RESUMO
Potassium-coupled chloride transporters (KCCs) play crucial roles in regulating cell volume and intracellular chloride concentration. They are characteristically inhibited under isotonic conditions via phospho-regulatory sites located within the cytoplasmic termini. Decreased inhibitory phosphorylation in response to hypotonic cell swelling stimulates transport activity, and dysfunction of this regulatory process has been associated with various human diseases. Here, we present cryo-EM structures of human KCC3b and KCC1, revealing structural determinants for phospho-regulation in both N- and C-termini. We show that phospho-mimetic KCC3b is arrested in an inward-facing state in which intracellular ion access is blocked by extensive contacts with the N-terminus. In another mutant with increased isotonic transport activity, KCC1Δ19, this interdomain interaction is absent, likely due to a unique phospho-regulatory site in the KCC1 N-terminus. Furthermore, we map additional phosphorylation sites as well as a previously unknown ATP/ADP-binding pocket in the large C-terminal domain and show enhanced thermal stabilization of other CCCs by adenine nucleotides. These findings provide fundamentally new insights into the complex regulation of KCCs and may unlock innovative strategies for drug development.
Assuntos
Cloretos/metabolismo , Nucleotídeos/metabolismo , Potássio/metabolismo , Simportadores/metabolismo , Animais , Linhagem Celular , Tamanho Celular , Humanos , Fosforilação/fisiologia , Células Sf9 , Transdução de Sinais/fisiologia , Cotransportadores de K e Cl-RESUMO
Bone turnover diseases are exceptionally prevalent in human and come with a high burden on physical health. While these diseases are associated with a variety of risk factors and causes, they are all characterized by common denominators, that is, abnormalities in the function or number of osteoblasts, osteoclasts, and/or osteocytes. As such, much effort has been deployed in the recent years to understand the signaling mechanisms of bone cell proliferation and differentiation with the objectives of exploiting the intermediates involved as therapeutic preys. Ion transport systems at the external and in the intracellular membranes of osteoblasts and osteoclasts also play an important role in bone turnover by coordinating the movement of Ca2+ , PO4 2- , and H+ ions in and out of the osseous matrix. Even if they sustain the terminal steps of osteoformation and osteoresorption, they have been the object of very little attention in the last several years. Members of the cation-Cl- cotransporter (CCC) family are among the systems at work as they are expressed in bone cells, are known to affect the activity of Ca2+ -, PO4 2- -, and H+ -dependent transport systems and have been linked to bone mass density variation in human. In this review, the roles played by the CCCs in bone remodeling will be discussed in light of recent developments and their potential relevance in the treatment of skeletal disorders.
Assuntos
Osteócitos , Simportadores , Humanos , Cátions/metabolismo , Transporte de Íons/fisiologia , Osteócitos/metabolismo , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Simportadores/metabolismo , Remodelação Óssea , Densidade ÓsseaRESUMO
Huntington's disease (HD) is a monogenic neurodegenerative disorder resulting from a mutation in the huntingtin gene. This leads to the expression of the mutant huntingtin protein (mHTT) which provokes pathological changes in both the central nervous system (CNS) and periphery. Accumulating evidence suggests that mHTT can spread between cells of the CNS but here, we explored the possibility that mHTT could also propagate and cause pathology via the bloodstream. For this, we used a parabiosis approach to join the circulatory systems of wild-type (WT) and zQ175 mice. After surgery, we observed mHTT in the plasma and circulating blood cells of WT mice and post-mortem analyses revealed the presence of mHTT aggregates in several organs including the liver, kidney, muscle and brain. The presence of mHTT in the brain was accompanied by vascular abnormalities, such as a reduction of Collagen IV signal intensity and altered vessel diameter in the striatum, and changes in expression of Glutamic acid decarboxylase 65/67 (GAD65-67) in the cortex. Conversely, we measured reduced pathology in zQ175 mice by decreased mitochondrial impairments in peripheral organs, restored vessel diameter in the cortex and improved expression of Dopamine- and cAMP-regulated phosphoprotein 32 (DARPP32) in striatal neurons. Collectively, these results demonstrate that circulating mHTT can disseminate disease, but importantly, that healthy blood can dilute pathology. These findings have significant implications for the development of therapies in HD.
