RESUMEN
Aquaporin 0 (AQP0) plays a key role in water circulation in the eye lens through a variety of functions. In contrast to mammalian genomes, zebrafish contains two aqp0 genes leading to a separation of AQP0 multiple functions between the two gene products, Aqp0a and Aqp0b. A notable feature of the zebrafish AQP0 paralogs is the increased water permeability of Aqp0b relative to Aqp0a as well as a severa lfold increase relative to mammalian AQP0. Here, we report equilibrium molecular dynamics (MD) simulations on the microsecond timescale to identify the structural basis underlying the differences in water permeability between zebrafish AQP0 paralogs and between AQP0 mammalian and fish orthologs. Our simulations are able to reproduce the experimental trends in water permeability. Our results suggest that a substitution of a key Y23 residue in mammalian AQP0 for F23 in fish AQP0 orthologs introduces significant changes in the conformational dynamics of the CS-I structural motif, which, in conjunction with different levels of hydration of the channel vestibule, can account for the differences in permeabilities between fish and mammalian AQP0 orthologs and between zebrafish AQP0 paralogs.
Asunto(s)
Acuaporinas , Proteínas del Ojo , Pez Cebra , Animales , Acuaporinas/química , Acuaporinas/metabolismo , Acuaporinas/genética , Proteínas del Ojo/química , Proteínas del Ojo/metabolismo , Proteínas del Ojo/genética , Cristalino/metabolismo , Cristalino/química , Simulación de Dinámica Molecular , Agua/química , Agua/metabolismo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Vesicle trafficking is a fundamental process that allows for the sorting and transport of specific proteins (i.e., "cargoes") to different compartments of eukaryotic cells. Cargo recognition primarily occurs through coats and the associated proteins at the donor membrane. However, it remains unclear whether cargoes can also be selected at other stages of vesicle trafficking to further enhance the fidelity of the process. The WDR11-FAM91A1 complex functions downstream of the clathrin-associated AP-1 complex to facilitate protein transport from endosomes to the TGN. Here, we report the cryo-EM structure of human WDR11-FAM91A1 complex. WDR11 directly and specifically recognizes a subset of acidic clusters, which we term super acidic clusters (SACs). WDR11 complex assembly and its binding to SAC-containing proteins are indispensable for the trafficking of SAC-containing proteins and proper neuronal development in zebrafish. Our studies thus uncover that cargo proteins could be recognized in a sequence-specific manner downstream of a protein coat.
Asunto(s)
Microscopía por Crioelectrón , Transporte de Proteínas , Pez Cebra , Humanos , Animales , Endosomas/metabolismo , Células HEK293 , Células HeLa , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/química , Unión ProteicaRESUMEN
Environmental contaminants, such as polycyclic aromatic hydrocarbons (PAHs), have raised concerns regarding their potential endocrine-disrupting effects on aquatic organisms, including fish. In this study, molecular docking and molecular dynamics techniques were employed to evaluate the endocrine-disrupting potential of PAHs in zebrafish, as a model organism. A virtual screening with 72 PAHs revealed a correlation between the number of PAH aromatic rings and their binding affinity to proteins involved in endocrine regulation. Furthermore, PAHs with the highest binding affinities for each protein were identified: cyclopenta[cd]pyrene for AR (-9.7 kcal/mol), benzo(g)chrysene for ERα (-11.5 kcal/mol), dibenzo(a,e)pyrene for SHBG (-8.7 kcal/mol), dibenz(a,h)anthracene for StAR (-11.2 kcal/mol), and 2,3-benzofluorene for TRα (-9.8 kcal/mol). Molecular dynamics simulations confirmed the stability of the protein-ligand complexes formed by the PAHs with the highest binding affinities throughout the simulations. Additionally, the effectiveness of the protocol used in this study was demonstrated by the receiver operating characteristic curve (ROC) analysis, which effectively distinguished decoys from true ligands. Therefore, this research provides valuable insights into the endocrine-disrupting potential of PAHs in fish, highlighting the importance of assessing their impact on aquatic ecosystems.
Asunto(s)
Disruptores Endocrinos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Hidrocarburos Policíclicos Aromáticos , Pez Cebra , Hidrocarburos Policíclicos Aromáticos/química , Hidrocarburos Policíclicos Aromáticos/metabolismo , Hidrocarburos Policíclicos Aromáticos/toxicidad , Animales , Disruptores Endocrinos/química , Disruptores Endocrinos/metabolismo , Disruptores Endocrinos/toxicidad , Unión Proteica , Sitios de Unión , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/química , Ligandos , Curva ROC , Contaminantes Químicos del Agua/metabolismo , Contaminantes Químicos del Agua/química , Contaminantes Químicos del Agua/toxicidad , Receptor alfa de Estrógeno/metabolismo , Receptor alfa de Estrógeno/químicaRESUMEN
Many animals exhibit remarkable colors that are produced by the constructive interference of light reflected from arrays of intracellular guanine crystals. These animals can fine-tune their crystal-based structural colors to communicate with each other, regulate body temperature, and create camouflage. While it is known that these changes in color are caused by changes in the angle of the crystal arrays relative to incident light, the cellular machinery that drives color change is not understood. Here, using a combination of 3D focused ion beam scanning electron microscopy (FIB-SEM), micro-focused X-ray diffraction, superresolution fluorescence light microscopy, and pharmacological perturbations, we characterized the dynamics and 3D cellular reorganization of crystal arrays within zebrafish iridophores during norepinephrine (NE)-induced color change. We found that color change results from a coordinated 20° tilting of the intracellular crystals, which alters both crystal packing and the angle at which impinging light hits the crystals. Importantly, addition of the dynein inhibitor dynapyrazole-a completely blocked this NE-induced red shift by hindering crystal dynamics upon NE addition. FIB-SEM and microtubule organizing center (MTOC) mapping showed that microtubules arise from two MTOCs located near the poles of the iridophore and run parallel to, and in between, individual crystals. This suggests that dynein drives crystal angle change in response to NE by binding to the limiting membrane surrounding individual crystals and walking toward microtubule minus ends. Finally, we found that intracellular cAMP regulates the color change process. Together, our results provide mechanistic insight into the cellular machinery that drives structural color change.
