREST (JP21gm1610005, to Kazuo Takayama; JP22gm1610008, to Takasuke Fukuhara; JST PRESTO (JPMJPR22R1, to Jumpei Ito); JST CREST (JPMJCR20H4, to Kei Sato); JSPS KAKENHI Fund for the Promotion of Joint International Research (International Leading Research) (JP23K20041, to G2P-Japan Consortium, Keita Matsuno, Takasuke Fukuhara and Kei Sato); JST SPRING (JPMJSP2108 to Shigeru Fujita); JSPS KAKENHI Grant-in-Aid for Scientific Research C (22K07103, to Terumasa Ikeda); JSPS KAKENHI Grant-in-Aid for Scientific Research B (21H02736, to Takasuke Fukuhara); JSPS KAKENHI Grant-in-Aid for Early-Career Scientists (22K16375, to Hesham Nasser; 20K15767, to Jumpei Ito); JSPS Core-to-Core Program (A. Advanced Research Networks) (JPJSCCA20190008, to Kei Sato); JSPS Research Fellow DC2 (22J11578, to Keiya Uriu); JSPS Research Fellow DC1 (23KJ0710, to Yusuke Kosugi); JSPS Leading Initiative for Excellent Young Researchers (LEADER) (to Terumasa Ikeda); World-leading Innovative and Smart Education (WISE) Program 1801 from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) (to Naganori Nao); Ministry of Health, Labour and Welfare (MHLW) under grant 23HA2010 (to Naganori Nao and Keita Matsuno); The Cooperative Research Program (Joint Usage/Research Center program) of Institute for Life and Medical Sciences, Kyoto University (to Kei Sato); International Joint Research Project of the Institute of Medical Science, the University of Tokyo (to Terumasa Ikeda and Takasuke Fukuhara); The Tokyo Biochemical Research Foundation (to Kei Sato); Takeda Science Foundation (to Terumasa Ikeda and Takasuke Fukuhara); Mochida Memorial Foundation for Medical and Pharmaceutical Research (to Terumasa Ikeda); The Naito Foundation (to Terumasa Ikeda); Hokuto Foundation for Bioscience (to Tomokazu Tamura); Hirose Foundation (to Tomokazu Tamura); and Mitsubishi Foundation (to Kei Sato).


Asunto(s)
COVID-19 , Quirópteros , SARS-CoV-2 , Animales , SARS-CoV-2/genética , SARS-CoV-2/fisiología , Humanos , COVID-19/virología , Quirópteros/virología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/genética , Organoides/virología , Organoides/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/virología , Cricetinae , Furina/metabolismo , Células Epiteliales/virología , Células Vero , Chlorocebus aethiops
3.
Sci Adv ; 10(23): eadj4735, 2024 Jun 07.
Artículo en Inglés | MEDLINE | ID: mdl-38838150

RESUMEN

Why individuals with Down syndrome (DS) are more susceptible to SARS-CoV-2-induced neuropathology remains elusive. Choroid plexus (ChP) plays critical roles in barrier function and immune response modulation and expresses the ACE2 receptor and the chromosome 21-encoded TMPRSS2 protease, suggesting its substantial role in establishing SARS-CoV-2 infection in the brain. To explore this, we established brain organoids from DS and isogenic euploid iPSC that consist of a core of functional cortical neurons surrounded by a functional ChP-like epithelium (ChPCOs). DS-ChPCOs recapitulated abnormal DS cortical development and revealed defects in ciliogenesis and epithelial cell polarity in ChP-like epithelium. We then demonstrated that the ChP-like epithelium facilitates infection and replication of SARS-CoV-2 in cortical neurons and that this is increased in DS. Inhibiting TMPRSS2 and furin activity reduced viral replication in DS-ChPCOs to euploid levels. This model enables dissection of the role of ChP in neurotropic virus infection and euploid forebrain development and permits screening of therapeutics for SARS-CoV-2-induced neuropathogenesis.


Asunto(s)
Encéfalo , COVID-19 , Plexo Coroideo , Síndrome de Down , Organoides , SARS-CoV-2 , Serina Endopeptidasas , Plexo Coroideo/virología , Plexo Coroideo/metabolismo , Plexo Coroideo/patología , Organoides/virología , Organoides/metabolismo , Organoides/patología , Humanos , SARS-CoV-2/fisiología , COVID-19/virología , COVID-19/patología , COVID-19/metabolismo , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/genética , Síndrome de Down/metabolismo , Síndrome de Down/patología , Síndrome de Down/genética , Encéfalo/virología , Encéfalo/patología , Encéfalo/metabolismo , Neuronas/metabolismo , Neuronas/virología , Neuronas/patología , Replicación Viral , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/virología , Furina/metabolismo , Furina/genética , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/genética , Tropismo Viral
4.
Int J Mol Sci ; 25(10)2024 May 07.
Artículo en Inglés | MEDLINE | ID: mdl-38791119

