Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 25
Filtrar
Mais filtros










Intervalo de ano de publicação
1.
Pediatr Transplant ; 25(4): e13992, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33813776

RESUMO

BACKGROUND: Medullary sponge kidney (MSK) disease predisposes patients to recurrent nephrolithiasis, which affects one in every 5000 people in the United States. METHODS: We report a rare case of a pediatric recipient of a living donor MSK transplant and discuss considerations when discussing risks and benefits of accepting MSK allografts for this population. RESULTS: The recipient was admitted due to concerns for nephrolithiasis, hydronephrosis, and urinary tract infection at 1-month post-transplant. The hydronephrosis was resolved by surgical removal of an encrusted ureteral stent; this was followed by supplementation with oral medications to prevent future episodes of nephrolithiasis. The recipient did not have any further episodes after this as seen at a 1-year follow-up. The donor has remained well through this period. CONCLUSIONS: With increasing organ shortages, the use of variety of donors may need to be considered to enlarge the organ pool.

2.
Plant Physiol ; 185(1): 240-255, 2021 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-33631806

RESUMO

In Arabidopsis (Arabidopsis thaliana), a hypersensitive-like response (HR-like response) is triggered underneath the eggs of the large white butterfly Pieris brassicae (P. brassicae), and this response is dependent on salicylic acid (SA) accumulation and signaling. Previous reports indicate that the clade I L-type LECTIN RECEPTOR KINASE-I.8 (LecRK-I.8) is involved in early steps of egg recognition. A genome-wide association study was used to better characterize the genetic structure of the HR-like response and discover loci that contribute to this response. We report here the identification of LecRK-I.1, a close homolog of LecRK-I.8, and show that two main haplotypes that explain part of the variation in HR-like response segregate among natural Arabidopsis accessions. Besides, signatures of balancing selection at this locus suggest that it may be ecologically important. Disruption of LecRK-I.1 results in decreased HR-like response and SA signaling, indicating that this protein is important for the observed responses. Furthermore, we provide evidence that LecRK-I.1 functions in the same signaling pathway as LecRK-I.8. Altogether, our results show that the response to eggs of P. brassicae is controlled by multiple LecRKs.


Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/imunologia , Morte Celular/genética , Morte Celular/imunologia , Imunidade Vegetal/genética , Imunidade Vegetal/imunologia , /imunologia , Animais , Arabidopsis/genética , Arabidopsis/imunologia , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Insetos/parasitologia , Óvulo
3.
Clin Transplant ; 34(9): e14040, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32654278

RESUMO

Prolonged warm (WIT) and cold (CIT) ischemia times are often important considerations in the discard of DCD kidneys, but their impact on post-transplant outcomes in the post-KAS era is unclear. We examined the association of ischemia time on delayed graft function (DGF) and death-censored graft failure for DCD kidneys. The 2018 SRTR SAF was utilized to identify post-KAS DCD kidney transplants occurring from 2015 to 2018. Relative risk and Cox regression were used to calculate risk of delayed graft function and hazard of death-censored graft failure, respectively. We identified 4,680 kidneys from DCD donors transplanted from 2015 to 2018 with recorded WIT and CIT times. Median WIT was 21.0 minutes (IQR 14.0-28.0), and CIT was 18.5 hours (IQR 13.9-23.5). The overall incidence of DGF was 42.7%. In a univariable relative risk regression model, extended CIT (24-30 hours:RR 1.37, 95% CI 1.15-1.77; >30 hours:RR 1.47, 95% CI 1.22-1.77) and WIT (20-40 minutes:RR 1.10, 95% CI 1.03-1.17) were associated with increased risk of DGF. When included in a multivariable model, neither prolonged CIT nor WIT were significantly associated with death-censored graft failure. Prolonged WIT and CIT are associated with increased DGF but not death-censored graft failure in recipients of DCD kidney transplants in the post-KAS era. Extended ischemia alone should not be used as a basis for discard or non-utilization of these organs.