Assuntos
Doença de Huntington , Animais , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Fosfoproteína 32 Regulada por cAMP e Dopamina , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/genética , Camundongos , Camundongos Transgênicos , Neurônios/metabolismoRESUMO
Na+ -K+ -Cl- cotransporter 2 (NKCC2; SLC12A1) is an integral membrane protein that comes as three splice variants and mediates the cotranslocation of Na+ , K+ , and Cl- ions through the apical membrane of the thick ascending loop of Henle (TALH). In doing so, and through the involvement of other ion transport systems, it allows this nephron segment to reclaim a large fraction of the ultrafiltered Na+ , Cl- , Ca2+ , Mg2+ , and HCO3- loads. The functional relevance of NKCC2 in human is illustrated by the many abnormalities that result from the inactivation of this transport system through the use of loop diuretics or in the setting of inherited disorders. The following presentation aims at discussing the physiological roles and molecular characteristics of Na+ -K+ -Cl- cotransport in the TALH and those of the individual NKCC2 splice variants more specifically. Many of the historical and recent data that have emerged from the experiments conducted will be outlined and their larger meaning will also be placed into perspective with the aid of various hypotheses.
Assuntos
Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Processamento Alternativo/genética , Sequência de Aminoácidos , Animais , Humanos , Transporte de Íons , Alça do Néfron/metabolismo , Modelos Biológicos , Membro 3 da Família 12 de Carreador de Soluto/química , Membro 3 da Família 12 de Carreador de Soluto/genéticaRESUMO
Silicon (Si) supplementation has been shown to improve plant tolerance to different stresses, and its accumulation in the aerial organs is mediated by NIP2;1 aquaporins (Lsi channels) and Lsi2-type exporters in roots. In the present study, we tested the hypothesis that grapevine expresses a functional NIP2;1 that accounts for root Si uptake and, eventually, Si accumulation in leaves. Own-rooted grapevine cuttings of the cultivar Vinhão accumulated >0.2% Si (DW) in leaves when irrigated with 1.5 mM Si for 1 month, while Si was undetected in control leaves. Real-time PCR showed that VvNIP2;1 was highly expressed in roots and in green berries. The transient transformation of tobacco leaf epidermal cells mediated by Agrobacterium tumefaciens confirmed VvNIP2;1 localization at the plasma membrane. Transport experiments in oocytes showed that VvNIP2;1 mediates Si and arsenite uptake, whereas permeability studies revealed that VvNIP2;1 expressed in yeast is unable to transport water and glycerol. Si supplementation to pigmented grape cultured cells (cv. Gamay Freáux) had no impact on the total phenolic and anthocyanin content, or on the growth rate and VvNIP2;1 expression. Long-term experiments should help determine the extent of Si uptake over time and whether grapevine can benefit from Si fertilization.
Assuntos
Aquaporinas , Vitis , Aquaporinas/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Silício/metabolismo , Vitis/genética , Vitis/metabolismoRESUMO
Na+-K+-Cl- cotransporter type 2 (NKCC2) is confined to the apical membrane of the thick ascending limb of Henle, where it reabsorbs a substantial fraction of the ultrafiltered NaCl load. It is expressed along this nephron segment as three main splice variants (called NKCC2A, NKCC2B, and NKCC2F) that differ in residue composition along their second transmembrane domain and first intracellular cytosolic connecting segment (CS2). NKCC2 is known to be activated by cell shrinkage and intracellular [Cl-] reduction. Although the with no lysine (WNK) kinases could play a role in this response, the mechanisms involved are ill defined, and the possibility of variant-specific responses has not been tested thus far. In this study, we have used the Xenopus laevis oocyte expression system to gain further insight in these regards. We have found for the first time that cell shrinkage could stimulate NKCC2A- and NKCC2B-mediated ion transport by increasing carrier abundance at the cell surface and that this response was achieved (at least in part) by the enzymatic function of a WNK kinase. Interestingly, we have also found that the activity and cell surface abundance of NKCC2F were less affected by cell shrinkage compared with the other variants and that ion transport by certain variants could be stimulated through WNK kinase expression in the absence of carrier redistribution. Taken together, these results suggest that the WNK kinase-dependent pathway can affect both the trafficking as well as intrinsic activity of NKCC2 and that CS2 plays an important role in carrier regulation.
