RESUMO
PURPOSE: To determine the prevalence of concomitant squamous metaplasia (SM), the initial histological change from normal urethra to urethral stricture, in bulbar urethral strictures and to investigate the associated clinical factors. METHODS: A retrospective review was conducted on 165 male patients with bulbar urethral strictures who underwent excision and primary anastomosis (EPA) between 2010 and 2020, for whom complete clinical data and excised urethral specimens were available. An experienced pathologist histologically evaluated concomitant SM in paraffin sections of the proximal end of the excised urethra blinded to the clinical data. Disease duration was calculated as the period from the initial diagnosis of urethral stricture to the date of EPA. The association between concomitant SM and clinical background was investigated. RESULTS: SM was identified in 86 (52.1%) patients. The median disease duration in patients with SM (38 months) was significantly longer than that in patients without SM (9 months, p < 0.0001). In multivariate analysis, the longer disease duration, non-traumatic stricture etiology, and failure to maintain urethral rest with urinary diversion via a suprapubic tube for more than 90 days were independent factors predicting concomitant SM. No significant difference was observed in success rates of EPA between patients with SM (93.2%) and those without SM (97.5%, p = 0.18). CONCLUSIONS: Reconstructive urologists need to be aware that concomitant SM is frequent in patients with bulbar urethral stricture, especially in those with long disease duration and those who were voiding volitionally during the period of urethral rest.
Assuntos
Metaplasia , Uretra , Estreitamento Uretral , Procedimentos Cirúrgicos Urológicos Masculinos , Humanos , Estreitamento Uretral/epidemiologia , Estreitamento Uretral/patologia , Estreitamento Uretral/cirurgia , Masculino , Estudos Retrospectivos , Pessoa de Meia-Idade , Idoso , Uretra/patologia , Adulto , Procedimentos Cirúrgicos Urológicos Masculinos/métodos , Tempo para o TratamentoRESUMO
Deep-sea organisms are subjected to extreme conditions; therefore, understanding their adaptive strategies is crucial. We utilize Saccharomyces cerevisiae as a model to investigate pressure-dependent protein regulation and piezo-adaptation. Using yeast deletion library analysis, we identified 6 poorly characterized genes that are crucial for high-pressure growth, forming novel functional modules associated with cell growth. In this study, we aimed to unravel the molecular mechanisms of high-pressure adaptation in S. cerevisiae, focusing on the role of MTC6. MTC6, the gene encoding the novel glycoprotein Mtc6/Ehg2, was found to stabilize tryptophan permease Tat2, ensuring efficient tryptophan uptake and growth under high pressure at 25 MPa. The loss of MTC6 led to promoted vacuolar degradation of Tat2, depending on the Rsp5-Bul1 ubiquitin ligase complex. These findings enhance our understanding of deep-sea adaptations and stress biology, with broad implications for biotechnology, environmental microbiology, and evolutionary insights across species.
Assuntos
Sistemas de Transporte de Aminoácidos , Retículo Endoplasmático , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Triptofano , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Triptofano/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Retículo Endoplasmático/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Estabilidade Proteica , Complexos Ubiquitina-Proteína Ligase/metabolismo , Complexos Ubiquitina-Proteína Ligase/genética , Vacúolos/metabolismo , Pressão Hidrostática , ProteóliseRESUMO
Mannosyl phosphorylceramide (MIPC) is a membrane lipid classified as a complex sphingolipid in Saccharomyces cerevisiae. MIPC is synthesized by 2 redundant enzymes, Sur1/Csg1 and Csh1, in the Golgi lumen. MIPC consists of 5 subtypes (A, B', B, C, and D-type) according to the position and number of hydroxyl groups on the ceramide moiety. Sur1 exerts higher impact on synthesis of MIPC-B and MIPC-C than Csh1. In this study, we elucidated the roles played by N-glycans attached to Sur1 and Csh1, and dissected the mechanisms underlying substrate recognition by these 2 enzymes. Sur1 carries an N-glycan on Asn-224, whereas Csh1 has N-glycans on Asn-51 and Asn-247. Although intracellular proteins usually harbor core-type N-glycans, the N-glycan on Asn-51 of Csh1 exhibited a unique mannan-like structure containing a long backbone of mannose. Sur1 N224Q and Csh1 N51Q mutants exhibited a decrease in the activity to synthesize specific MIPC subtypes for each enzyme, suggesting that these N-glycans play a role in substrate recognition through their catalytic domains. Moreover, ectopic insertion of an N-glycosylation consensus sequence (NST) at codon 51 of Sur1 (Sur1-NST51) resulted in an artificial modification with mannan, which markedly decreased protein stability. Our results suggest that the diminished stability of the Sur1-NST51 mutant protein could be attributable to potential structural alterations by the mannan. Collectively, the present study reveals essential luminal domains of Sur1 and Csh1 that dictate substrate specificity and/or the protein stabilities via mannan modification.