Asunto(s)
Pez Cebra , Animales , Norepinefrina/metabolismo , Norepinefrina/farmacología , Color , Pigmentación/fisiología , Microscopía Electrónica de Rastreo , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/químicaRESUMEN
Guanylate cyclase activating protein-5 (GCAP5) in zebrafish photoreceptors promotes the activation of membrane receptor retinal guanylate cyclase (GC-E). Previously, we showed the R22A mutation in GCAP5 (GCAP5R22A) abolishes dimerization of GCAP5 and activates GC-E by more than 3-fold compared to that of wild-type GCAP5 (GCAP5WT). Here, we present ITC, NMR, and functional analysis of GCAP5R22A to understand how R22A causes a decreased dimerization affinity and increased cyclase activation. ITC experiments reveal GCAP5R22A binds a total of 3 Ca2+, including two sites in the nanomolar range followed by a single micromolar site. The two nanomolar sites in GCAP5WT were not detected by ITC, suggesting that R22A may affect the binding of Ca2+ to these sites. The NMR-derived structure of GCAP5R22A is overall similar to that of GCAP5WT (RMSD = 2.3 Å), except for local differences near R22A (Q19, W20, Y21, and K23) and an altered orientation of the C-terminal helix near the N-terminal myristate. GCAP5R22A lacks an intermolecular salt bridge between R22 and D71 that may explain the weakened dimerization. We present a structural model of GCAP5 bound to GC-E in which the R22 side-chain contacts exposed hydrophobic residues in GC-E. Cyclase assays suggest that GC-E binds to GCAP5R22A with â¼25% higher affinity compared to GCAP5WT, consistent with more favorable hydrophobic contact by R22A that may help explain the increased cyclase activation.
Asunto(s)
Proteínas Activadoras de la Guanilato-Ciclasa , Guanilato Ciclasa , Modelos Moleculares , Animales , Calcio/metabolismo , Activación Enzimática/genética , Guanilato Ciclasa/genética , Guanilato Ciclasa/metabolismo , Guanilato Ciclasa/química , Proteínas Activadoras de la Guanilato-Ciclasa/metabolismo , Proteínas Activadoras de la Guanilato-Ciclasa/genética , Proteínas Activadoras de la Guanilato-Ciclasa/química , Mutación , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Multimerización de Proteína , Retina/metabolismo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/metabolismoRESUMEN
TRAF7 serves as a crucial intracellular adaptor and E3 ubiquitin ligase involved in signal transduction pathways, contributing to immune responses, tumor progression, and embryonic development. Somatic mutations within the coiled-coil (CC) domain and WD40 repeat domain of TRAF7 could cause brain tumors, while germline pathogenic mutations contribute to severe developmental abnormalities. However, the precise molecular mechanism underlying TRAF7 involvement in embryonic development remains unclear. In this study, we employed zebrafish as an in vivo model system. TRAF7 knock down caused defects in zebrafish embryonic development. We determined the crystal structure of TRAF7 CC domain at 3.3 Å resolution and found that the CC region trimerization was essential for TRAF7 functionality during zebrafish embryonic development. Additionally, disease-causing mutations in TRAF7 CC region could impair the trimer formation, consequently impacting early embryonic development of zebrafish. Therefore, our study sheds light on the molecular mechanism of TRAF7 CC trimer formation and its pivotal role in embryonic development.
Asunto(s)
Desarrollo Embrionario , Proteínas de Pez Cebra , Pez Cebra , Animales , Pez Cebra/metabolismo , Pez Cebra/embriología , Desarrollo Embrionario/genética , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/química , Multimerización de Proteína , Mutación , Modelos Moleculares , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/metabolismo , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/genética , Humanos , Cristalografía por Rayos XRESUMEN
Introduction Thyroid cancer incidence has increased in the previous 2 decades. Preoperative identification of lymph node metastasis is a suggested risk factor associated with recurrence following thyroidectomy. Objectives We aimed to evaluate the accuracy of preoperative radiologic investigations of nodal status in determining the postoperative risk of regional nodal recurrence in cases of well-differentiated thyroid cancer. Methods This is a case series. We retrospectively reviewed data, including preoperative ultrasonography and/or computed tomography results, on patients who underwent total thyroidectomy for thyroid cancer at our hospital between 2006 and 2012. Prognostic factors for predicting recurrence, including age, sex, tumor diameter, and nodal diameter, were evaluated. Results Total thyroidectomy was performed on 24 male and 74 female patients (median age, 43 years). The median follow-up time was 21 months. Sixty-eight patients had papillary thyroid cancer, and 30 had follicular cancer. Nodal recurrence was evident in 30% of patients, and 4% of patients died. Identification of lymph node involvement during preoperative radiologic investigations was strongly prognostic for recurrence: 35.3% of patients with positive preoperative ultrasonography findings and 62.5% of those with positive preoperative computed tomography findings had recurrence (p = 0.01). Conclusions Preoperative identification of lymph node metastasis on radiologic studies was correlated with an increased risk of regional nodal recurrence in well-differentiated thyroid cancer. Computed tomography was superior to ultrasonography in detecting metastatic nodal involvement preoperatively and is therefore recommended for preoperative assessment and postoperative follow-up. .