Assuntos
Rim/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Reabsorção Renal , Membro 1 da Família 12 de Carreador de Soluto/metabolismo , Proteína Quinase 1 Deficiente de Lisina WNK/metabolismo , Animais , Tamanho Celular , Endocitose , Glicosilação , Transporte de Íons , Cinética , Camundongos , Oócitos , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico , Coelhos , Membro 1 da Família 12 de Carreador de Soluto/genética , Proteína Quinase 1 Deficiente de Lisina WNK/genética , Xenopus laevisRESUMO
KEY POINTS: Na+ -K+ -Cl- cotransporter type 2 (NKCC2) is a 27-exon membrane protein that is expressed in the thick ascending limb (TAL) of Henle where it is involved in reabsorption of the ultrafiltered NaCl load. It comes as three splice variants that are identical to each other except for the residue composition of exon 4 and that differ in their transport characteristics, functional roles and distributions along the TAL. In this report, it is shown that the variants also differ in their trafficking properties and that two residues in exon 4 play a key role in this regard. One of these residues was also shown to sustain carrier internalization. Through these results, a novel function for the alternatively spliced exon of NKCC2 has been identified and a domain that is involved in carrier trafficking has been uncovered for the first time in a cation-Cl- cotransporter family member. ABSTRACT: Na+ -K+ -Cl- cotransporter type 2 (NKCC2) is a 12-transmembrane (TM) domain cell surface glycoprotein that is expressed in the thick ascending limb (TAL) of Henle and stimulated during cell shrinkage. It comes as three splice variants (A, B and F) that are identical to each other except for TM2 and the following connecting segment (CS2). Yet, these variants do not share the same localization, transport characteristics and physiological roles along the TAL. We have recently found that while cell shrinkage could exert its activating effect by increasing NKCC2 expression at the cell surface, the variants also responded differentially to this stimulus. In the current work, a mutagenic approach was exploited to determine whether CS2 could play a role in carrier trafficking and identify the residues potentially involved. We found that when the residue of position 238 in NKCC2A (F) and NKCC2B (Y) was replaced by the corresponding residue in NKCC2F (V), carrier activity increased by over 3-fold and endocytosis decreased concomitantly. We also found that when the residue of position 230 in NKCC2F (M) was replaced by the one in NKCC2B (T), carrier activity and affinity for ions both increased substantially whereas expression at the membrane decreased. Taken together, these results suggest that CS2 is involved in carrier trafficking and that two of its residues, those of positions 238 and 230, are part of an internalization motif. They also indicate that the divergent residue of position 230 plays the dual role of specifying ion affinity and sustaining carrier internalization.
Assuntos
Simportadores de Cloreto de Sódio-Potássio/metabolismo , Processamento Alternativo , Animais , Sequência de Bases , Membrana Celular , Éxons , Regulação da Expressão Gênica/fisiologia , Oócitos , Conformação Proteica , Transporte Proteico/fisiologia , Simportadores de Cloreto de Sódio-Potássio/classificação , Simportadores de Cloreto de Sódio-Potássio/genética , Xenopus laevisRESUMO
The K+ -Cl- cotransporters (KCCs) belong to the cation-Cl- cotransporter family and consist of four isoforms and many splice variants. Their main role is to promote electroneutral efflux of K+ and Cl- ions across the surface of many cell types and, thereby, to regulate intracellular ion concentration, cell volume, and epithelial salt movement. These transport systems are induced by an increase in cell volume and are less active at lower intracellular [Cl- ] (Cli ), but the mechanisms at play are still ill-defined. In this work, we have exploited the Xenopus laevis expression system to study the role of lysine-deficient protein kinases (WNKs), protein phosphatases 1 (PP1s), and SPS1-related proline/alanine-rich kinase (SPAK) in KCC4 regulation during cell swelling. We have found that WNK4 and PP1 regulate KCC4 activity as part of a common signaling module, but that they do not exert their effects through SPAK or carrier dephosphorylation. We have also found that the phosphatases at play include PP1α and PP1γ1, but that WNK4 acts directly on the PP1s instead of the opposite. Unexpectedly, however, both cell swelling and a T926A substitution in the C-terminus of full-length KCC4 led to higher levels of heterologous K+ -Cl- cotransport and overall carrier phosphorylation. These results imply that the response to cell swelling must also involve allosteric-sensitive kinase-dependent phosphoacceptor sites in KCC4. They are thus partially inconsistent with previous models of KCC regulation.