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Carbamatos , Glicosiltransferases/metabolismo , Mananas/metabolismo , Manosiltransferases/metabolismo , Proteínas de Membrana/química , Mutação , Polissacarídeos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Mechanical stresses, including high hydrostatic pressure, elicit diverse physiological effects on organisms. Gtr1, Gtr2, Ego1 (also known as Meh1) and Ego3 (also known as Slm4), central regulators of the TOR complex 1 (TORC1) nutrient signaling pathway, are required for the growth of Saccharomyces cerevisiae cells under high pressure. Here, we showed that a pressure of 25â MPa (â¼250â kg/cm2) stimulates TORC1 to promote phosphorylation of Sch9, which depends on the EGO complex (EGOC) and Pib2. Incubation of cells at this pressure aberrantly increased glutamine and alanine levels in the ego1Δ, gtr1Δ, tor1Δ and pib2Δ mutants, whereas the polysome profiles were unaffected. Moreover, we found that glutamine levels were reduced by combined deletions of EGO1, GTR1, TOR1 and PIB2 with GLN3 These results suggest that high pressure leads to the intracellular accumulation of amino acids. Subsequently, Pib2 loaded with glutamine stimulates the EGOC-TORC1 complex to inactivate Gln3, downregulating glutamine synthesis. Our findings illustrate the regulatory circuit that maintains intracellular amino acid homeostasis and suggest critical roles for the EGOC-TORC1 and Pib2-TORC1 complexes in the growth of yeast under high hydrostatic pressure.
Assuntos
Proteínas Monoméricas de Ligação ao GTP , Proteínas de Saccharomyces cerevisiae , Aminoácidos , Homeostase , Pressão Hidrostática , Alvo Mecanístico do Complexo 1 de Rapamicina , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas Serina-Treonina Quinases , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Lymphatic recanalization failure after lymphadenectomy constitutes a major risk of lymphedema in cancer surgery. It has been reported that GATA2, a zinc finger transcription factor, is expressed in lymphatic endothelial cells and is involved in the development of fetal lymphatic vessels. GATA3, another member of the GATA family of transcription factors, is required for the differentiation of lymphoid tissue inducer (LTi) cells and is essential for lymph node formation. However, how GATA2 and GATA3 function in recanalization after the surgical extirpation of lymphatic vessels has not been elucidated. Employing a new model of lymphatic recanalization, we examined the lymphatic reconnection process in Gata2 heterozygous deficient (Gata2+/- ) and Gata3 heterozygous deficient (Gata3+/- ) mice. We found that lymphatic recanalization was significantly impaired in Gata2+/- mice, while Gata3+/- mice rarely showed such abnormalities. Notably, the perturbed lymphatic recanalization in the Gata2+/- mice was partially restored by crossing with the Gata3+/- mice. Our results demonstrate for the first time that GATA2 participates in the regeneration of damaged lymphatic vessels and the unexpected suppressive activity of GATA3 against lymphatic recanalization processes.
Assuntos
Fator de Transcrição GATA2/metabolismo , Excisão de Linfonodo/efeitos adversos , Vasos Linfáticos/metabolismo , Linfedema/metabolismo , Complicações Pós-Operatórias/metabolismo , Animais , Fator de Transcrição GATA2/genética , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Heterozigoto , Vasos Linfáticos/fisiologia , Linfedema/etiologia , Camundongos , Complicações Pós-Operatórias/etiologia , RegeneraçãoRESUMO
Zinc-finger transcription factors GATA2 and GATA3 are both expressed in the developing inner ear, although their overlapping versus distinct activities in adult definitive inner ear are not well understood. We show here that GATA2 and GATA3 are co-expressed in cochlear spiral ganglion cells and redundantly function in the maintenance of spiral ganglion cells and auditory neural circuitry. Notably, Gata2 and Gata3 compound heterozygous mutant mice had a diminished number of spiral ganglion cells due to enhanced apoptosis, which resulted in progressive hearing loss. The decrease in spiral ganglion cellularity was associated with lowered expression of neurotrophin receptor TrkC that is an essential factor for spiral ganglion cell survival. We further show that Gata2 null mutants that additionally bear a Gata2 YAC (yeast artificial chromosome) that counteracts the lethal hematopoietic deficiency due to complete Gata2 loss nonetheless failed to complement the deficiency in neonatal spiral ganglion neurons. Furthermore, cochlea-specific Gata2 deletion mice also had fewer spiral ganglion cells and resultant hearing impairment. These results show that GATA2 and GATA3 redundantly function to maintain spiral ganglion cells and hearing. We propose possible mechanisms underlying hearing loss in human GATA2- or GATA3-related genetic disorders.
