RESUMEN

Bone-resorbing osteoclasts (OCLs) are formed by differentiation and fusion of monocyte precursor cells, generating large multi-nucleated cells. Tightly-regulated cell fusion during osteoclastogenesis leads to formation of resorption-competent OCLs, whose sizes fall within a predictable physiological range. The molecular mechanisms that regulate the onset of OCL fusion and its subsequent arrest are, however, largely unknown. We have previously shown that OCLs cultured from mice homozygous for the R51Q mutation in the vesicle trafficking-associated protein sorting nexin 10, a mutation that induces autosomal recessive osteopetrosis in humans and in mice, display deregulated and continuous fusion that generates gigantic, inactive OCLs. Fusion of mature OCLs is therefore arrested by an active, genetically-encoded, cell-autonomous, and SNX10-dependent mechanism. In order to directly examine whether SNX10 performs a similar role in vivo, we generated SNX10-deficient (SKO) mice and demonstrated that they display massive osteopetrosis and that their OCLs fuse uncontrollably in culture, as do homozygous R51Q SNX10 (RQ/RQ) mice. OCLs that lack SNX10 exhibit persistent presence of DC-STAMP protein at their periphery, which may contribute to their uncontrolled fusion. In order to visualize endogenous SNX10-mutant OCLs in their native bone environment we genetically labelled the OCLs of wild-type, SKO and RQ/RQ mice with EGFP, and then visualized the three-dimensional organization of resident OCLs and the pericellular bone matrix by two-photon, confocal, and second harmonics generation microscopy. We show that the volumes, surface areas and, in particular, the numbers of nuclei in the OCLs of both mutant strains were on average 2-6 fold larger than those of OCLs from wild-type mice, indicating that deregulated, excessive fusion occurs in the mutant mice. We conclude that the fusion of OCLs, and consequently their size, are regulated in vivo by SNX10-dependent arrest of fusion of mature OCLs.

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Osteoclasts (OCLs) are cells that degrade bone. These cells are generated by fusion of monocyte precursor cells, but the mechanisms that regulate this process and eventually arrest it are unknown. We had previously shown that OCLs cultured from mice carrying the R51Q mutation in the protein sorting nexin 10 (SNX10) lose their resorptive capacity and become gigantic due to uncontrolled fusion. To examine whether SNX10 is required for OCL fusion arrest also in vivo, we inactivated the Snx10 gene in mice and fluorescently labelled their OCLs and OCLs of R51Q SNX10 mice, isolated their femurs, and used advanced 3D microscopy methods to visualize OCLs within the bone matrix. As expected, mice lacking SNX10 exhibited excessive bone mass, indicating that their OCLs are inactive. OCLs within bones of both mutant mouse strains were on average 2-6-fold larger than in control mice, and contained proportionally more nuclei. We conclude that OCL fusion is arrested in control, but not SNX10 mutant, mice, indicating that the sizes of mature OCLs are limited in vivo by an active, SNX10-dependent mechanism that suppresses cell fusion.

RESUMEN

Endosomal membrane trafficking is mediated by specific protein coats and formation of actin-rich membrane domains. The Retromer complex coordinates with sorting nexin (SNX) cargo adaptors including SNX27, and the SNX27-Retromer assembly interacts with the Wiskott-Aldrich syndrome protein and SCAR homolog (WASH) complex which nucleates actin filaments establishing the endosomal recycling domain. Crystal structures, modeling, biochemical, and cellular validation reveal how the FAM21 subunit of WASH interacts with both Retromer and SNX27. FAM21 binds the FERM domain of SNX27 using acidic-Asp-Leu-Phe (aDLF) motifs similar to those found in the SNX1 and SNX2 subunits of the ESCPE-1 complex. Overlapping FAM21 repeats and a specific Pro-Leu containing motif bind three distinct sites on Retromer involving both the VPS35 and VPS29 subunits. Mutation of the major VPS35-binding site does not prevent cargo recycling; however, it partially reduces endosomal WASH association indicating that a network of redundant interactions promote endosomal activity of the WASH complex. These studies establish the molecular basis for how SNX27-Retromer is coupled to the WASH complex via overlapping and multiplexed motif-based interactions required for the dynamic assembly of endosomal membrane recycling domains.