RESUMEN

SARS-CoV-2 is the pathogen responsible for the most recent global pandemic, which has claimed hundreds of thousands of victims worldwide. Despite remarkable efforts to develop an effective vaccine, concerns have been raised about the actual protection against novel variants. Thus, researchers are eager to identify alternative strategies to fight against this pathogen. Like other opportunistic entities, a key step in the SARS-CoV-2 lifecycle is the maturation of the envelope glycoprotein at the RARR685↓ motif by the cellular enzyme Furin. Inhibition of this cleavage greatly affects viral propagation, thus representing an ideal drug target to contain infection. Importantly, no Furin-escape variants have ever been detected, suggesting that the pathogen cannot replace this protease by any means. Here, we designed a novel fluorogenic SARS-CoV-2-derived substrate to screen commercially available and custom-made libraries of small molecules for the identification of new Furin inhibitors. We found that a peptide substrate mimicking the cleavage site of the envelope glycoprotein of the Omicron variant (QTQTKSHRRAR-AMC) is a superior tool for screening Furin activity when compared to the commercially available Pyr-RTKR-AMC substrate. Using this setting, we identified promising novel compounds able to modulate Furin activity in vitro and suitable for interfering with SARS-CoV-2 maturation. In particular, we showed that 3-((5-((5-bromothiophen-2-yl)methylene)-4-oxo-4,5 dihydrothiazol-2-yl)(3-chloro-4-methylphenyl)amino)propanoic acid (P3, IC50 = 35 µM) may represent an attractive chemical scaffold for the development of more effective antiviral drugs via a mechanism of action that possibly implies the targeting of Furin secondary sites (exosites) rather than its canonical catalytic pocket. Overall, a SARS-CoV-2-derived peptide was investigated as a new substrate for in vitro high-throughput screening (HTS) of Furin inhibitors and allowed the identification of compound P3 as a promising hit with an innovative chemical scaffold. Given the key role of Furin in infection and the lack of any Food and Drug Administration (FDA)-approved Furin inhibitor, P3 represents an interesting antiviral candidate.


Asunto(s)
Furina , SARS-CoV-2 , Bibliotecas de Moléculas Pequeñas , Furina/antagonistas & inhibidores , Furina/metabolismo , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/metabolismo , Humanos , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/química , Antivirales/farmacología , Antivirales/química , COVID-19/virología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/antagonistas & inhibidores , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Evaluación Preclínica de Medicamentos/métodos
5.
Drug Discov Today ; 29(7): 104026, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38762086

RESUMEN

SARS-CoV-2 has triggered an international outbreak of the highly contagious acute respiratory disease known as COVID-19. Identifying key targets in the virus infection lifecycle is crucial for developing effective prevention and therapeutic strategies against it. Furin is a serine endoprotease that belongs to the family of proprotein convertases and plays a critical role in the entry of host cells by SARS-CoV-2. Furin can cleave a specific S1/S2 site, PRRAR, on the spike protein of SARS-CoV-2, which promotes viral transmission by facilitating membrane fusion. Hence, targeting furin could hold clinical implications for the prevention and treatment of COVID-19. This review offers an overview of furin's structure, substrates, function, and inhibitors, with a focus on its potential role in SARS-CoV-2 infection.


Asunto(s)
Antivirales , Tratamiento Farmacológico de COVID-19 , COVID-19 , Furina , SARS-CoV-2 , Furina/metabolismo , Humanos , COVID-19/prevención & control , Antivirales/farmacología , Antivirales/uso terapéutico , SARS-CoV-2/efectos de los fármacos , Internalización del Virus/efectos de los fármacos , Animales , Glicoproteína de la Espiga del Coronavirus/metabolismo
6.
Nat Commun ; 15(1): 4162, 2024 May 16.
Artículo en Inglés | MEDLINE | ID: mdl-38755139

RESUMEN

The multibasic furin cleavage site at the S1/S2 boundary of the spike protein is a hallmark of SARS-CoV-2 and plays a crucial role in viral infection. However, the mechanism underlying furin activation and its regulation remain poorly understood. Here, we show that GalNAc-T3 and T7 jointly initiate clustered O-glycosylations in the furin cleavage site of the SARS-CoV-2 spike protein, which inhibit furin processing, suppress the incorporation of the spike protein into virus-like-particles and affect viral infection. Mechanistic analysis reveals that the assembly of the spike protein into virus-like particles relies on interactions between the furin-cleaved spike protein and the membrane protein of SARS-CoV-2, suggesting a possible mechanism for furin activation. Interestingly, mutations in the spike protein of the alpha and delta variants of the virus confer resistance against glycosylation by GalNAc-T3 and T7. In the omicron variant, additional mutations reverse this resistance, making the spike protein susceptible to glycosylation in vitro and sensitive to GalNAc-T3 and T7 expression in human lung cells. Our findings highlight the role of glycosylation as a defense mechanism employed by host cells against SARS-CoV-2 and shed light on the evolutionary interplay between the host and the virus.