Assuntos
Tamanho Celular , Proteína Fosfatase 1/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Simportadores/metabolismo , Animais , Tamanho Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Toxinas Marinhas , Mutação , Oxazóis/farmacologia , Fosforilação , Proteína Fosfatase 1/antagonistas & inibidores , Proteína Fosfatase 1/genética , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Simportadores/efeitos dos fármacos , Simportadores/genética , Xenopus laevis , Cotransportadores de K e Cl-RESUMO
Silicon (Si) is increasingly recognized as an essential trace element in animals, especially since the identification of mammalian Si transport systems and Si responsive genes not long ago. During many years, however, efforts to gain substantial insight into the biological role of this element in animals have achieved partial success due in part to the unavailability of validated protocols to study Si movement across biological membranes. To circumvent such limitations, we have developed a general transport assay in which cellular Si content was determined by automated electrothermal atomic absorption spectrophotometry. We have found this assay to provide great analytic sensitivity with Si detection thresholds of less than 1 µM, that is, below or very close to the concentration range of animal cells. We have also found this assay to provide valid and cost-effective determinations in Si transport studies while requiring workable quantities of samples. The protocol described here should thus become gold standard toward accelerated progress in the field of Si transport.
Assuntos
Aquaporinas/genética , Membrana Celular/metabolismo , Silício/metabolismo , Oligoelementos/metabolismo , Animais , Transporte Biológico/genética , Membrana Celular/genética , Regulação da Expressão Gênica , Células HEK293 , Humanos , Oócitos/citologia , Silício/química , Espectrofotometria Atômica , Oligoelementos/química , Xenopus laevis/metabolismoRESUMO
Silicon (Si) has long been known to play a major physiological and structural role in certain organisms, including diatoms, sponges, and many higher plants, leading to the recent identification of multiple proteins responsible for Si transport in a range of algal and plant species. In mammals, despite several convincing studies suggesting that silicon is an important factor in bone development and connective tissue health, there is a critical lack of understanding about the biochemical pathways that enable Si homeostasis. Here we report the identification of a mammalian efflux Si transporter, namely Slc34a2 (also termed NaPiIIb), a known sodium-phosphate cotransporter, which was upregulated in rat kidney following chronic dietary Si deprivation. Normal rat renal epithelium demonstrated punctate expression of Slc34a2, and when the protein was heterologously expressed in Xenopus laevis oocytes, Si efflux activity (i.e., movement of Si out of cells) was induced and was quantitatively similar to that induced by the known plant Si transporter OsLsi2 in the same expression system. Interestingly, Si efflux appeared saturable over time, but it did not vary as a function of extracellular [Formula: see text] or Na+ concentration, suggesting that Slc34a2 harbors a functionally independent transport site for Si operating in the reverse direction to the site for phosphate. Indeed, in rats with dietary Si depletion-induced upregulation of transporter expression, there was increased urinary phosphate excretion. This is the first evidence of an active Si transport protein in mammals and points towards an important role for Si in vertebrates and explains interactions between dietary phosphate and silicon.
Assuntos
Fosfatos/metabolismo , Silício/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/química , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/metabolismo , Sódio/metabolismo , Animais , Feminino , Ratos , Ratos Sprague-Dawley , Especificidade da EspécieRESUMO
The controversy surrounding silicon (Si) benefits and essentiality in plants is exacerbated by the differential ability of species to absorb this element. This property is seemingly enhanced in species carrying specific nodulin 26-like intrinsic proteins (NIPs), a subclass of aquaporins. In this work, our aim was to characterize plant aquaporins to define the features that confer Si permeability. Through comparative analysis of 985 aquaporins in 25 species with differing abilities to absorb Si, we were able to predict 30 Si transporters and discovered that Si absorption is exclusively confined to species that possess NIP-III aquaporins with a GSGR selectivity filter and a precise distance of 108 amino acids (AA) between the asparagine-proline-alanine (NPA) domains. The latter feature is of particular significance since it had never been reported to be essential for Si selectivity. Functionality assessed in the Xenopus oocyte expression system showed that NIPs with 108 AA spacing exhibited Si permeability, while proteins differing in that distance did not. In subsequent functional studies, a Si transporter from poplar mutated into variants with 109- or 107-AA spacing failed to import, and a tomato NIP gene mutated from 109 to 108 AA exhibited a rare gain of function. These results provide a precise molecular basis to classify higher plants into Si accumulators or excluders.
