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1.
Artigo em Inglês | MEDLINE | ID: mdl-31405865

RESUMO

VL-2397 (previously termed ASP2397) is an antifungal, aluminum-chelating cyclic hexapeptide with a structure analogous to that of ferrichrome-type siderophores, whereby replacement of aluminum by iron was shown to decrease the antifungal activity of this compound. Here, we found that inactivation of an importer for ferrichrome-type siderophores, termed Sit1, renders Aspergillus fumigatus resistant to VL-2397. Moreover, expression of the endogenous sit1 gene under the control of a xylose-inducible promoter (to uncouple sit1 expression from iron repression) combined with C-terminal tagging with a fluorescent protein demonstrated localization of Sit1 in the plasma membrane and xylose-dependent VL-2397 susceptibility. This underlines that Sit1-mediated uptake is essential for VL-2397 susceptibility. Under xylose-induced sit1 expression, VL-2397 also retained antifungal activity after replacing aluminum with iron, which demonstrates that VL-2397 bears antifungal activity independent of cellular aluminum importation. Analysis of sit1 expression indicated that the reduced antifungal activity of the iron-chelated VL-2397 is caused by downregulation of sit1 expression by the imported iron. Furthermore, we demonstrate that defects in iron homeostatic mechanisms modulate the activity of VL-2397. In contrast to A. fumigatus and Candida glabrata, Saccharomyces cerevisiae displays intrinsic resistance to VL-2397 antifungal activity. However, expression of sit1 from A. fumigatus, or its homologue from C. glabrata, resulted in susceptibility to VL-2397, which suggests that the intrinsic resistance of S. cerevisiae is based on lack of uptake and that A. fumigatus, C. glabrata, and S. cerevisiae share an intracellular target for VL-2397.


Assuntos
Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/metabolismo , Complexos de Coordenação/farmacologia , Proteínas Fúngicas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Peptídeos Cíclicos/farmacologia , Sideróforos/metabolismo , Antifúngicos/farmacologia , Transporte Biológico/efeitos dos fármacos , Candida glabrata/efeitos dos fármacos , Candida glabrata/metabolismo , Compostos Férricos/farmacologia , Ferricromo/metabolismo , Ferro/metabolismo , Quelantes de Ferro/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo
2.
Am J Physiol Gastrointest Liver Physiol ; 315(5): G887-G895, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30160974

RESUMO

The expression of amino acid transporters in small intestine epithelia of human newborns has not been studied yet. It is further not known whether the maturation of imino acid (proline) transport is delayed as in the kidney proximal tubule. The possibility to obtain small intestinal tissue from patients undergoing surgery for jejunal or ileal atresia during their first days after birth was used to address these questions. As control, adult terminal ileum tissue was sampled during routine endoscopies. Gene expression of luminal imino and amino acid transporter SIT1 (SLC6A20) was approximately threefold lower in newborns versus adults. mRNA levels of all other luminal and basolateral amino acid transporters and accessory proteins tested were similar in newborn mucosa compared with adults. At the protein level, the major luminal neutral amino acid transporter B0AT1 (SLC6A19) and its accessory protein angiotensin-converting enzyme 2 were shown by immunofluorescence to be expressed similarly in newborns and in adults. SIT1 protein was not detectable in the small intestine of human newborns, in contrast to adults. The morphology of newborn intestinal mucosa proximal and distal to the obstruction was generally normal, but a decreased proliferation rate was visualized distally of the atresia by lower levels of the mitosis marker Ki-67. The mRNA level of the 13 tested amino acid transporters and accessory proteins was nonetheless similar, suggesting that the intestinal obstruction and interruption of amniotic fluid passage through the small intestinal lumen did not affect amino acid transporter expression. NEW & NOTEWORTHY System IMINO transporter SIT1 is not expressed in the small intestine of human newborns. This new finding resembles the situation in the proximal kidney tubule leading to iminoglycinuria. Lack of amniotic fluid passage in small intestinal atresia does not affect amino acid transporter expression distal to intestinal occlusion.