Assuntos
Surdez/etiologia , Fatores de Transcrição GATA/metabolismo , Gânglio Espiral da Cóclea/metabolismo , Animais , Apoptose/genética , Contagem de Células , Cóclea/metabolismo , Cóclea/patologia , Surdez/metabolismo , Surdez/fisiopatologia , Modelos Animais de Doenças , Fatores de Transcrição GATA/genética , Expressão Gênica , Genes Reporter , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mutação , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/patologia , Gânglio Espiral da Cóclea/patologiaRESUMO
Glycosphingolipids (GSLs) are abundant in plasma membranes of mammalian cells, and their synthesis is strictly regulated in the Golgi apparatus. Disruption of GSL homeostasis is the cause of numerous diseases. Hundreds of molecular species of GSLs exist, and the detailed mechanisms underlying their homeostasis remain unclear. We investigated the physiological significance of isoform production for ß1,4-N-acetyl-galactosaminyl transferase 1/B4GALNT1 (B4GN1), an enzyme involved in synthesis of ganglio-series GSLs GM2/GD2/GA2. We discovered a new mRNA variant (termed variant 2) of B4GN1 through EST clone search. A new isoform, M1-B4GN1, which has an NH2-terminal cytoplasmic tail longer than that of previously-known isoform M2-B4GN1, is translated from variant 2. M1-B4GN1 has R-based motif (a retrograde transport signal) in the cytoplasmic tail. M1-B4GN1 is partially localized in the endoplasmic reticulum (ER) depending on the R-based motif, whereas M2-B4GN1 is localized in the Golgi. Stability of M1-B4GN1 is higher than that of M2-B4GN1 because of the R-based motif. M2-B4GN1 forms a homodimer via disulfide bonding. When M1-B4GN1 and M2-B4GN1 were co-expressed in CHO-K1 cells, the two isoforms formed a heterodimer. The M1/M2-B4GN1 heterodimer was more stable than the M2-B4GN1 homodimer, but the heterodimer was not transported from the Golgi to the ER. Our findings indicate that stabilization of M1-B4GN1 homodimer and M1/M2-B4GN1 heterodimer by R-based motif is related to prolongation of Golgi retention, but not to retrograde transport from the Golgi to the ER. Coexistence of several B4GN1 isoforms having distinctive characteristics presumably helps maintain overall enzyme stability and GSL homeostasis.
Assuntos
Arginina/metabolismo , Estabilidade Enzimática/fisiologia , N-Acetilgalactosaminiltransferases/metabolismo , Isoformas de Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Linhagem Celular , Cricetulus , Retículo Endoplasmático/metabolismo , Glicoesfingolipídeos/metabolismo , Complexo de Golgi/metabolismo , Transporte Proteico/fisiologiaRESUMO
Tryptophan is an essential amino acid in humans and an important serotonin and melatonin precursor. Monocarboxylate transporter MCT10 is a member of the SLC16A family proteins that mediates low-affinity tryptophan transport across basolateral membranes of kidney, small intestine, and liver epithelial cells, although the precise transport mechanism remains unclear. Here we developed a simple functional assay to analyze tryptophan transport by human MCT10 using a deletion mutant for the high-affinity tryptophan permease Tat2 in Saccharomyces cerevisiae. tat2Δtrp1 cells are defective in growth in YPD medium because tyrosine present in the medium competes for the low-affinity tryptophan permease Tat1 with tryptophan. MCT10 appeared to allow growth of tat2Δtrp1 cells in YPD medium, and accumulate in cells deficient for Rsp5 ubiquitin ligase. These results suggest that MCT10 is functional in yeast, and is subject to ubiquitin-dependent quality control. Whereas growth of Tat2-expressing cells was significantly impaired by neutral pH, that of MCT10-expressing cells was nearly unaffected. This property is consistent with the transport mechanism of MCT10 via facilitated diffusion without a need for pH gradient across the plasma membrane. Single-nucleotide polymorphisms (SNPs) are known to occur in the human MCT10 coding region. Among eight SNP amino acid changes in MCT10, the N81K mutation completely abrogated tryptophan import without any abnormalities in the expression or localization. In the MCT10 modeled structure, N81 appeared to protrude into the putative trajectory of tryptophan. Plasma membrane localization of MCT10 and the variant proteins was also verified in human embryonic kidney 293T cells.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Linhagem Celular , Membrana Celular/metabolismo , Células HEK293 , Humanos , Polimorfismo de Nucleotídeo Único/genética , Triptofano/metabolismo , Tirosina/metabolismo , Ubiquitina/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismoRESUMO
GM3, a major lipid component of the plasma membrane outer leaflet in mammalian cells, is synthesized in the luminal side of Golgi by ST3GAL5 protein (ST3G5), a type II membrane protein. Two strains of St3Gal5 knockout mice have been established for studies of GM3 physiological function: St3Gal5-Ex5-KO (lacking exon 5, which contains the catalytic domain of ST3G5), and St3Gal5-Ex3-KO (lacking exon 3, which contains the initiation codons). Results of the present study demonstrate that GM3 synthesis is still present, at a low level, in liver of St3Gal5-Ex3-KO mice. St3Gal5 has two mRNA transcriptional variants: a-type and b-type. When exon 3 is deleted, ST3G5 is not translated from a-type or b-type, as a result of initiation codon deletion or frame shift. Through NCBI database search and real-time PCR analyses of various mouse tissues, we identified a liver-specific St3Gal5 transcriptional variant (c-type) capable of producing artificial ST3G5 (M*-ST3G5) having GM3 synthase activity in the absence of exon 3. St3Gal5-Ex3-KO mice expressed c-type mRNA without exon 3 (c-type-/-) in liver. The transmembrane and catalytic domains of M*-ST3G5 translated from c-type-/- were identical to those from wild-type, although the cytoplasmic regions differed. Expression of M*-ST3G5 in embryonic fibroblasts derived from St3Gal5-Ex3-KO mice led to GM3 synthesis; M*-ST3G5 thus displayed enzyme activity in vivo. Taken together, our findings indicate that expression of liver-specific c-type variant accounts for the residual GM3 synthase activity observed in liver of St3Gal5-Ex3-KO mice.
Assuntos
Processamento Alternativo , Gangliosídeo G(M3)/biossíntese , Fígado/enzimologia , RNA Mensageiro/genética , Sialiltransferases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Éxons , Fibroblastos/citologia , Fibroblastos/enzimologia , Gangliosídeo G(M3)/genética , Expressão Gênica , Complexo de Golgi/enzimologia , Complexo de Golgi/genética , Íntrons , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Camundongos Knockout , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Sialiltransferases/metabolismoRESUMO
In the Golgi maturation model, the Golgi cisternae dynamically mature along a secretory pathway. In this dynamic process, glycosyltransferases are transported from the endoplasmic reticulum (ER) to the Golgi apparatus where they remain and function. The precise mechanism behind this maturation process remains unclear. We investigated two glycosyltransferases, ST3Gal5 (ST3G5) and B4GalNAcT1 (B4GN1), involved in ganglioside synthesis and examined their signal sequences for ER export and Golgi retention. Reports have suggested that the [R/K](X)[R/K] motif functions as an ER exporting signal; however, this signal sequence is insufficient in stably expressed, full-length ST3G5. Through further analysis, we have clarified that the (2)R(3)R(X)(5) (9)K(X)(3) (13)K sequence in ST3G5 is essential for ER export. We have named the sequence the R/K-based motif. On the other hand, for ER export of B4GN1, the homodimer formation in addition to the R/K-based motif is required for ER export suggesting the importance of unidentified lumenal side interaction. We found that ST3G5 R2A/R3A and K9A/K13A mutants localized not only in Golgi apparatus but also in endosomes. Furthermore, the amounts of mature type asparagine-linked (N)-glycans in ST3G5 R2A/R3A and K9A/K13A mutants were decreased compared with those in wild-type proteins, and the stability of the mutants was lower. These results suggest that the R/K-based motif is necessary for the Golgi retention of ST3G5 and that the retention is involved in the maturation of N-glycans and in stability. Thus, several basic amino acids located on the cytoplasmic tail of ST3G5 play important roles in both ER export and Golgi retention.
Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , N-Acetilgalactosaminiltransferases/metabolismo , Sinais Direcionadores de Proteínas , Sialiltransferases/metabolismo , Motivos de Aminoácidos , Animais , Arginina/genética , Células CHO , Cricetinae , Cricetulus , Lisina/genética , Camundongos , Mutação , N-Acetilgalactosaminiltransferases/química , N-Acetilgalactosaminiltransferases/genética , Estrutura Terciária de Proteína , Transporte Proteico , Sialiltransferases/química , Sialiltransferases/genéticaRESUMO
In Saccharomyces cerevisiae, high-affinity tryptophan import is performed by subtle mechanisms involving tryptophan permease Tat2. We have shown that Tat2 requires 15 amino acid residues in the transmembrane domains (TMDs) for its import activity, whereas leucine permease Bap2 requires only seven corresponding residues for its leucine import. For this reason, the structure of Tat2 is elaborately designed to transport the hydrophobic and bulky tryptophan. Newly synthesized cell surface proteins first undergo endoplasmic reticulum (ER)-associated quality check before entering the secretory pathway. In this study, we used domain replacement with general amino acid permease Gap1 to show that Tat2 chimeric proteins were dysfunctional when TMD10 or TMD11 was replaced. These chimeras formed large 270-800-kDa protein complexes and were stably retained in the ER membrane without efficient degradation. In contrast, Tat2 chimeras of TMD9 or TMD12 retained some of their tryptophan import activity and underwent vacuolar degradation as observed with wild-type Tat2. Thus, ours results suggest that TMD10 and TMD11 are essential for the correct folding of Tat2, probably because of their interdomain interactions. Notably, overexpression of Tat2-Gap1 chimera of TMD10 activated the unfolded protein response (UPR) element-lacZ reporter, suggesting that ER retention of the protein aggregates induces the UPR.
Assuntos
Sistemas de Transporte de Aminoácidos/genética , Dobramento de Proteína , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Resposta a Proteínas não Dobradas/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Retículo Endoplasmático/metabolismo , Estrutura Terciária de Proteína/genética , Transporte Proteico/genética , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The peptide transporter Ptr2 plays a central role in di- or tripeptide import in Saccharomyces cerevisiae. Although PTR2 transcription has been extensively analyzed in terms of upregulation by the Ubr1-Cup9 circuit, the structural and functional information for this transporter is limited. Here we identified 14 amino acid residues required for peptide import through Ptr2 based on the crystallographic information of Streptococcus thermophilus peptide transporter PepTst and based on the conservation of primary sequences among the proton-dependent oligopeptide transporters (POTs). Expression of Ptr2 carrying one of the 14 mutations of which the corresponding residues of PepTst are involved in peptide recognition, salt bridge interaction, or peptide translocation failed to enable ptr2Δtrp1 cell growth in alanyl-tryptophan (Ala-Trp) medium. We observed that Ptr2 underwent rapid degradation after cycloheximide treatment (half-life, approximately 1 h), and this degradation depended on Rsp5 ubiquitin ligase. The ubiquitination of Ptr2 most likely occurs at the N-terminal lysines 16, 27, and 34. Simultaneous substitution of arginine for the three lysines fully prevented Ptr2 degradation. Ptr2 mutants of the presumed peptide-binding site (E92Q, R93K, K205R, W362L, and E480D) exhibited severe defects in peptide import and were subjected to Rsp5-dependent degradation when cells were moved to Ala-Trp medium, whereas, similar to what occurs in the wild-type Ptr2, mutant proteins of the intracellular gate were upregulated. These results suggest that Ptr2 undergoes quality control and the defects in peptide binding and the concomitant conformational change render Ptr2 subject to efficient ubiquitination and subsequent degradation.