Asunto(s)

Endosomas , Nexinas de Clasificación , Proteínas de Transporte Vesicular , Humanos , Endosomas/metabolismo , Nexinas de Clasificación/metabolismo , Nexinas de Clasificación/genética , Nexinas de Clasificación/química , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/química , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/química , Unión Proteica , Cristalografía por Rayos X , Sitios de Unión , Modelos Moleculares

RESUMEN

BACKGROUNDS: Pathological cardiac hypertrophy is considered one of the independent risk factors for heart failure, with a rather complex pathogenic machinery. Sorting nexins (SNXs), denoting a diverse family of cytoplasmic- and membrane-associated phosphoinositide-binding proteins, act as a pharmacological target against specific cardiovascular diseases including heart failure. Family member SNX5 was reported to play a pivotal role in a variety of biological processes. However, contribution of SNX5 to the development of cardiac hypertrophy, remains unclear. METHODS: Mice underwent transverse aortic constriction (TAC) to induce cardiac hypertrophy and simulate pathological conditions. TAC model was validated using echocardiography and histological staining. Expression of SNX5 was assessed by western blotting. Then, SNX5 was delivered through intravenous administration of an adeno-associated virus serotype 9 carrying cTnT promoter (AAV9-cTnT-SNX5) to achieve SNX5 cardiac-specific overexpression. To assess the impact of SNX5, morphological analysis, echocardiography, histological staining, hypertrophic biomarkers, and cardiomyocyte contraction were evaluated. To unravel potential molecular events associated with SNX5, interactome analysis, fluorescence co-localization, and membrane protein profile were evaluated. RESULTS: Our results revealed significant downregulated protein level of SNX5 in TAC-induced hypertrophic hearts in mice. Interestingly, cardiac-specific overexpression of SNX5 improved cardiac function, with enhanced left ventricular ejection fraction, fraction shortening, as well as reduced cardiac fibrosis. Mechanistically, SNX5 directly bound to Rab11a, increasing membrane accumulation of Rab11a (a Rab GTPase). Afterwards, this intricate molecular interaction upregulated the membrane content of low-density lipoprotein receptor-related protein 6 (LRP6), a key regulator against cardiac hypertrophy. Our comprehensive assessment of siRab11a expression in HL-1 cells revealed its role in antagonism of LRP6 membrane accumulation under SNX5 overexpression. CONCLUSIONS: This study revealed that binding of SNX5 with LRP6 triggers their membrane translocation through Rab11a assisting, defending against cardiac remodeling and cardiac dysfunction under pressure overload. These findings provide new insights into the previously unrecognized role of SNX5 in the progression of cardiac hypertrophy.

Asunto(s)

Cardiomegalia , Miocitos Cardíacos , Transporte de Proteínas , Nexinas de Clasificación , Proteínas de Unión al GTP rab , Animales , Masculino , Ratones , Cardiomegalia/metabolismo , Cardiomegalia/patología , Cardiomegalia/genética , Membrana Celular/metabolismo , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Miocitos Cardíacos/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/genética , Nexinas de Clasificación/metabolismo , Nexinas de Clasificación/genética

RESUMEN

SNX32 is a member of the evolutionarily conserved Phox (PX) homology domain- and Bin/Amphiphysin/Rvs (BAR) domain- containing sorting nexin (SNX-BAR) family of proteins, which play important roles in sorting and membrane trafficking of endosomal cargoes. Although SNX32 shares the highest amino acid sequence homology with SNX6, and has been believed to function redundantly with SNX5 and SNX6 in retrieval of the cation-independent mannose-6-phosphate receptor (CI-MPR) from endosomes to the trans-Golgi network (TGN), its role(s) in intracellular protein trafficking remains largely unexplored. Here, we report that it functions in parallel with SNX1 in mediating epidermal growth factor (EGF)-stimulated postendocytic trafficking of the epidermal growth factor receptor (EGFR). Moreover, SNX32 interacts directly with EGFR, and recruits SNX5 to promote sorting of EGF-EGFR into multivesicular bodies (MVBs) for lysosomal degradation. Thus, SNX32 functions distinctively from other SNX-BAR proteins to mediate signaling-coupled endolysosomal trafficking of EGFR.