Asunto(s)
COVID-19 , Furina , Mutación , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/química , Humanos , SARS-CoV-2/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/fisiología , Glicosilación , Furina/metabolismo , Furina/genética , COVID-19/virología , COVID-19/metabolismo , Células HEK293 , N-Acetilgalactosaminiltransferasas/metabolismo , N-Acetilgalactosaminiltransferasas/genética , Animales , Chlorocebus aethiops , Polipéptido N-Acetilgalactosaminiltransferasa
7.
Cells ; 13(7)2024 Mar 28.
Artículo en Inglés | MEDLINE | ID: mdl-38607027

RESUMEN

The pro-protein convertase FURIN (PCSK3) is implicated in a wide range of normal and pathological biological processes such as infectious diseases, cancer and cardiovascular diseases. Previously, we performed a systemic inhibition of FURIN in a mouse model of atherosclerosis and demonstrated significant plaque reduction and alterations in macrophage function. To understand the cellular mechanisms affected by FURIN inhibition in myeloid cells, we optimized a CRISPR-mediated gene deletion protocol for successfully deriving hemizygous (HZ) and nullizygous (NZ) FURIN knockout clones in U937 monocytic cells using lipotransfection-based procedures and a dual guide RNA delivery strategy. We observed differences in monocyte and macrophage functions involving phagocytosis, lipid accumulation, cell migration, inflammatory gene expression, cytokine release patterns, secreted proteomics (cytokines) and whole-genome transcriptomics between wild-type, HZ and NZ FURIN clones. These studies provide a mechanistic basis on the possible roles of myeloid cell FURIN in cardiovascular disorders.


Asunto(s)
Furina , Edición Génica , Monocitos , Animales , Humanos , Ratones , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Citocinas/genética , Furina/genética , Furina/metabolismo , Monocitos/metabolismo , Multiómica , ARN Guía de Sistemas CRISPR-Cas , Células U937
8.
Am J Physiol Renal Physiol ; 326(6): F1066-F1077, 2024 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-38634134

RESUMEN

The epithelial Na+ channel (ENaC) γ subunit is essential for homeostasis of Na+, K+, and body fluid. Dual γ subunit cleavage before and after a short inhibitory tract allows dissociation of this tract, increasing channel open probability (PO), in vitro. Cleavage proximal to the tract occurs at a furin recognition sequence (143RKRR146, in the mouse γ subunit). Loss of furin-mediated cleavage prevents in vitro activation of the channel by proteolysis at distal sites. We hypothesized that 143RKRR146 mutation to 143QQQQ146 (γQ4) in 129/Sv mice would reduce ENaC PO, impair flow-stimulated flux of Na+ (JNa) and K+ (JK) in perfused collecting ducts, reduce colonic amiloride-sensitive short-circuit current (ISC), and impair Na+, K+, and body fluid homeostasis. Immunoblot of γQ4/Q4 mouse kidney lysates confirmed loss of a band consistent in size with the furin-cleaved proteolytic fragment. However, γQ4/Q4 male mice on a low Na+ diet did not exhibit altered ENaC PO or flow-induced JNa, though flow-induced JK modestly decreased. Colonic amiloride-sensitive ISC in γQ4/Q4 mice was not altered. γQ4/Q4 males, but not females, exhibited mildly impaired fluid volume conservation when challenged with a low Na+ diet. Blood Na+ and K+ were unchanged on a regular, low Na+, or high K+ diet. These findings suggest that biochemical evidence of γ subunit cleavage should not be used in isolation to evaluate ENaC activity. Furthermore, factors independent of γ subunit cleavage modulate channel PO and the influence of ENaC on Na+, K+, and fluid volume homeostasis in 129/Sv mice, in vivo.NEW & NOTEWORTHY The epithelial Na+ channel (ENaC) is activated in vitro by post-translational proteolysis. In vivo, low Na+ or high K+ diets enhance ENaC proteolysis, and proteolysis is hypothesized to contribute to channel activation in these settings. Using a mouse expressing ENaC with disruption of a key proteolytic cleavage site, this study demonstrates that impaired proteolytic activation of ENaC's γ subunit has little impact upon channel open probability or the ability of mice to adapt to low Na+ or high K+ diets.