Assuntos
Obtenção de Tecidos e Órgãos , Isquemia Quente , Morte , Função Retardada do Enxerto/etiologia , Sobrevivência de Enxerto , Humanos , Rim , Fatores de Risco , Doadores de Tecidos , Resultado do Tratamento , Isquemia Quente/efeitos adversos
4.
Biochim Biophys Acta Proteins Proteom ; 1868(11): 140512, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32731033

RESUMO

The genome of Entamoeba histolytica encodes approximately 50 Cysteine Proteases (CPs) whose activity is regulated by two Inhibitors of Cysteine Proteases (ICPs), EhICP1 and EhICP2. The main difference between both EhICPs is the acquisition of a 17 N-terminal targeting signal in EhICP2 and three exposed cysteine residues in EhICP1. The three exposed cysteines in EhICP1 potentiate the formation of cross-linking species that drive heterogeneity. Here we solved the NMR structure of EhICP1 using a mutant protein without accessible cysteines. Our structural data shows that EhICP1 adopts an immunoglobulin fold composed of seven ß-strands, and three solvent exposed loops that resemble the structures of EhICP2 and chagasin. EhICP1 and EhICP2 are able to inhibit the archetypical cysteine protease papain by intercalating their BC loops into the protease active site independently of the character of the residue (serine or threonine) responsible to interact with the active site of papain. EhICP1 and EhICP2 present signals of functional divergence as they clustered in different clades. Two of the three exposed cysteines in EhICP1 are located at the DE loop that intercalates into the CP substrate-binding cleft. We propose that the solvent exposed cysteines of EhICP1 play a role in regulating its inhibitory activity and that in oxidative conditions, the cysteines of EhICP1 react to form intra and intermolecular disulfide bonds that render an inactive inhibitor. EhICP2 is not subject to redox regulation, as this inhibitor does not contain a single cysteine residue. This proposed redox regulation may be related to the differential cellular localization between EhICP1 and EhICP2.


Assuntos
Entamoeba histolytica , Proteínas de Protozoários/química , Clonagem Molecular , Inibidores de Cisteína Proteinase , Entamoeba histolytica/genética , Escherichia coli/genética , Mutagênese Sítio-Dirigida , Papaína/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Soluções
5.
Methods Mol Biol ; 2151: 211-218, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32452007

RESUMO

Protein structure determination by X-ray crystallography guides structure-function and rational drug design studies. Helminths cause devastating diseases, including schistosomiasis that affects over one-third of the human population. Trematodes from the genus Schistosoma heavily depend on glycolysis; thus enzymes involved in this metabolic pathway are potential drug targets. Here we present a protocol to obtain crystal structures of recombinantly expressed triosephosphate isomerase from S. mansoni (SmTPI) that diffracted in house to a resolution of 2 Å.


Assuntos
Cristalografia por Raios X/métodos , Schistosoma mansoni/enzimologia , Triose-Fosfato Isomerase/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Cristalização , Expressão Gênica , Vetores Genéticos/metabolismo , Triose-Fosfato Isomerase/genética , Triose-Fosfato Isomerase/isolamento & purificação
6.
Biochim Biophys Acta Gen Subj ; 1864(7): 129608, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32234506