Assuntos
Aquaporinas/genética , Oligopeptídeos/genética , Silício/metabolismo , Animais , Genômica , Xenopus laevisAssuntos
Doenças Renais Policísticas/diagnóstico , Receptores de Superfície Celular/genética , Insuficiência Renal Crônica/terapia , Idoso , Análise Mutacional de DNA , Humanos , Rim/diagnóstico por imagem , Rim/cirurgia , Imageamento por Ressonância Magnética , Masculino , Mutação , Nefrectomia , Doenças Renais Policísticas/complicações , Doenças Renais Policísticas/genética , Insuficiência Renal Crônica/etiologiaRESUMO
Alport syndrome has been linked to three different genes, that is, COL4A3, COL4A4 and COL4A5. It is characterized by progressive and non-specific glomerulosclerosis with irregular thickening of the glomerular basement membrane (GBM). At times, the histopathologic picture is dominated by lesions that are consistent with focal and segmental glomerulosclerosis or IgA nephropathy. Here, we report the cases of two related individuals (mother and son) who were diagnosed with COL4A5-related Alport syndrome due to a missense variant (p.Gly1170Ser) in a G-X-Y repeat and found to present the same highly unusual histopathological abnormalities on their kidney biopsies. One of the abnormalities shared, which does not appear to have been reported, was reduced COL4A5 immunolabeling that was limited to Bowman's capsule even though the ultrastructure of the GBM was distorted. The other abnormality was superimposed segmental IgA deposition in both individuals, accompanied by mesangial changes in the mother. We feel that these findings provide novel insight into the mechanisms of disease manifestation in Alport syndrome. They suggest, in particular, that collagen expression and/or assemblies in Bowman's capsule is more vulnerable to missense mutations in COL4A5 than elsewhere in the kidney. Our findings also suggest that certain coinherited gene polymorphisms act as unexpectedly important phenotypic determinants in COL4A-related disorders.
Assuntos
Colágeno Tipo IV , Membrana Basal Glomerular , Mutação de Sentido Incorreto , Nefrite Hereditária , Adulto , Humanos , Pessoa de Meia-Idade , Biópsia , Cápsula Glomerular/patologia , Colágeno Tipo IV/genética , Predisposição Genética para Doença , Membrana Basal Glomerular/patologia , Membrana Basal Glomerular/ultraestrutura , Imunoglobulina A , Nefrite Hereditária/genética , Nefrite Hereditária/patologia , Linhagem , FenótipoRESUMO
Sporadic venous malformations are genetic conditions primarily caused by somatic gain-of-function mutation of PIK3CA or TEK, an endothelial transmembrane receptor signaling through PIK3CA. Venous malformations are associated with pain, bleedings, thrombosis, pulmonary embolism, esthetic deformities and, in severe cases, life-threatening situations. No authorized medical treatment exists for patients with venous malformations. Here, we created a genetic mouse model of PIK3CA-related capillary venous malformations that replicates patient phenotypes. We showed that these malformations only partially signal through AKT proteins. We compared the efficacy of different drugs, including rapamycin, a mTORC1 inhibitor, miransertib, an AKT inhibitor and alpelisib, a PI3Kα inhibitor at improving the lesions seen in the mouse model. We demonstrated the effectiveness of alpelisib in preventing vascular malformations' occurrence, improving the already established ones, and prolonging survival. Considering these findings, we were authorized to treat 25 patients with alpelisib, including 7 children displaying PIK3CA (n = 16) or TEK (n = 9)-related capillary venous malformations resistant to usual therapies including sirolimus, debulking surgical procedures or percutaneous sclerotherapies. We assessed the volume of vascular malformations using magnetic resonance imaging (MRI) for each patient. Alpelisib demonstrated improvement in all 25 patients. Vascular malformations previously considered intractable were reduced and clinical symptoms were attenuated. MRI showed a decrease of 33.4% and 27.8% in the median volume of PIK3CA and TEK malformations respectively, over 6 months on alpelisib. In conclusion, this study supports PI3Kα inhibition as a promising therapeutic strategy in patients with PIK3CA or TEK-related capillary venous malformations.