Asunto(s)
Canales Epiteliales de Sodio , Proteolisis , Sodio , Animales , Canales Epiteliales de Sodio/metabolismo , Canales Epiteliales de Sodio/genética , Masculino , Femenino , Sodio/metabolismo , Túbulos Renales Colectores/metabolismo , Homeostasis , Furina/metabolismo , Furina/genética , Ratones , Colon/metabolismo , Potasio/metabolismo , Dieta Hiposódica , Ratones de la Cepa 129 , Mutación , Amilorida/farmacología
9.
Sci Rep ; 14(1): 7822, 2024 04 03.
Artículo en Inglés | MEDLINE | ID: mdl-38570613

RESUMEN

SARS CoV-2, the causative agent for the ongoing COVID-19 pandemic, it enters the host cell by activating the ACE2 receptor with the help of two proteasesi.e., Furin and TMPRSS2. Therefore, variations in these genes may account for differential susceptibility and severity between populations. Previous studies have shown that the role of ACE2 and TMPRSS2 gene variants in understanding COVID-19 susceptibility among Indian populations. Nevertheless, a knowledge gap exists concerning the COVID-19 susceptibility of Furin gene variants among diverse South Asian ethnic groups. Investigating the role of Furin gene variants and their global phylogeographic structure is essential to comprehensively understanding COVID-19 susceptibility in these populations. We have used 450 samples from diverse Indian states and performed linear regression to analyse the Furin gene variant's with COVID-19 Case Fatality Rate (CFR) that could be epidemiologically associated with disease severity outcomes. Associated genetic variants were further evaluated for their expression and regulatory potential through various Insilco analyses. Additionally, we examined the Furin gene using next-generation sequencing (NGS) data from 393 diverse global samples, with a particular emphasis on South Asia, to investigate its Phylogeographic structure among diverse world populations. We found a significant positive association for the SNP rs1981458 with COVID-19 CFR (p < 0.05) among diverse Indian populations at different timelines of the first and second waves. Further, QTL and other regulatory analyses showed various significant associations for positive regulatory roles of rs1981458 and Furin gene, mainly in Immune cells and virus infection process, highlighting their role in host immunity and viral assembly and processing. The Furin protein-protein interaction suggested that COVID-19 may contribute to Pulmonary arterial hypertension via a typical inflammation mechanism. The phylogeographic architecture of the Furin gene demonstrated a closer genetic affinity of South Asia with West Eurasian populations. Therefore, it is worth proposing that for the Furin gene, the COVID-19 susceptibility of South Asians will be more similar to the West Eurasian population. Our previous studies on the ACE2 and TMPRSS2 genes showed genetic affinity of South Asian with East Eurasians and West Eurasians, respectively. Therefore, with the collective information from these three important genes (ACE2, TMPRSS2 and Furin) we modelled COVID-19 susceptibilityof South Asia in between these two major ancestries with an inclination towards West Eurasia. In conclusion, this study, for the first time, concluded the role of rs1981458 in COVID-19 severity among the Indian population and outlined its regulatory potential.This study also highlights that the genetic structure for COVID-19 susceptibilityof South Asia is distinct, however, inclined to the West Eurasian population. We believe this insight may be utilised as a genetic biomarker to identify vulnerable populations, which might be directly relevant for developing policies and allocating resources more effectively during an epidemic.


Asunto(s)
COVID-19 , Humanos , Enzima Convertidora de Angiotensina 2/genética , COVID-19/epidemiología , COVID-19/genética , Furina/genética , Pandemias , Polimorfismo Genético
10.
PLoS One ; 19(4): e0302436, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38662786

RESUMEN

Severe cases of COVID-19 are characterized by development of acute respiratory distress syndrome (ARDS). Water accumulation in the lungs is thought to occur as consequence of an exaggerated inflammatory response. A possible mechanism could involve decreased activity of the epithelial Na+ channel, ENaC, expressed in type II pneumocytes. Reduced transepithelial Na+ reabsorption could contribute to lung edema due to reduced alveolar fluid clearance. This hypothesis is based on the observation of the presence of a novel furin cleavage site in the S protein of SARS-CoV-2 that is identical to the furin cleavage site present in the alpha subunit of ENaC. Proteolytic processing of αENaC by furin-like proteases is essential for channel activity. Thus, competition between S protein and αENaC for furin-mediated cleavage in SARS-CoV-2-infected cells may negatively affect channel activity. Here we present experimental evidence showing that coexpression of the S protein with ENaC in a cellular model reduces channel activity. In addition, we show that bidirectional competition for cleavage by furin-like proteases occurs between 〈ENaC and S protein. In transgenic mice sensitive to lethal SARS-CoV-2, however, a significant decrease in gamma ENaC expression was not observed by immunostaining of lungs infected as shown by SARS-CoV2 nucleoprotein staining.