Assuntos
Sítios de Ligação/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Transporte Proteico/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Proteínas de Transporte/genética , Cristalografia por Raios X , Cicloeximida/farmacologia , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas de Membrana Transportadoras/ultraestrutura , Inibidores da Síntese de Proteínas/farmacologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Streptococcus thermophilus/genética , Streptococcus thermophilus/metabolismo , Ubiquitina/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , UbiquitinaçãoRESUMO
In the yeast Saccharomyces cerevisiae, structural diversities of complex sphingolipids [inositol phosphorylceramide (IPC), mannosylinositol phosphorylceramide, and mannosyldiinositol phosphorylceramide] are often observed in the presence or absence of hydroxyl groups on the C-4 position of long-chain base (C4-OH) and the C-2 position of very long-chain fatty acids (C2-OH), but the biological significance of these groups remains unclear. Here, we evaluated cellular membrane fluidity in hydroxyl group-defective yeast mutants by fluorescence recovery after photobleaching. The lateral diffusion of enhanced green fluorescent protein-tagged hexose transporter 1 (Hxt1-EGFP) was influenced by the absence of C4-OH and/or C2-OH. Notably, the fluorescence recovery of Hxt1-EGFP was dramatically decreased in the sur2Δ mutant (absence of C4-OH) under the csg1Δcsh1Δ background, in which mannosylation of IPC is blocked leading to IPC accumulation, while the recovery in the scs7Δ mutant (absence of C2-OH) under the same background was modestly decreased. In addition, the amount of low affinity tryptophan transporter 1 (Tat1)-EGFP was markedly decreased in the sur2Δcsg1Δcsh1Δ mutant and accumulated in intracellular membranes in the scs7Δcsg1Δcsh1Δ mutant without altering its protein expression. These results suggest that C4-OH and C2-OH are most probably critical factors for maintaining membrane fluidity and proper turnover of membrane molecules in yeast containing complex sphingolipids with only one hydrophilic head group.
Assuntos
Membrana Celular/metabolismo , Ceramidas/metabolismo , Proteínas de Membrana/metabolismo , Oxigenases de Função Mista/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Membrana Celular/genética , Ceramidas/genética , Proteínas de Membrana/genética , Oxigenases de Função Mista/genética , Mutação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The ganglioside GM4 is a sialic acid-containing glycosphingolipid mainly expressed in mammalian brain and erythrocytes. GM4 is synthesized by the sialylation of galactosylceramide (GalCer), while the ganglioside GM3 is synthesized by the sialylation of lactosylceramide (LacCer). Recently, the enzyme GM3 synthase was found to be responsible for the synthesis of GM4 in vitro and in vivo, yet the mechanism behind GM4 expression in cells remains unclear. In this study, we attempted to establish GM4-reconstituted cells to reveal the regulation of GM4 synthesis. Interestingly, GM4 was not detected in RPMI 1846 cells expressing LacCer, GalCer, and GM3. Similarly, GM4 was not detected in CHO-K1 cells, even when such cells expressing LacCer and GM3 were stably transfected with the GalCer synthase (GalCerS) gene. GM4 became detectable only when the GM3/GM4 synthase (GM3/GM4S, ST3GAL5) gene was overexpressed in either RPMI 1846 or CHO-K1/GalCerS cells. A mutant of the B16 melanoma cell line, GM-95, lacks GlcCer and LacCer, due to an absence of GlcCer synthase, but carries endogenous LacCer synthase and GM3/GM4S. GalCer became detectable after transfection of GalCerS into GM95 cells, but the GM95/GalCerS reconstituted cells did not express GM4, indicating that competition between the substrates LacCer and GalCer for GM3/GM4S does not cause the failure of GM4 synthesis. These results suggest that the expression machinery of GM4 under physiological conditions is independent from that of GM3.
Assuntos
Gangliosídeos/genética , Gangliosídeos/metabolismo , Regulação Enzimológica da Expressão Gênica , Sialiltransferases/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Técnicas de Transferência de Genes , Vetores Genéticos , Humanos , Microscopia de Fluorescência , Transdução de SinaisRESUMO
Cells of Saccharomyces cerevisiae express two tryptophan permeases, Tat1 and Tat2, which have different characteristics in terms of their affinity for tryptophan and intracellular localization. Although the high-affinity permease Tat2 has been well documented in terms of its ubiquitin-dependent degradation, the low-affinity permease Tat1 has not yet been characterized fully. Here we show that a high hydrostatic pressure of 25 MPa triggers a degradation of Tat1 which depends on Rsp5 ubiquitin ligase and the EH domain-containing protein End3. Tat1 was resistant to a 3-h cycloheximide treatment, suggesting that it is highly stable under normal growth conditions. The ubiquitination of Tat1 most likely occurs at N-terminal lysines 29 and 31. Simultaneous substitution of arginine for the two lysines prevented Tat1 degradation, but substitution of either of them alone did not, indicating that the roles of lysines 29 and 31 are redundant. When cells were exposed to high pressure, Tat1-GFP was completely lost from the plasma membrane, while substantial amounts of Tat1(K29R-K31R)-GFP remained. The HPG1-1 (Rsp5(P514T)) and rsp5-ww3 mutations stabilized Tat1 under high pressure, but any one of the rsp5-ww1, rsp5-ww2, and bul1Δ bul2Δ mutations or single deletions of genes encoding arrestin-related trafficking adaptors did not. However, simultaneous loss of 9-arrestins and Bul1/Bul2 prevented Tat1 degradation at 25 MPa. The results suggest that multiple PPxY motif proteins share some essential roles in regulating Tat1 ubiquitination in response to high hydrostatic pressure.
Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Endocitose , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Pressão Hidrostática , Proteólise , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Complexos Ubiquitina-Proteína Ligase/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Pressão Atmosférica , Espaço Intracelular/metabolismo , Lisina/metabolismo , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Triptofano/metabolismo , UbiquitinaçãoRESUMO
Lymphedema has become a global health issue following the growing number of cancer surgeries. Curative or supportive therapeutics have long been awaited for this refractory condition. Transcription factor GATA2 is crucial in lymphatic development and maintenance, as GATA2 haploinsufficient disease often manifests as lymphedema. We recently demonstrated that Gata2 heterozygous deficient mice displayed delayed lymphatic recanalization upon lymph node resection. However, whether GATA2 contributes to lymphatic regeneration by functioning in the damaged lymph vessels' microenvironment remains explored. In this study, our integrated analysis demonstrated that dermal collagen fibers were more densely accumulated in the Gata2 heterozygous deficient mice. The collagen metabolism-related transcriptome was perturbed, and collagen matrix contractile activity was aberrantly increased in Gata2 heterozygous embryonic fibroblasts. Notably, soluble collagen placement ameliorated delayed lymphatic recanalization, presumably by modulating the stiffness of the extracellular matrix around the resection site of Gata2 heterozygous deficient mice. Our results provide valuable insights into mechanisms underlying GATA2-haploinsufficiency-mediated lymphedema and shed light on potential therapeutic avenues for this intractable disease.
Assuntos
Colágeno , Fator de Transcrição GATA2 , Heterozigoto , Linfedema , Animais , Camundongos , Fator de Transcrição GATA2/metabolismo , Fator de Transcrição GATA2/genética , Linfedema/metabolismo , Linfedema/genética , Linfedema/patologia , Colágeno/metabolismo , Vasos Linfáticos/metabolismo , Vasos Linfáticos/patologia , Camundongos Knockout , Haploinsuficiência , Deficiência de GATA2/metabolismo , Deficiência de GATA2/genética , Camundongos Endogâmicos C57BLRESUMO
Hydrostatic pressure is a common mechanical stressor that modulates metabolism and reduces cell viability. Eukaryotic cells have genetic programs to cope with hydrostatic pressure stress and maintain intracellular homeostasis. However, the mechanism underlying hydrostatic pressure tolerance remains largely unknown. We have recently demonstrated that maintenance of telomere capping protein 6 (Mtc6) plays a protective role in the survival of the budding yeast Saccharomyces cerevisiae under hydrostatic pressure stress by supporting the integrity of nutrient permeases. The current study demonstrates that Mtc6 acts as an endoplasmic reticulum (ER) membrane protein. Mtc6 comprises two transmembrane domains, a C-terminal cytoplasmic domain and a luminal region with 12 Asn (N)-linked glycans attached to it. Serial mutational analyses showed that the cytoplasmic C-terminal amino acid residues GVPS Mtc6 activity. Multiple N-linked glycans in the luminal region are involved in the structural conformation of Mtc6. Moreover, deletion of MTC6 led to increased degradation of the leucine permease Bap2 under hydrostatic pressure, suggesting that Mtc6 facilitates the proper folding of nutrient permeases in the ER under stress conditions. We propose a novel model of molecular function in which the glycosylated luminal domain and cytoplasmic GVPS sequences of Mtc6 cooperatively support the nutrient permease activity.