RESUMO

BACKGROUND: Mutations in human gene encoding the mitochondrial DNA polymerase γ (HsPolγ) are associated with a broad range of mitochondrial diseases. Here we studied the impact on DNA replication by disease variants clustered around residue HsPolγ-K1191, a residue that in several family-A DNA polymerases interacts with the 3' end of the primer. METHODS: Specifically, we examined the effect of HsPolγ carrying pathogenic variants in residues D1184, I1185, C1188, K1191, D1196, and a stop codon at residue T1199, using as a model the yeast mitochondrial DNA polymerase protein, Mip1p. RESULTS: The introduction of pathogenic variants C1188R (yV945R), and of a stop codon at residue T1199 (yT956X) abolished both polymerization and exonucleolysis in vitro. HsPolγ substitutions in residues D1184 (yD941), I1185 (yI942), K1191 (yK948) and D1196 (yD953) shifted the balance between polymerization and exonucleolysis in favor of exonucleolysis. HsPolγ pathogenic variants at residue K1191 (yK948) and D1184 (yD941) were capable of nucleotide incorporation albeit with reduced processivity. Structural analysis of mitochondrial DNAPs showed that residue HsPolγ-N864 is placed in an optimal distance to interact with the 3' end of the primer and the phosphate backbone previous to the 3' end. Amino acid changes in residue HsPolγ-N864 to Ala, Ser or Asp result in enzymes that did not decrease their polymerization activity on short templates but exhibited a substantial decrease for processive DNA synthesis. CONCLUSION: Our data suggest that in mitochondrial DNA polymerases multiple amino acids are involved in the primer-stand stabilization.


Assuntos
DNA Polimerase gama/genética , DNA Mitocondrial/metabolismo , Doenças Mitocondriais/metabolismo , DNA Polimerase gama/química , DNA Polimerase gama/metabolismo , Replicação do DNA/genética , DNA Mitocondrial/química , Humanos , Modelos Moleculares , Mutação
8.
PLoS Negl Trop Dis ; 14(1): e0007815, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31923219

RESUMO

Triosephosphate isomerases (TPIs) from Taenia solium (TsTPI) and Schistosoma mansoni (SmTPI) are potential vaccine and drug targets against cysticercosis and schistosomiasis, respectively. This is due to the dependence of parasitic helminths on glycolysis and because those proteins elicit an immune response, presumably due to their surface localization. Here we report the crystal structures of TsTPI and SmTPI in complex with 2-phosphoglyceric acid (2-PGA). Both TPIs fold into a dimeric (ß-α)8 barrel in which the dimer interface consists of α-helices 2, 3, and 4, and swapping of loop 3. TPIs from parasitic helminths harbor a region of three amino acids knows as the SXD/E insert (S155 to E157 and S157 to D159 in TsTPI and SmTPI, respectively). This insert is located between α5 and ß6 and is proposed to be the main TPI epitope. This region is part of a solvent-exposed 310-helix that folds into a hook-like structure. The crystal structures of TsTPI and SmTPI predicted conformational epitopes that could be used for vaccine design. Surprisingly, the epitopes corresponding to the SXD/E inserts are not the ones with the greatest immunological potential. SmTPI, but not TsTPI, habors a sole solvent exposed cysteine (SmTPI-S230) and alterations in this residue decrease catalysis. The latter suggests that thiol-conjugating agents could be used to target SmTPI. In sum, the crystal structures of SmTPI and TsTPI are a blueprint for targeted schistosomiasis and cysticercosis drug and vaccine development.


Assuntos
Schistosoma mansoni/enzimologia , Taenia solium/enzimologia , Triose-Fosfato Isomerase/química , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Desenho de Fármacos , Epitopos/química , Proteínas de Helminto/química , Vacinas
9.
Clin Transplant ; 33(12): e13715, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31541580

RESUMO

Therapeutic living donor nephrectomy is defined as a nephrectomy that is performed as therapy for an underlying medical condition. The patient directly benefits from having their kidney removed, but the kidney is deemed transplantable. The kidney is subsequently used as an allograft for an individual with advanced renal disease. Therapeutic donor nephrectomy can be successfully utilized for a heterogenous cohort of disease processes as both treatment for the donor and to increase the number of suitable organs available for transplantation. We describe four cases of therapeutic donor nephrectomy that were performed at our institution. Of the four cases, two patients elected to undergo therapeutic donor nephrectomy as treatment for loin pain hematuria syndrome; one after blunt abdominal trauma that resulted in complete proximal ureteral avulsion; and the fourth after being diagnosed with a small renal mass. Based on our data presented to the United Network for Organ Sharing Board of Directors (UNOS) in December 2015, living donor evaluation has been made simpler for patients electing to undergo therapeutic donor nephrectomy. UNOS eliminated the requirement for a psychosocial evaluation for these patients. As the organ shortage continues to limit transplantation, therapeutic donor nephrectomy should be considered when appropriate.