Asunto(s)
COVID-19 , Canales Epiteliales de Sodio , Furina , Ratones Transgénicos , Proteolisis , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Canales Epiteliales de Sodio/metabolismo , Animales , Humanos , Ratones , Furina/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/metabolismo , COVID-19/metabolismo , COVID-19/virología , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/virología , Pulmón/metabolismo , Pulmón/virología , Pulmón/patología , Células HEK293
11.
Nutrients ; 16(8)2024 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-38674868

RESUMEN

Vitamin A deficiency (VAD) induced TGF-ß hyperactivation and reduced expression of cell adhesion proteins in the lung, suggesting that the disruption of retinoic acid (RA) signaling leads to epithelial-mesenchymal transition (EMT). To elucidate the role of lung vitamin A status in EMT, several EMT markers and the expression of the proprotein convertase furin, which activates TGF-ß, were analyzed in two experimental models. Our in vivo model included control rats, VAD rats, and both control rats and VAD rats, treated with RA. For the in vitro studies, human bronchoalveolar epithelial cells treated with RA were used. Our data show that EMT and furin are induced in VAD rats. Furthermore, furin expression continues to increase much more markedly after treatment of VAD rats with RA. In control rats and cell lines, an acute RA treatment induced a significant increase in furin expression, concomitant with changes in EMT markers. A ChIP assay demonstrated that RA directly regulates furin transcription. These results emphasize the importance of maintaining vitamin A levels within the physiological range since both levels below and above this range can cause adverse effects that, paradoxically, could be similar. The role of furin in EMT is discussed.


Asunto(s)
Transición Epitelial-Mesenquimal , Furina , Pulmón , Deficiencia de Vitamina A , Vitamina A , Furina/metabolismo , Transición Epitelial-Mesenquimal/efectos de los fármacos , Animales , Humanos , Pulmón/metabolismo , Pulmón/efectos de los fármacos , Vitamina A/farmacología , Vitamina A/metabolismo , Ratas , Deficiencia de Vitamina A/metabolismo , Masculino , Tretinoina/farmacología , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismo , Línea Celular , Ratas Wistar
12.
Anal Chem ; 96(17): 6707-6714, 2024 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-38631336

RESUMEN

Molecular magnetic resonance imaging (mMRI) of biomarkers is essential for accurate cancer detection in precision medicine. However, the current clinically used contrast agents provide structural magnetic resonance imaging (sMRI) information only and rarely provide mMRI information. Here, a tumor-specific furin-catalyzed nanoprobe (NP) was reported for differential diagnosis of malignant breast cancers (BCs) in vivo. This NP with a compact structure of Fe3O4@Gd-DOTA NPs (FFG NPs) contains an "always-on" T2-weighted MR signal provided by the magnetic Fe3O4 core and a furin-catalyzed enhanced T1-weighted MR signal provided by the Gd-DOTA moiety. The FFG NPs were found to produce an activated T1 signal in the presence of furin catalysis and an "always-on" T2 signal, providing mMRI and sMRI information simultaneously. Ratiometric mMRI:sMRI intensity can be used for differential diagnosis of malignant BCs MDA-MB-231 and MCF-7, where the furin levels relatively differ. The proposed probe not only provides structural imaging but also enables real-time molecular differential visualization of BC through enzymatic activities of cancer tissues.


Asunto(s)
Neoplasias de la Mama , Furina , Imagen por Resonancia Magnética , Furina/metabolismo , Furina/análisis , Humanos , Neoplasias de la Mama/diagnóstico por imagen , Femenino , Diagnóstico Diferencial , Animales , Catálisis , Ratones , Medios de Contraste/química , Línea Celular Tumoral
13.
Front Endocrinol (Lausanne) ; 15: 1379231, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38638139

RESUMEN

Receptor tyrosine kinases (RTKs) mediate the actions of growth factors in metazoans. In decapod crustaceans, RTKs are implicated in various physiological processes, such molting and growth, limb regeneration, reproduction and sexual differentiation, and innate immunity. RTKs are organized into two main types: insulin receptors (InsRs) and growth factor receptors, which include epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), vascular endothelial growth factor receptor (VEGFR), and platelet-derived growth factor receptor (PDGFR). The identities of crustacean RTK genes are incomplete. A phylogenetic analysis of the CrusTome transcriptome database, which included all major crustacean taxa, showed that RTK sequences segregated into receptor clades representing InsR (72 sequences), EGFR (228 sequences), FGFR (129 sequences), and PDGFR/VEGFR (PVR; 235 sequences). These four receptor families were distinguished by the domain organization of the extracellular N-terminal region and motif sequences in the protein kinase catalytic domain in the C-terminus or the ligand-binding domain in the N-terminus. EGFR1 formed a single monophyletic group, while the other RTK sequences were divided into subclades, designated InsR1-3, FGFR1-3, and PVR1-2. In decapods, isoforms within the RTK subclades were common. InsRs were characterized by leucine-rich repeat, furin-like cysteine-rich, and fibronectin type 3 domains in the N-terminus. EGFRs had leucine-rich repeat, furin-like cysteine-rich, and growth factor IV domains. N-terminal regions of FGFR1 had one to three immunoglobulin-like domains, whereas FGFR2 had a cadherin tandem repeat domain. PVRs had between two and five immunoglobulin-like domains. A classification nomenclature of the four RTK classes, based on phylogenetic analysis and multiple sequence alignments, is proposed.