Asunto(s)

Factor de Crecimiento Epidérmico , Receptores ErbB , Lisosomas , Transporte de Proteínas , Nexinas de Clasificación , Nexinas de Clasificación/metabolismo , Nexinas de Clasificación/genética , Receptores ErbB/metabolismo , Lisosomas/metabolismo , Humanos , Transporte de Proteínas/fisiología , Factor de Crecimiento Epidérmico/metabolismo , Células HeLa , Endosomas/metabolismo , Red trans-Golgi/metabolismo , Cuerpos Multivesiculares/metabolismo

RESUMEN

The complete molecular mechanism underlying doxorubicin-induced cardiomyopathy remains incompletely elucidated. In this investigation, we engineered mice with cardiomyocyte-specific sorting nexin 3 knockout (SNX3Cko ) to probe the potential protective effects of SNX3 ablation on doxorubicin-triggered myocardial injury, focusing on GPX4-dependent ferroptosis. Our findings indicate that SNX3 deletion normalized heart contractile/relaxation function and thwarted the escalation of cardiac injury biomarkers following doxorubicin exposure. Additionally, SNX3 deletion in the heart mitigated the inflammatory response and oxidative stress in the presence of doxorubicin. At the molecular level, the detrimental effects of doxorubicin-induced cell death, endoplasmic reticulum (ER) stress, and mitochondrial dysfunction were alleviated by SNX3 deficiency. Molecular analysis revealed the activation of GPX4-mediated ferroptosis by doxorubicin, whereas loss of SNX3 prevented the initiation of GPX4-dependent ferroptosis. Furthermore, treatment with erastin, a ferroptosis inducer, markedly reduced cell viability, exacerbated ER stress, and induced mitochondrial dysfunction in SNX3-depleted cardiomyocytes upon doxorubicin exposure. In summary, our results demonstrate that SNX3 deficiency shielded the heart from doxorubicin-induced myocardial dysfunction by modulating GPX4-associated ferroptosis.

Asunto(s)

Cardiomiopatías , Doxorrubicina , Ferroptosis , Ratones Noqueados , Miocitos Cardíacos , Fosfolípido Hidroperóxido Glutatión Peroxidasa , Nexinas de Clasificación , Ferroptosis/efectos de los fármacos , Ferroptosis/genética , Animales , Doxorrubicina/efectos adversos , Doxorrubicina/toxicidad , Cardiomiopatías/inducido químicamente , Cardiomiopatías/patología , Cardiomiopatías/genética , Nexinas de Clasificación/genética , Nexinas de Clasificación/metabolismo , Ratones , Fosfolípido Hidroperóxido Glutatión Peroxidasa/metabolismo , Fosfolípido Hidroperóxido Glutatión Peroxidasa/genética , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Humanos , Estrés Oxidativo/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos

RESUMEN

Oncogenic HPV E6 proteins have a PDZ-binding motif (PBM) which plays important roles in both the viral life cycle and tumor development. The PBM confers interaction with a large number of different PDZ domain-containing substrates, one of which is Sorting Nexin 27. This protein is part of the retromer complex and plays an important role in endocytic sorting pathways. It has been shown that at least two SNX27 interacting partners, GLUT1 and TANC2, are aberrantly trafficked due to the E6 PBM-dependent interaction with SNX27. To investigate further which other components of the endocytic trafficking pathway might be affected by the SNX27-HPV E6 interaction, we analyzed the SNX27 proteome interaction profile in a previously described HeLa cell line expressing GFP-SNX27, both in the presence and absence of the HPV-18 E6 oncoprotein. In this study, we identify a novel interacting partner of SNX27, secreted glycoprotein EMILIN2, whose release is blocked by HPV18 E6 in a PBM-dependent manner. Mechanistically, E6 can block EMILIN2 interaction with the WNT1 ligand, thereby enhancing WNT1 signaling and promoting cell proliferation. IMPORTANCE: This study demonstrates that HPV E6 blocks EMILIN2 inhibition of WNT1 signaling, thereby enhancing cell proliferation in HPV-positive tumor cells. This involves a novel mechanism whereby the E6 PBM actually contributes toward enhancing the interaction between SNX27 and EMILIN2, suggesting that the mode of recognition of SNX27 by E6 and EMILIN2 is different. This is the first example of the E6 PBM altering a PDZ domain-containing protein to enhance potential substrate recognition.