Assuntos
Transplante de Rim/métodos , Doadores Vivos/provisão & distribuição , Nefrectomia/métodos , Coleta de Tecidos e Órgãos/métodos , Feminino , Humanos , Pessoa de Meia-Idade , Usos Terapêuticos
10.
Protein J ; 38(4): 435-446, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31435809

RESUMO

Protease inhibitors are crucial for the control of proteolytic activity in different physiological processes. However, some inhibitors do not show canonical enzyme recognition of the enzyme under certain conditions. In this work, we present evidence that indicates the formation of an active complex between the protease bovine α-chymotrypsin and the Tepary bean protease inhibitor (TBPI). The composition of the active chymotrypsin-TBPI complex (AC) was confirmed by three different methods: size-exclusion chromatography, polyacrylamide gel electrophoresis (PAGE), and mass spectrometry. The kinetic parameters for the AC were similar to those of the enzyme alone, indicating that TBPI binding does not produce any large changes in chymotrypsin. The molecular model proposed here postulates that TBPI binds outside the active cleft of the protease, but near enough to hinder the binding of high molecular weight substrates into the active site. This model was experimentally supported by the inhibitory effect on casein as a substrate, and the unaltered protease activity when a small synthetic substrate was used. We also found that the formation of this complex provided the enzyme with extra stability in denaturing conditions or in the presence of a reducing agent. The chymotrypsin-TBPI complex exhibited higher stability, indicating that autolysis can be partially prevented. When the enzyme was first inactivated followed by the addition of the inhibitor, the activity of the protease was restored. We described a possible mechanism where a plant protease inhibitor binds outside the active site of the enzyme while increasing its stability.


Assuntos
Quimotripsina/química , Inibidores de Serino Proteinase/química , Animais , Bovinos , Quimotripsina/metabolismo , Cinética , Modelos Moleculares , Phaseolus/metabolismo , Ligação Proteica , Inibidores de Serino Proteinase/metabolismo
11.
Mitochondrion ; 49: 166-177, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31445096

RESUMO

Human and yeast mitochondrial DNA polymerases (DNAPs), POLG and Mip1, are related by evolution to bacteriophage DNAPs. However, mitochondrial DNAPs contain unique amino and carboxyl-terminal extensions that physically interact. Here we describe that N-terminal deletions in Mip1 polymerases abolish polymerization and decrease exonucleolytic degradation, whereas moderate C-terminal deletions reduce polymerization. Similarly, to the N-terminal deletions, an extended C-terminal deletion of 298 amino acids is deficient in nucleotide addition and exonucleolytic degradation of double and single-stranded DNA. The latter observation suggests that the physical interaction between the amino and carboxyl-terminal regions of Mip1 may be related to the spread of pathogenic POLG mutant along its primary sequence.


Assuntos
DNA Polimerase I/metabolismo , DNA Fúngico/biossíntese , DNA Mitocondrial/biossíntese , Proteínas Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Domínio Catalítico , DNA Polimerase I/genética , DNA Polimerase gama/genética , DNA Polimerase gama/metabolismo , DNA Fúngico/genética , DNA Mitocondrial/genética , Humanos , Proteínas Mitocondriais/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
12.
Plant J ; 99(5): 950-964, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31034710