Assuntos
Intestino Delgado/metabolismo , Proteínas de Membrana Transportadoras/genética , Adulto , Idoso , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Recém-Nascido , Intestino Delgado/crescimento & desenvolvimento , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Pessoa de Meia-Idade
3.
Biochem J ; 473(9): 1203-13, 2016 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-26929401

RESUMO

Aspergillus fumigatus is an opportunistic fungal pathogen for immunocompromised patients, and genes involved in siderophore metabolism have been identified as virulence factors. Recently, we identified the membrane transporters sit1 and sit2, which are putative virulence factors of A. fumigatus; sit1 and sit2 are homologous to yeast Sit1, and sit1 and sit2 gene expression was up-regulated after iron depletion. When expressed heterologously in Saccharomyces cerevisiae, sit1 and sit2 were localized to the plasma membrane; sit1 efficiently complemented ferrichrome (FC) and ferrioxamine B (FOB) uptake in yeast cells, whereas sit2 complemented only FC uptake. Deletion of sit1 resulted in a decrease in FOB and FC uptake, and deletion of sit2 resulted in a decrease in FC uptake in A. fumigatus It is of interest that a sit1 and sit2 double-deletion mutant resulted in a synergistic decrease in FC uptake activity. Both sit1 and sit2 were localized to the plasma membrane in A. fumigatus The expression levels of the sit1 and sit2 genes were dependent on hapX under low-but not high-iron conditions. Furthermore, mirB, and sidA gene expression was up-regulated and sreA expression down-regulated when sit1 and sit2 were deleted. Although sit1 and sit2 failed to affect mouse survival rate, these genes affected conidial killing activity. Taken together, our results suggest that sit1 and sit2 are siderophore transporters and putative virulence factors localized to the plasma membrane.


Assuntos
Aspergillus fumigatus/metabolismo , Membrana Celular/metabolismo , Desferroxamina/metabolismo , Compostos Férricos/metabolismo , Ferricromo/metabolismo , Ferro/metabolismo , Fatores de Virulência/metabolismo , Animais , Aspergillus fumigatus/genética , Aspergillus fumigatus/patogenicidade , Membrana Celular/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Fatores de Virulência/genética
4.
FEMS Yeast Res ; 15(7)2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26323600

RESUMO

Previously, we reported that Aft1 regulates Sit1 by modulating the ubiquitination of Sit1 in Saccharomyces cerevisiae. Here, we report the function of the physical interaction between Sit1 and Aft1 in ferrioxamine B (FOB) uptake. The interaction between Sit1 and Aft1 induced protein localization of Sit1 to the plasma membrane, and more Sit1 was detected in the plasma membrane when Sit1 and Aft1 were coexpressed compared with Sit1 expression alone. The MSN5-deletion mutant, which failed to translocate Aft1 to the cytosolic compartment, showed lower FOB uptake activity than the wild type. However, higher free iron uptake activity was detected in the MSN5-deletion mutant. Furthermore, the strain transformed with AFT1-1(up) plasmid, which failed to regulate Aft1 via iron concentration and accumulated Aft1 in the nucleus, showed lower FOB uptake activity. The Aft1 Y179F mutant, which contained a tyrosine residue that was changed to phenylalanine, failed to interact physically with Sit1 and showed more degradation of the Sit1 and, ultimately, lower FOB uptake activity. Additionally, we found that MG132 and PMSF, which are inhibitors of proteasomes and serine proteases, respectively, increased the Sit1 protein level. Taken together, these results suggest that the protein-protein interaction between Sit1 and Aft1 is an important factor in the FOB uptake activity of Sit1.


Assuntos
Desferroxamina/metabolismo , Compostos Férricos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteólise , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Membrana Celular/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas
5.
Immunol Res ; 72(4): 754-765, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38691318

RESUMO

This study aims to elucidate the expression and functionality of SIT1 in circulating CD8/CD4 + T cells in humans and to delineate its significance in systemic lupus erythematosus (SLE) patients. We employed multiparametric flow cytometry to investigate the expression of SIT1 in circulating CD8/CD4 + T cells and their respective subsets, comparing healthy controls (HCs) with SLE patients. Furthermore, we assessed the levels of granzyme B, perforin, IL-17, and IFN-γ in SIT1-related CD8/CD4 + T cells from both HCs and SLE patients, both before and after PMA stimulation. Clinically, we conducted receiver operating characteristic curve analysis and correlation analysis to evaluate the clinical relevance of SIT1-related CD8/CD4 + T cells in SLE patients. SIT1 exhibited higher expression in CD4 + T cells, with SIT1 - T cells demonstrating elevated levels of granzyme B, perforin, and IFN-γ compared to SIT1 + T cells. PMA-stimulated T cells exhibited reduced SIT1 expression compared to unstimulated T cells. SLE patients displayed increased SIT1 + proportions in CD8 + T cells and decreased SIT1 + CD4 + T cell numbers. Additionally, SIT1 + cells in SLE patients exhibited significantly higher levels of granzyme B and perforin compared to HCs. SIT1 + cells demonstrated significant associations with clinical indicators in SLE patients, with indicators related to SIT1 proving valuable in the diagnosis of SLE patients. SIT1 is inversely correlated with T cell activation. In SLE patients, SIT1 expression is altered in T cells concomitant with an augmented secretion of cytotoxic molecules. This upregulation may contribute to the pathogenesis of SLE and enhance its diagnostic potential.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Granzimas , Lúpus Eritematoso Sistêmico , Glicoproteínas de Membrana , Perforina , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Citotoxicidade Imunológica , Citometria de Fluxo , Granzimas/metabolismo , Interferon gama/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/diagnóstico , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/metabolismo , Ativação Linfocitária/imunologia , Perforina/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo
6.
J Pers Med ; 13(1)2022 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-36675674