Assuntos
Capilares , Classe I de Fosfatidilinositol 3-Quinases , Malformações Vasculares , Classe I de Fosfatidilinositol 3-Quinases/genética , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Animais , Camundongos , Humanos , Malformações Vasculares/genética , Malformações Vasculares/tratamento farmacológico , Malformações Vasculares/patologia , Capilares/efeitos dos fármacos , Capilares/patologia , Feminino , Masculino , Sirolimo/farmacologia , Sirolimo/uso terapêutico , Criança , Modelos Animais de Doenças , Terapia de Alvo Molecular , TiazóisAssuntos
Dor no Flanco/diagnóstico por imagem , Insuficiência Renal/cirurgia , Obstrução Ureteral/cirurgia , Idoso , Feminino , Dor no Flanco/etiologia , Dor no Flanco/patologia , Humanos , Nefrotomia , Radiografia Abdominal , Insuficiência Renal/diagnóstico por imagem , Insuficiência Renal/etiologia , Resultado do Tratamento , Obstrução Ureteral/complicações , Obstrução Ureteral/diagnóstico por imagemRESUMO
Atypical hemolytic uremic syndrome (aHUS) is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and renal impairment. Complement and coagulation gene variants have been associated with aHUS susceptibility. We assessed the diagnostic yield of a next-generation sequencing (NGS) panel in a large cohort of Canadian patients with suspected aHUS. Molecular testing was performed on peripheral blood DNA samples from 167 patients, collected between May 2019 and December 2021, using a clinically validated NGS pipeline. Coding exons with 20 base pairs of flanking intronic regions for 21 aHUS-associated or candidate genes were enriched using a custom hybridization protocol. All sequence and copy number variants were assessed and classified following American College of Medical Genetics guidelines. Molecular diagnostic results were reported for four variants in three individuals (1.8%). Twenty-seven variants of unknown significance were identified in 25 (15%) patients, and 34 unique variants in candidate genes were identified in 28 individuals. An illustrative patient case describing two genetic alterations in complement genes is presented, highlighting that variable expressivity and incomplete penetrance must be considered when interpreting genetic data in patients with complement-mediated disease, alongside the potential additive effects of genetic variants on aHUS pathophysiology. In this cohort of patients with suspected aHUS, using clinical pipelines for genetic testing and variant classification, pathogenic/likely pathogenic variants occurred in a very small percentage of patients. Our results highlight the ongoing challenges in variant classification following NGS panel testing in patients with suspected aHUS, alongside the need for clear testing guidance in the clinical setting. KEY MESSAGES: ⢠Clinical molecular testing for disease associated genes in aHUS is challenging. ⢠Challenges include patient selection criteria, test validation, and interpretation. ⢠Most variants were of uncertain significance (31.7% of patients; VUS + candidates). ⢠Their clinical significance may be elucidated as more evidence becomes available. ⢠Low molecular diagnostic rate (1.8%), perhaps due to strict classification criteria. ⢠Case study identified two likely pathogenic variants; one each in MCP/CD46 and CFI.
Assuntos
Síndrome Hemolítico-Urêmica Atípica , Genótipo , Mutação , Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Seleção de Pacientes , Síndrome Hemolítico-Urêmica Atípica/diagnóstico , Síndrome Hemolítico-Urêmica Atípica/genética , Estudos de Coortes , Reprodutibilidade dos Testes , IncertezaRESUMO
Nodulin 26-like intrinsic protein (NIP) subfamily of aquaporins (AQPs) in plants, is known to be involved in the uptake of metalloids including boron, germanium (Ge), arsenic (As), and silicon (Si). In the present study, a thorough evaluation of 55 AQPs found in the mungbean genome, including phylogenetic distribution, sequence homology, expression profiling, and structural characterization, contributed to the identification of VrNIP2-1 as a metalloid transporter. The pore-morphology of VrNIP2-1 was studied using molecular dynamics simulation. Interestingly, VrNIP2-1 was found to harbor an aromatic/arginine (ar/R) selectivity filter formed with ASGR amino acids instead of GSGR systematically reported in metalloid transporters (NIP2s) in higher plants. Evaluation of diverse cultivars showed a high level of Si accumulation in leaves indicating functional Si transport in mungbean. In addition, heterologous expression of VrNIP2-1 in yeast revealed As(III) and GeO2 transport activity. Similarly, VrNIP2-1 expression in Xenopus oocytes confirmed its Si transport ability. The metalloid transport activity with unique structural features will be helpful to better understand the solute specificity of NIP2s in mungbean and related pulses. The information provided here will also serve as a basis to improve Si uptake while restricting hazardous metalloids like As in plants.