RESUMO

Reactive oxidative species (ROS) and S-glutathionylation modulate the activity of plant cytosolic triosephosphate isomerases (cTPI). Arabidopsis thaliana cTPI (AtcTPI) is subject of redox regulation at two reactive cysteines that function as thiol switches. Here we investigate the role of these residues, AtcTPI-Cys13 and At-Cys218, by substituting them with aspartic acid that mimics the irreversible oxidation of cysteine to sulfinic acid and with amino acids that mimic thiol conjugation. Crystallographic studies show that mimicking AtcTPI-Cys13 oxidation promotes the formation of inactive monomers by reposition residue Phe75 of the neighboring subunit, into a conformation that destabilizes the dimer interface. Mutations in residue AtcTPI-Cys218 to Asp, Lys, or Tyr generate TPI variants with a decreased enzymatic activity by creating structural modifications in two loops (loop 7 and loop 6) whose integrity is necessary to assemble the active site. In contrast with mutations in residue AtcTPI-Cys13, mutations in AtcTPI-Cys218 do not alter the dimeric nature of AtcTPI. Therefore, modifications of residues AtcTPI-Cys13 and AtcTPI-Cys218 modulate AtcTPI activity by inducing the formation of inactive monomers and by altering the active site of the dimeric enzyme, respectively. The identity of residue AtcTPI-Cys218 is conserved in the majority of plant cytosolic TPIs, this conservation and its solvent-exposed localization make it the most probable target for TPI regulation upon oxidative damage by reactive oxygen species. Our data reveal the structural mechanisms by which S-glutathionylation protects AtcTPI from irreversible chemical modifications and re-routes carbon metabolism to the pentose phosphate pathway to decrease oxidative stress.


Assuntos
Arabidopsis/enzimologia , Citosol/enzimologia , Citosol/metabolismo , Triose-Fosfato Isomerase/química , Triose-Fosfato Isomerase/metabolismo , Sequência de Aminoácidos , Arabidopsis/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oxirredução , Conformação Proteica , Espécies Reativas de Oxigênio , Triose-Fosfato Isomerase/genética
13.
Sci Rep ; 9(1): 2591, 2019 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-30796308

RESUMO

There is no structural information about any chitinase synthesized by Bacillus thuringiensis, the most successful microbial insect larvicide used worldwide. In this study, we solved the 3D structure of the chitinase ChiA74 at 2.26 Å. The crystal structure shows that ChiA74 is composed of a modular arrangement formed by (i) a catalytic region (CD), (ii) a chitinase insertion domain (CID), (iii) a fibronectin type III domain (FnIII), and (iv) a chitin binding domain (CBD). The location of the CBD with respect to the CD has no structural similarity to other chitinases with known structures. The activity of a ChiA74 lacking its secretion signal peptide (ChiA74Δsp) and a truncated version lacking its CBD/FnIII domains (ChiA74Δsp-50) did not have statistical differences in activity against colloidal chitin. However, ChiA74Δsp exhibits 4.5 and 2.0 higher activity than versions lacking the CBD (ChiA74Δsp-60) and CBD/FnIII domains (ChiA74Δsp-50), respectively, when crystalline chitin was used as substrate. Our data suggest that the CBD might plays a significant role in crystalline chitin hydrolysis. We also demonstrated the importance of the catalytic E211 in the CD, as mutants ChiA74ΔspE211N and ChiA74ΔspD207N, E211N were inactive against colloidal and crystalline chitins, chitosan and 4-MU-GlcNAc3. ChiA74 has a processive activity producing oligosaccharides with degree of polymerization (DP) of 1 (GlcNAc) and 2 (GlcNAc2).