Asunto(s)
Furina , Insulina , Furina/genética , Filogenia , Insulina/genética , Transcriptoma , Cisteína , Leucina/genética , Factor A de Crecimiento Endotelial Vascular/genética , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores ErbB/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/genética , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Tirosina
14.
Biochem Cell Biol ; 102(3): 275-284, 2024 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-38484367

RESUMEN

Neutrophil myeloperoxidase/H2O2/chloride system is a key mechanism to control pathogen infection. This enzyme, myeloperoxidase, plays a pivotal role in the arsenal of azurophilic granules that are released through degranulation upon neutrophil activation, which trigger local hypochlorous acid production. Myeloperoxidase gene encodes a protein precursor named promyeloperoxidase that arbors a propeptide that gets cleaved later during secretory routing in post-endoplasmic reticulum compartments. Although evidence suggested that this processing event was performed by one or different enzymes from the proprotein convertases family, the identity of this enzyme was never investigated. In this work, the naturally producing myeloperoxidase promyelocytic cell line HL-60 was used to investigate promyeloperoxidase cleavage during granulocytic differentiation in response to proprotein convertase inhibitors decanoyl-RVKR-chloromethylketone and hexa-d-arginine. Stable PC knockdown of endogenously expressed proprotein convertases, furin and PC7, was achieved using lentiviral delivery of shRNAs. None of the knockdown cell line could reproduce the effect of the pan-proprotein convertases inhibitor decanoyl-RVKR-chloromethylketone that accumulated intracellular promyeloperoxidase stores in HL-60 cells, therefore illustrating that both furin and PC7 redundantly process this proprotein.


Asunto(s)
Furina , Peroxidasa , Humanos , Células HL-60 , Furina/metabolismo , Furina/genética , Peroxidasa/metabolismo , Granulocitos/metabolismo , Granulocitos/citología , Diferenciación Celular , Subtilisinas/metabolismo , Precursores Enzimáticos/metabolismo , Precursores Enzimáticos/genética , Clorometilcetonas de Aminoácidos/farmacología
15.
Int J Mol Sci ; 25(5)2024 Feb 23.
Artículo en Inglés | MEDLINE | ID: mdl-38473856

RESUMEN

Myelin basic protein (MBP) is the second most abundant protein in the central nervous system and is responsible for structural maintenance of the myelin sheath covering axons. Previously, we showed that MBP has a more proactive role in the oligodendrocyte homeostasis, interacting with membrane-associated proteins, including integral membrane protein 2B (ITM2B or Bri2) that is associated with familial dementias. Here, we report that the molecular dynamics of the in silico-generated MBP-Bri2 complex revealed that MBP covers a significant portion of the Bri2 ectodomain, assumingly trapping the furin cleavage site, while the surface of the BRICHOS domain, which is responsible for the multimerization and activation of the Bri2 high-molecular-weight oligomer chaperone function, remains unmasked. These observations were supported by the co-expression of MBP with Bri2, its mature form, and disease-associated mutants, which showed that in mammalian cells, MBP indeed modulates the post-translational processing of Bri2 by restriction of the furin-catalyzed release of its C-terminal peptide. Moreover, we showed that the co-expression of MBP and Bri2 also leads to an altered cellular localization of Bri2, restricting its membrane trafficking independently of the MBP-mediated suppression of the Bri2 C-terminal peptide release. Further investigations should elucidate if these observations have physiological meaning in terms of Bri2 as a MBP chaperone activated by the MBP-dependent postponement of Bri2 membrane trafficking.


Asunto(s)
Furina , Glicoproteínas de Membrana , Animales , Furina/metabolismo , Proteína Básica de Mielina , Proteínas de la Membrana/metabolismo , Péptidos , Mamíferos/metabolismo
16.
Microbiol Res ; 282: 127659, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38430890

RESUMEN

The presence of a multibasic cleavage site in the Spike protein of SARS-CoV-2 makes it prone to be cleaved by Furin at the S1/S2 junction (aa. 685-686), which enhances the usage of TMPRSS2 to promote cell-cell fusion to form syncytia. Syncytia may contribute to pathology by facilitating viral dissemination, cytopathicity, immune evasion, and inflammation. However, the role of other SARS-CoV-2 encoding viral proteins in syncytia formation remains largely unknown. Here, we report that SARS-CoV-2 M protein effectively inhibits syncytia formation triggered by Spike or its variants (Alpha, Delta, Omicron, etc.) and prevents Spike cleavage into S1 and S2 based on a screen assay of 20 viral proteins. Mechanistically, M protein interacts with Furin and inhibits its enzymatic activity, preventing the cleavage of Spike. In addition, M interacts with Spike independent of its cytoplasmic tail, retaining it within the cytoplasm and reducing cell membrane localization. Our findings offer new insights into M protein's role in regulating Spike's function and underscore the importance of functional interplay among viral proteins, highlighting potential avenues for SARS-CoV-2 therapy development.