RESUMO

Signaling threshold regulating transmembrane adaptor 1 (SIT1) encodes a disulfide-linked homodimeric lymphocyte-specific glycoprotein involved in immune cell activation. However, the relationship between SIT1 and the prognosis of skin cutaneous melanoma (SKCM) and tumor-infiltrating lymphocytes remains elusive. Here, we first compared the differences in SIT1 expression levels between SKCM tissues and adjacent normal tissues. Next, we found that the immune cell infiltration levels and signature pattern of immune infiltration were positively associated with the SIT1 gene mRNA levels. TCGA_SKCM RNA-seq data unveiled that the SIT1 upregulated several immune-associated signaling pathways in GSEA analysis. The high expression of SIT1 was closely related to improved survival in patients with SKCM. A pathway enrichment analysis of SIT1-associated immunomodulators indicated the involvement of the NF-κB signaling pathways. Based on SIT1-associated immunomodulators, we built a 13-gene signature by LASSO Cox regression which served as an independent prognostic factor for the survival of melanoma patients. By using the signature risk score, we achieved a good prediction result for the immunotherapy response and survival of SKCM patients. Our findings provided evidence for SIT1's implication in tumor immunity and survival of SKCM patients. The nominated immune signature is a promising predictive model for prognosis and immunotherapy sensitivity in SKCM patients.

7.
J Fungi (Basel) ; 8(9)2022 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-36135634

RESUMO

GR-2397 (previously VL-2397, ASP2397) is a first-in-class antifungal agent for the treatment of invasive aspergillosis. This siderophore-like molecule resembles ferrichrome; however, it is differentiated by three amino acid changes and an aluminum rather than iron chelate. GR-2397 is transported into fungal cells via the Sit1 transporter, which is not found in humans, leading to fungal specificity. Although the precise mechanism of action is currently unknown, GR-2397 is active against Aspergillus spp. including azole-resistant strains, Fusarium solani, and Candida glabrata in addition to other organisms. Efficacy has been demonstrated in several animal models of invasive aspergillosis, including a 24 h delayed-treatment model where rapid fungicidal activity was observed. Phase 1 single- and multiple-ascending intravenous dose studies showed that GR-2397 was safe and well-tolerated in humans. No signs of GR-2397 accumulation were observed following IV infusions of 300, 600, and 1200 mg every 24 h (q24h) for 7 days. The favorable safety, tolerability and drug-drug interaction profile, along with good tissue distribution, support further development of GR-2397 as a new treatment option for patients with invasive aspergillosis. This systematic review summarizes the published findings of GR-2397.

8.
Cell Host Microbe ; 27(3): 454-466.e8, 2020 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-32075740

RESUMO

Type I interferons (IFNs-I) fulfil multiple protective functions during pathogenic infections, but they can also cause detrimental effects and enhance immunopathology. Here, we report that IFNs-I promote the dysregulation of iron homeostasis in macrophages during systemic infections with the intracellular pathogen Candida glabrata, leading to fungal survival and persistence. By engaging JAK1, IFNs-I disturb the balance of the transcriptional activator NRF2 and repressor BACH1 to induce downregulation of the key iron exporter Fpn1 in macrophages. This leads to enhanced iron accumulation in the phagolysosome and failure to restrict fungal access to iron pools. As a result, C. glabrata acquires iron via the Sit1/Ftr1 iron transporter system, facilitating fungal intracellular replication and immune evasion. Thus, IFNs-I are central regulators of iron homeostasis, which can impact infection, and restricting iron bioavailability may offer therapeutic strategies to combat invasive fungal infections.


Assuntos
Candida glabrata/patogenicidade , Homeostase , Interferon Tipo I/imunologia , Ferro/fisiologia , Macrófagos/microbiologia , Adulto , Animais , Fatores de Transcrição de Zíper de Leucina Básica/imunologia , Candidíase/imunologia , Proteínas de Transporte de Cátions/imunologia , Células Cultivadas , Feminino , Humanos , Evasão da Resposta Imune , Janus Quinase 1/imunologia , Macrófagos/imunologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 2 Relacionado a NF-E2/imunologia , Fagossomos/microbiologia , Baço/imunologia
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