Assuntos
Bacillus thuringiensis/enzimologia , Proteínas de Bactérias/química , Quitinases/química , Quitina/metabolismo , Hidrólise , Cinética , Domínios Proteicos , Especificidade por Substrato
14.
Transpl Int ; 32(7): 702-709, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30721545

RESUMO

Most transplant centers decline morbidly obese people for living kidney donation. Their inclusion in the living donor pool after weight loss and reversal of comorbidities by bariatric surgery could reverse the downward living donation trend. We investigated whether bariatric surgery in the morbidly obese altered their candidacy for donation, complicated their subsequent donor nephrectomy, and impacted their early postoperative outcomes in a series of 22 donors who had bariatric surgery 0.7-22 years prior to laparoscopic living donor nephrectomy. Eighteen would have been excluded from donation prior to bariatric surgery based on a body mass index (BMI) > 40. Seventeen reached a BMI < 35 after bariatric surgery. One had hypertension that resolved after bariatric surgery. Prior bariatric surgery did not influence port placement and laterality of donor nephrectomy. None required open conversion or blood transfusion. In an exploratory comparison with 37 donors with a BMI 35-40, length of stay and warm ischemic time were shorter, blood loss and postoperative complications were similar, and operative time was longer. We therefore advocate the consideration of bariatric surgery in preparation for donation in morbidly obese people since it positively alters their candidacy without major impact on the subsequent living donor nephrectomy and early outcomes.


Assuntos
Cirurgia Bariátrica , Seleção do Doador , Transplante de Rim/métodos , Doadores Vivos , Nefrectomia , Obesidade Mórbida/complicações , Obesidade Mórbida/cirurgia , Adulto , Índice de Massa Corporal , Feminino , Humanos , Laparoscopia , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias , Período Pós-Operatório , Período Pré-Operatório , Estudos Retrospectivos , Coleta de Tecidos e Órgãos , Resultado do Tratamento
15.
Clin Transplant ; 33(3): e13483, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30661276

RESUMO

Chylous ascites is a difficult, albeit uncommon complication of laparoscopic live donor nephrectomy (LLDN). Lymphatic leak is believed to be a result of injury to the cisterna chyli, regional lymph nodes, or other peri-aortic lymphatics intraoperatively. Recommended management with dietary modifications can result in malnutrition and immunodeficiency. We present four patients who developed chylous ascites following LLDN. Approach to these patients evolved over time. Our initial two patients were successfully treated with a combination of surgical intervention followed by drain placement, after the failure of conservative management. The latter two cases were successfully treated with prompt intra-abdominal drain placement, without dietary modifications. Our cohort challenges the standard of care for treatment of chylous ascites after LLDN. We believe that prompt diagnosis and placement of an intra-abdominal drain can be used safely in select patients that develop this complication. We hypothesize that continuously draining the lymphatic leak, thus avoiding the re-accumulation of ascites, allows bowel and mesentery to make contact and adhere to the retroperitoneal tissue. We believe that prompt, initial, percutaneous drain placement is a viable alternative to both conservative and reoperative management in the treatment of chylous ascites after LLDN and should be considered as a reasonable first-line therapy.


Assuntos
Ascite Quilosa/terapia , Drenagem , Laparoscopia/efeitos adversos , Doadores Vivos/provisão & distribuição , Nefrectomia/efeitos adversos , Reoperação , Coleta de Tecidos e Órgãos/métodos , Adulto , Idoso , Ascite Quilosa/etiologia , Ascite Quilosa/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico
16.
Front Mol Biosci ; 5: 103, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30538993

RESUMO

In plants, the ancestral cyanobacterial triosephosphate isomerase (TPI) was replaced by a duplicated version of the cytosolic TPI. This isoform acquired a transit peptide for chloroplast localization and functions in the Calvin-Benson cycle. To gain insight into the reasons for this gene replacement in plants, we characterized the TPI from the photosynthetic bacteria Synechocystis (SyTPI). SyTPI presents typical TPI enzyme kinetics profiles and assembles as a homodimer composed of two subunits that arrange in a (ß-α)8 fold. We found that oxidizing agents diamide (DA) and H2O2, as well as thiol-conjugating agents such as oxidized glutathione (GSSG) and methyl methanethiosulfonate (MMTS), do not inhibit the catalytic activity of SyTPI at concentrations required to inactivate plastidic and cytosolic TPIs from the plant model Arabidopsis thaliana (AtpdTPI and AtcTPI, respectively). The crystal structure of SyTPI revealed that each monomer contains three cysteines, C47, C127, and C176; however only the thiol group of C176 is solvent exposed. While AtcTPI and AtpdTPI are redox-regulated by chemical modifications of their accessible and reactive cysteines, we found that C176 of SyTPI is not sensitive to redox modification in vitro. Our data let us postulate that SyTPI was replaced by a eukaryotic TPI, because the latter contains redox-sensitive cysteines that may be subject to post-translational modifications required for modulating TPI's enzymatic activity.