Asunto(s)

Papillomavirus Humano 18 , Proteínas Oncogénicas Virales , Nexinas de Clasificación , Vía de Señalización Wnt , Humanos , Proteínas de Unión al ADN , Células HEK293 , Células HeLa , Papillomavirus Humano 18/metabolismo , Papillomavirus Humano 18/genética , Proteínas Oncogénicas Virales/metabolismo , Proteínas Oncogénicas Virales/genética , Infecciones por Papillomavirus/virología , Infecciones por Papillomavirus/metabolismo , Dominios PDZ , Unión Proteica , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Nexinas de Clasificación/metabolismo , Nexinas de Clasificación/genética

RESUMEN

High risk human papillomavirus (HPV) infection is responsible for 99 % of cervical cancers and 5 % of all human cancers worldwide. HPV infection requires the viral genome (vDNA) to gain access to nuclei of basal keratinocytes of epithelium. After virion endocytosis, the minor capsid protein L2 dictates the subcellular retrograde trafficking and nuclear localization of the vDNA during mitosis. Prior work identified a cell-permeable peptide termed SNX1.3, derived from the BAR domain of sorting nexin 1 (SNX1), that potently blocks the retrograde and nuclear trafficking of EGFR in triple negative breast cancer cells. Given the importance of EGFR and retrograde trafficking pathways in HPV16 infection, we set forth to study the effects of SNX1.3 within this context. SNX1.3 inhibited HPV16 infection by both delaying virion endocytosis, as well as potently blocking virion retrograde trafficking and Golgi localization. SNX1.3 had no effect on cell proliferation, nor did it affect post-Golgi trafficking of HPV16. Looking more directly at L2 function, SNX1.3 was found to impair membrane spanning of the minor capsid protein. Future work will focus on mechanistic studies of SNX1.3 inhibition, and the role of EGFR signaling and SNX1-mediated endosomal tubulation, cargo sorting, and retrograde trafficking in HPV infection.

RESUMEN

Recently, emerging evidence has suggested that obesity become a prevalent health threat worldwide. Reportedly, CTRP9 can ameliorate HFD induced obesity. However, the molecular mechanism underlying the role of CTRP9 in obesity remains elusive. In this study, we reported its major function in the regulation of lipolysis. First, we found that the expression of CTRP9 was decreased in mature adipocytes and white adipose tissue of obese mice. Then, we showed that overexpression adipose tissue CTRP9 alleviated diet-induced obesity and adipocytes hypertrophy, improved glucose intolerance and raised energy expenditure. Moreover, CTRP9 increased the lipolysis in vitro and vivo. Additionally, we determined that CTRP9 enhanced autophagy flux in adipocytes. Intriguingly, knock down Beclin1 by SiRNA abolished the effect of CTRP9 on lipolysis. Mechanically, CTRP9 enhanced the expression of SNX26. We demonstrated that SNX26 was a component of the ATG14L-Beclin1-VPS34 complex and enhanced the assembly of the autophagy-initiation complex. Collectively, our results suggested that CTRP9 alleviated diet induced obesity through enhancing lipolysis mediated by autophagy-initiation complex formation.

Asunto(s)

Adipocitos , Autofagia , Beclina-1 , Lipólisis , Ratones Endogámicos C57BL , Obesidad , Animales , Obesidad/metabolismo , Ratones , Masculino , Adipocitos/metabolismo , Beclina-1/metabolismo , Dieta Alta en Grasa/efectos adversos , Células 3T3-L1 , Proteínas Relacionadas con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Adiponectina/metabolismo , Metabolismo Energético , Glicoproteínas

RESUMEN

BACKGROUND: Sorting nexin 14 (SNX14) is a member of the sorting junction protein family. Its specific roles in cancer development remain unclear. Therefore, in this study, we aimed to determine the effects and underlying mechanisms of SNX14 on autophagy of breast cancer cells to aid in the therapeutic treatment of breast cancer. METHODS: In this study, we performed in vitro experiments to determine the effect of SNX14 on breast cancer cell growth. Moreover, we used an MCF7 breast cancer tumor-bearing mouse model to confirm the effect of SNX14 on tumor cell growth in vivo. We also performed western blotting and quantitative polymerase chain reaction to identify the mechanism by which SNX14 affects breast cancer MCF7 cells. RESULTS: We found that SNX14 regulated the onset and progression of breast cancer by promoting the proliferation and inhibiting the autophagy of MCF7 breast cancer cells. In vivo experiments further confirmed that SNX14 knockdown inhibited the tumorigenicity and inhibited the growth of tumor cells in tumor tissues of nude mice. In addition, western blotting analysis revealed that SNX14 modulate the autophagy of MCF7 breast cancer cells via the phosphoinositide 3-kinase/protein kinase B/mechanistic target of rapamycin kinase signaling pathway. CONCLUSION: Our findings indicate that SNX14 is an essential tumor-promoting factor in the development of breast cancer.