Asunto(s)
COVID-19 , Furina , Humanos , SARS-CoV-2 , Membrana Celular , Proteínas de la Membrana , Glicoproteína de la Espiga del Coronavirus
17.
Mol Biol Rep ; 51(1): 437, 2024 Mar 23.
Artículo en Inglés | MEDLINE | ID: mdl-38520572

RESUMEN

Polycystic ovary syndrome (PCOS) is a disorder resulted from interactions between genetic and environmental factors. Based on the importance of epigenetic factors in the pathoetiology of PCOS, the current review focused on identification of circular RNAs (circRNAs) that are involved in PCOS through acting as molecular sponges for microRNAs (miRNAs). The literature search led to identification of circ_0043533/miR-1179, circ_0030018/miR-136, circ_FURIN/miR-423-5p, circ-FURIN/miR-195-5p, circ_0043532/miR-182, circ_RANBP9/miR-136-5p, circRHBG/miR-515-5p, circMTO1/miR-320b, circASPH/miR-375, circPSMC3/miR-296-3p, circLDLR/miR-1294, circPUM1/miR-760, and hsa_circ_0118530/miR-136 as molecular axes contributing to the pathogenesis of PCOS. To set the stage for future research on the role of the ceRNA network in PCOS, in-silico analyses were performed using miRWalk, miRNet, and miRDIP databases. miRWalk identified 80 genes regulated by 5 miRNAs, miRNet revealed 6449 circRNAs potentially controlling 11 miRNAs, and miRDIP identified 11 miRNAs associated with 35 human pathways. These targets can be used in the treatment options, design of personalized medicine and prediction of prognosis of PCOS.


Asunto(s)
MicroARNs , Síndrome del Ovario Poliquístico , Femenino , Humanos , ARN Circular/genética , ARN Circular/metabolismo , Furina , Síndrome del Ovario Poliquístico/genética , MicroARNs/genética , MicroARNs/metabolismo , ARN Endógeno Competitivo
18.
J Virol ; 98(4): e0010224, 2024 Apr 16.
Artículo en Inglés | MEDLINE | ID: mdl-38470058

RESUMEN

The transmembrane serine protease 2 (TMPRSS2) activates the outer structural proteins of a number of respiratory viruses including influenza A virus (IAV), parainfluenza viruses, and various coronaviruses for membrane fusion. Previous studies showed that TMPRSS2 interacts with the carboxypeptidase angiotensin-converting enzyme 2 (ACE2), a cell surface protein that serves as an entry receptor for some coronaviruses. Here, by using protease activity assays, we determine that ACE2 increases the enzymatic activity of TMPRSS2 in a non-catalytic manner. Furthermore, we demonstrate that ACE2 knockdown inhibits TMPRSS2-mediated cleavage of IAV hemagglutinin (HA) in Calu-3 human airway cells and suppresses virus titers 100- to 1.000-fold. Transient expression of ACE2 in ACE2-deficient cells increased TMPRSS2-mediated HA cleavage and IAV replication. ACE2 knockdown also reduced titers of MERS-CoV and prevented S cleavage by TMPRSS2 in Calu-3 cells. By contrast, proteolytic activation and multicycle replication of IAV with multibasic HA cleavage site typically cleaved by furin were not affected by ACE2 knockdown. Co-immunoprecipitation analysis revealed that ACE2-TMPRSS2 interaction requires the enzymatic activity of TMPRSS2 and the carboxypeptidase domain of ACE2. Together, our data identify ACE2 as a new co-factor or stabilizer of TMPRSS2 activity and as a novel host cell factor involved in proteolytic activation and spread of IAV in human airway cells. Furthermore, our data indicate that ACE2 is involved in the TMPRSS2-catalyzed activation of additional respiratory viruses including MERS-CoV.IMPORTANCEProteolytic cleavage of viral envelope proteins by host cell proteases is essential for the infectivity of many viruses and relevant proteases provide promising drug targets. The transmembrane serine protease 2 (TMPRSS2) has been identified as a major activating protease of several respiratory viruses, including influenza A virus. TMPRSS2 was previously shown to interact with angiotensin-converting enzyme 2 (ACE2). Here, we report the mechanistic details of this interaction. We demonstrate that ACE2 increases or stabilizes the enzymatic activity of TMPRSS2. Furthermore, we describe ACE2 involvement in TMPRSS2-catalyzed cleavage of the influenza A virus hemagglutinin and MERS-CoV spike protein in human airway cells. These findings expand our knowledge of the activation of respiratory viruses by TMPRSS2 and the host cell factors involved. In addition, our results could help to elucidate a physiological role for TMPRSS2.