17.
Clin Transplant ; 32(12): e13427, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30329179

RESUMO

BACKGROUND: Discard rate of Public Health Service Increased Risk (PHS-IR) organs is high despite the absence of worse kidney transplant outcomes. METHODS: We conducted a retrospective, single-center study of PHS-IR kidney offers made to kidney transplant-only potential recipients from 6/2004 to 5/2015. Overall mortality and transplant outcomes between potential recipients were stratified by response to PHS-IR kidney offers. Cox regression and Kaplan-Meier analyses of mortality and allograft failure were performed. RESULTS: A total of 2423 potential recipients were offered a PHS-IR kidney, with 1502 transplanted, with or without a PHS-IR kidney. Predictors of accepting a PHS-IR kidney included higher Estimated Post Transplant Survival (EPTS) score, prior kidney transplant, and lower educational achievement on multivariable analysis (P = 0.025, P = 0.004, P = 0.023). A positive response to a PHS-IR kidney was associated with lower risk of mortality (3.63% vs 11.6%; aHR 0.467, P = 0.0008). PHS-IR kidney recipients had decreased risk of allograft loss compared to non-PHS-IR recipients (P = 0.007), though mortality outcomes were not significantly different based on PHS-IR status (P = 0.38). No transmission of HIV, HBV, or HCV occurred from PHS-IR kidney donors in this cohort. CONCLUSIONS: Efforts must be made to increase awareness of the beneficial outcomes of PHS-IR organs to maximize appropriate donor allocation.


Assuntos
Seleção do Doador/normas , Falência Renal Crônica/mortalidade , Transplante de Rim/mortalidade , Transplante de Rim/normas , Doadores de Tecidos/provisão & distribuição , Obtenção de Tecidos e Órgãos/normas , Listas de Espera/mortalidade , Adolescente , Adulto , Feminino , Seguimentos , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Falência Renal Crônica/cirurgia , Masculino , Pessoa de Meia-Idade , Segurança do Paciente , Prognóstico , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , Taxa de Sobrevida , Transplantados/estatística & dados numéricos , Estados Unidos , United States Public Health Service , Adulto Jovem
18.
Proteins ; 86(7): 802-812, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29696695

RESUMO

Antibodies recognize protein targets with great affinity and specificity. However, posttranslational modifications and the presence of intrinsic disulfide-bonds pose difficulties for their industrial use. The immunoglobulin fold is one of the most ubiquitous folds in nature and it is found in many proteins besides antibodies. An example of a protein family with an immunoglobulin-like fold is the Cysteine Protease Inhibitors (ICP) family I42 of the MEROPs database for protease and protease inhibitors. Members of this protein family are thermostable and do not present internal disulfide bonds. Crystal structures of several ICPs indicate that they resemble the Ig-like domain of the human T cell co-receptor CD8α As ICPs present 2 flexible recognition loops that vary accordingly to their targeted protease, we hypothesize that members of this protein family would be ideal to design peptide aptamers that mimic protein-protein interactions. Herein, we use an ICP variant from Entamoeba histolytica (EhICP1) to mimic the interaction between p53 and MDM2. We found that a 13 amino-acid peptide derived from p53 can be introduced in 2 variable loops (DE, FG) but not the third (BC). Chimeric EhICP1-p53 form a stable complex with MDM2 at a micromolar range. Crystal structure of the EhICP1-p53(FG)-loop variant in complex with MDM2 reveals a swapping subdomain between 2 chimeric molecules, however, the p53 peptide interacts with MDM2 as in previous crystal structures. The structural details of the EhICP1-p53(FG) interaction with MDM2 resemble the interaction between an antibody and MDM2.