Assuntos
Retículo Endoplasmático , Pressão Hidrostática , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Retículo Endoplasmático/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Glicoproteínas/metabolismo , Glicoproteínas/genéticaRESUMO
Hereditary spastic paraplegia (HSP) is a heterogeneous group of neurological disorders that are characterized by progressive spasticity and weakness in the lower limbs. SPG26 is a complicated form of HSP, which includes not only weakness in the lower limbs, but also cognitive impairment, developmental delay, cerebellar ataxia, dysarthria, and peripheral neuropathy, and is caused by biallelic mutations in the B4GALNT1 (beta-1,4-N-acetylgalactosaminyltransferase 1) gene. The B4GALNT1 gene encodes ganglioside GM2/GD2 synthase (GM2S), which catalyzes the transfer of N-acetylgalactosamine to lactosylceramide, GM3, and GD3 to generate GA2, GM2, and GD2, respectively. The present study attempted to characterize a novel B4GALNT1 variant (NM_001478.5:c.937G>A p.Asp313Asn) detected in a patient with progressive multi-system neurodegeneration as well as deleterious variants found in the general population in Japan. Peripheral blood T cells from our patient lacked the ability for activation-induced ganglioside expression assessed by cell surface cholera toxin binding. Structural predictions suggested that the amino acid substitution, p.Asp313Asn, impaired binding to the donor substrate UDP-GalNAc. An in vitro enzyme assay demonstrated that the variant protein did not exhibit GM2S activity, leading to the diagnosis of HSP26. This is the first case diagnosed with SPG26 in Japan. We then extracted 10 novel missense variants of B4GALNT1 from the whole-genome reference panel jMorp (8.3KJPN) of the Tohoku medical megabank organization, which were predicted to be deleterious by Polyphen-2 and SIFT programs. We performed a functional evaluation of these variants and demonstrated that many showed perturbed subcellular localization. Five of these variants exhibited no or significantly decreased GM2S activity with less than 10% activity of the wild-type protein, indicating that they are carrier variants for HSP26. These results provide the basis for molecular analyses of B4GALNT1 variants present in the Japanese population and will help improve the molecular diagnosis of patients suspected of having HSP.
RESUMO
Mast cells serve as a first-line defense of innate immunity. Interleukin-6 (IL-6) induced by bacterial lipopolysaccharide (LPS) in mast cells plays a crucial role in antibacterial protection. The zinc finger transcription factor GATA2 cooperatively functions with the ETS family transcription factor PU.1 in multiple mast cell activities. However, the regulatory landscape directed by GATA2 and PU.1 under inflammation remains elusive. We herein showed that a large proportion of GATA2-binding peaks were closely located with PU.1-binding peaks in distal cis-regulatory regions of inflammatory cytokine genes in mast cells. Notably, GATA2 and PU.1 played crucial roles in promoting LPS-mediated inflammatory cytokine production. Genetic ablation of GATA2-PU.1-clustered binding sites at the Il6 -39 kb region revealed its central role in LPS-induced Il6 expression in mast cells. We demonstrate a novel collaborative activity of GATA2 and PU.1 in cytokine induction upon inflammatory stimuli via the GATA2-PU.1 overlapping sites in the distal cis-regulatory regions.
RESUMO
We have previously reported that fish pathogens causing vibriosis specifically adhere to GM4 on the epithelial cells of fish intestinal tracts (Chisada, S., Horibata, Y., Hama, Y., Inagaki, M., Furuya, N., Okino, N., and Ito, M. (2005) Biochem. Biophys. Res. Commun. 333, 367-373). To identify the gene encoding the enzyme for GM4 synthesis in the fish intestinal tract, a phylogenetic tree of vertebrate ST3GalVs, including Danio rerio and Oryzias latipes, was generated in which two putative subfamilies of fish ST3GalVs were found. Two putative ST3GalVs of zebrafish (zST3GalV-1 and -2), each belonging to different subfamilies, were cloned from the zebrafish cDNA library. Interestingly, zST3GalV-1 synthesized GM3 (NeuAcalpha2-3Galbeta1-4Glcbeta1-1'Cer) but not GM4, whereas zSTGalV-2 synthesized both gangliosides in vitro when expressed in CHO-K1 and RPMI1846 cells. Flow cytometric analysis using anti-GM4 antibody revealed that the transformation of RPMI1846 cells with zST3GalV-2 but not zST3GalV-1 cDNA increased the cell-surface expression of GM4. Whole mount in situ hybridization showed that the zST3GalV-2 transcript was strongly expressed in the gastrointestinal tract, whereas zST3GalV-1 was expressed in the brain and esophagus but not gastrointestinal tract in 3-day post-fertilization embryos. It has long been a matter of controversy which enzyme is responsible for the synthesis of GM4 in mammals. We found that three isoforms of mouse ST3GalV (mST3GalV) having different N-terminal sequences can synthesize GM4 as well as GM3 when expressed in RPMI1846 and CHO-K1 cells. Furthermore, mST3GalV knock-out mice were found to lack GM4 synthase activity and GM4 in contrast to wild-type mice. These results clearly indicate that zST3GalV-2 and mST3GalV are the enzymes responsible for the synthesis of GM4 in zebrafish and mice, respectively.