Asunto(s)
Enzima Convertidora de Angiotensina 2 , Virus de la Influenza A , Pulmón , Proteolisis , Serina Endopeptidasas , Animales , Perros , Humanos , Enzima Convertidora de Angiotensina 2/deficiencia , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , Biocatálisis , Línea Celular , Furina/metabolismo , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Virus de la Influenza A/crecimiento & desarrollo , Virus de la Influenza A/metabolismo , Pulmón/citología , Pulmón/virología , Coronavirus del Síndrome Respiratorio de Oriente Medio/metabolismo , Unión Proteica , Serina Endopeptidasas/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Internalización del Virus , Replicación Viral
19.
Biochim Biophys Acta Mol Cell Res ; 1871(3): 119682, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38301907

RESUMEN

Dengue virus (DENV) envelope protein plays crucial role in virus entry and maturation of virus during infection. Maturation of DENV occurs in the trans Golgi network at slightly acidic pH which is close to pKa of histidine. When exposed to the acidic environment of the late secretory pathway, dengue virus particles go through a significant conformational change, whereby interactions of structural proteins envelope (E) and prM proteins are reorganised and enable furin protease to cleave prM resulting in mature virus. In order to study the role of histidine of E protein in DENV maturation, we mutated 7 conserved histidine residues of envelope protein and assessed the percent of budding using viral like particle (VLP) system. Histidine mutants; H144A, H244A, H261A and H282A severely disrupted VLP formation without any significant change in expression in cell and its oligomerization ability. Treatment with acidotropic amine reversed the defect for all 4 mutants suggesting that these histidines could be involved in maturation and release. Over expression of capsid protein slightly enhanced VLP release of H244A and H261A. Similarly, furin over expression increased VLP release of these mutants. Co-immunoprecipitation studies revealed that prM and E interaction is lost for H244A, H261A and H282A mutants at acidic pH but not at neutral pH indicating that they could be involved in histidine switch during maturation at acidic pH. Detailed analysis of the mutants could provide novel insights on the interplay of envelop protein during maturation and aid in target for drug development.


Asunto(s)
Dengue , Proteínas del Envoltorio Viral , Humanos , Proteínas del Envoltorio Viral/genética , Furina/genética , Histidina/genética , Mutación
20.
mBio ; 15(4): e0336023, 2024 Apr 10.
Artículo en Inglés | MEDLINE | ID: mdl-38411986

RESUMEN

SARS-CoV-2 infects both the upper and lower respiratory tracts, which are characterized by different temperatures (33°C and 37°C, respectively). In addition, fever is a common COVID-19 symptom. SARS-CoV-2 has been shown to replicate more efficiently at low temperatures, but the effect of temperature on different viral proteins remains poorly understood. Here, we investigate how temperature affects the SARS-CoV-2 spike function and evolution. We first observed that increasing temperature from 33°C to 37°C or 39°C increased spike-mediated cell-cell fusion. We then experimentally evolved a recombinant vesicular stomatitis virus expressing the SARS-CoV-2 spike at these different temperatures. We found that spike-mediated cell-cell fusion was maintained during evolution at 39°C but was lost in a high proportion of viruses that evolved at 33°C or 37°C. Consistently, sequencing of the spikes evolved at 33°C or 37°C revealed the accumulation of mutations around the furin cleavage site, a region that determines cell-cell fusion, whereas this did not occur in spikes evolved at 39°C. Finally, using site-directed mutagenesis, we found that disruption of the furin cleavage site had a temperature-dependent effect on spike-induced cell-cell fusion and viral fitness. Our results suggest that variations in body temperature may affect the activity and diversification of the SARS-CoV-2 spike. IMPORTANCE: When it infects humans, SARS-CoV-2 is exposed to different temperatures (e.g., replication site and fever). Temperature has been shown to strongly impact SARS-CoV-2 replication, but how it affects the activity and evolution of the spike protein remains poorly understood. Here, we first show that high temperatures increase the SARS-CoV-2 spike fusogenicity. Then, we demonstrate that the evolution of the spike activity and variants depends on temperature. Finally, we show that the functional effect of specific spike mutations is temperature-dependent. Overall, our results suggest that temperature may be a factor influencing the activity and adaptation of the SARS-CoV-2 spike in vivo, which will help understanding viral tropism, pathogenesis, and evolution.


Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , Temperatura , SARS-CoV-2/genética , Furina , Frío , Glicoproteína de la Espiga del Coronavirus/genética
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