Assuntos
Domínios de Imunoglobulina , Modelos Moleculares , Proteínas Proto-Oncogênicas c-mdm2/química , Proteína Supressora de Tumor p53/química , Inibidores de Cisteína Proteinase/metabolismo , Entamoeba histolytica/química , Humanos , Ligação Proteica , Conformação Proteica , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
19.
Biochim Biophys Acta Proteins Proteom ; 1865(11 Pt A): 1423-1432, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28803140

RESUMO

The protozoan parasite Trichomonas vaginalis contains two nearly identical triosephosphate isomerases (TvTIMs) that dissociate into stable monomers and dimerize upon substrate binding. Herein, we compare the role of the "ball and socket" and loop 3 interactions in substrate assisted dimer assembly in both TvTIMs. We found that point mutants at the "ball" are only 39 and 29-fold less catalytically active than their corresponding wild-type counterparts, whereas Δloop 3 deletions are 1502 and 9400-fold less active. Point and deletion mutants dissociate into stable monomers. However, point mutants assemble as catalytic competent dimers upon binding of the transition state substrate analog PGH, whereas loop 3 deletions remain monomeric. A comparison between crystal structures of point and loop 3 deletion monomeric mutants illustrates that the catalytic residues in point mutants and wild-type TvTIMs are maintained in the same orientation, whereas the catalytic residues in deletion mutants show an increase in thermal mobility and present structural disorder that may hamper their catalytic role. The high enzymatic activity present in monomeric point mutants correlates with the formation of dimeric TvTIMs upon substrate binding. In contrast, the low activity and lack of dimer assembly in deletion mutants suggests a role of loop 3 in promoting the formation of the active site as well as dimer assembly. Our results suggest that in TvTIMs the active site is assembled during dimerization and that the integrity of loop 3 and ball and socket residues is crucial to stabilize the dimer.


Assuntos
Sequência de Bases , Ácidos Hidroxâmicos/química , Proteínas de Protozoários/química , Deleção de Sequência , Trichomonas vaginalis/enzimologia , Triose-Fosfato Isomerase/química , Motivos de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Teste de Complementação Genética , Ácidos Hidroxâmicos/metabolismo , Cinética , Modelos Moleculares , Mutação Puntual , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Secundária de Proteína , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Termodinâmica , Trichomonas vaginalis/química , Triose-Fosfato Isomerase/genética , Triose-Fosfato Isomerase/metabolismo
20.
Dev Cell ; 41(5): 555-570.e3, 2017 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-28586647

RESUMO

Low inorganic phosphate (Pi) availability causes terminal differentiation of the root apical meristem (RAM), a phenomenon known as root meristem exhaustion or determined growth. Here, we report that the CLE14 peptide acts as a key player in this process. Low Pi stress induces iron mobilization in the RAM through the action of LPR1/LPR2, causing expression of CLE14 in the proximal meristem region. CLV2 and PEPR2 receptors perceive CLE14 and trigger RAM differentiation, with concomitant downregulation of SHR/SCR and PIN/AUXIN pathway. Our results reveal multiple steps of the molecular mechanism of one of the most physiologically important root nutrient responses.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Diferenciação Celular , Ferro/metabolismo , Proteínas de Membrana/metabolismo , Meristema/crescimento & desenvolvimento , Fosfatos/deficiência , Raízes de Plantas/crescimento & desenvolvimento , /metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Membrana/genética , Meristema/metabolismo , Raízes de Plantas/metabolismo , Transdução de Sinais
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...