Asunto(s)

Autofagia , Neoplasias de la Mama , Proliferación Celular , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Nexinas de Clasificación , Serina-Treonina Quinasas TOR , Animales , Femenino , Humanos , Ratones , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Células MCF-7 , Ratones Desnudos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Nexinas de Clasificación/metabolismo , Nexinas de Clasificación/genética , Serina-Treonina Quinasas TOR/metabolismo

RESUMEN

The distortion of the cellular membrane transport pathway has a profound impact on cell dynamics and can drive serious physiological consequences during the process of cell sorting. SNX17 is a member of the Sorting Nexin (SNX) family and plays a crucial role in protein sorting and transport in the endocytic pathway. SNX17, SNX27, and SNX31 belong to the SNX-FERM subfamily and possess the FERM domain, which can assist in endocytic transport and lysosomal degradation. The binding partners of SNX27 have been discovered to number over 100, and SNX27 has been linked to the development of Alzheimer's disease progression, tumorigenesis, cancer progression, and metastasis. However, the role and potential mechanisms of SNX17 in human health and disease remain poorly understood, and the function of SNX17 has not been fully elucidated. In this review, we summarize the structure and basic functions of SNX protein, focusing on providing current evidence of the role and possible mechanism of SNX17 in human neurodegenerative diseases and cardiovascular diseases.

RESUMEN

Sorting nexin 16 (SNX16), a pivotal sorting nexin, emerges in tumor progression complexity, fueling research interest. However, SNX16's biological impact and molecular underpinnings in hepatocellular carcinoma (HCC) remain elusive. This study probes SNX16's function, clinical relevance via mRNA, and protein expression in HCC. Overexpression/knockdown assays of SNX16 were employed to elucidate impacts on HCC cell invasion, proliferation, and EMT. Additionally, the study delved into SNX16's regulation of the EGFR-AKT signaling cascade mechanism. SNX16 overexpression in HCC correlates with poor patient survival; enhancing proliferation, migration, invasion, and tumorigenicity, while SNX16 knockdown suppresses these processes. SNX16 downregulation curbs phospho-EGFR, dampening AKT signaling. EGFR suppression counters SNX16-overexpression-induced HCC proliferation, motility, and invasiveness. Our findings delineate SNX16's regulatory role in HCC, implicating it as a prospective therapeutic target.

Asunto(s)

Carcinoma Hepatocelular , Movimiento Celular , Proliferación Celular , Receptores ErbB , Neoplasias Hepáticas , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Nexinas de Clasificación , Animales , Humanos , Ratones , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/mortalidad , Línea Celular Tumoral , Movimiento Celular/genética , Transición Epitelial-Mesenquimal/genética , Receptores ErbB/metabolismo , Receptores ErbB/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/mortalidad , Invasividad Neoplásica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Nexinas de Clasificación/metabolismo , Nexinas de Clasificación/genética

RESUMEN

High risk human papillomavirus (HPV) infection is responsible for 99% of cervical cancers and 5% of all human cancers worldwide. HPV infection requires the viral genome (vDNA) to gain access to nuclei of basal keratinocytes of epithelium. After virion endocytosis, the minor capsid protein L2 dictates the subcellular retrograde trafficking and nuclear localization of the vDNA during mitosis. Prior work identified a cell-permeable peptide termed SNX1.3, derived from the BAR domain of sorting nexin 1 (SNX1), that potently blocks the retrograde and nuclear trafficking of EGFR in triple negative breast cancer cells. Given the importance of EGFR and retrograde trafficking pathways in HPV16 infection, we set forth to study the effects of SNX1.3 within this context. SNX1.3 inhibited HPV16 infection by both delaying virion endocytosis, as well as potently blocking virion retrograde trafficking and Golgi localization. SNX1.3 had no effect on cell proliferation, nor did it affect post-Golgi trafficking of HPV16. Looking more directly at L2 function, SNX1.3 was found to impair membrane spanning of the minor capsid protein. Future work will focus on mechanistic studies of SNX1.3 inhibition, and the role of EGFR signaling and SNX1- mediated endosomal tubulation, cargo sorting, and retrograde trafficking in HPV infection.

RESUMEN

Proteins of the sorting nexin (SNX) family present a modular structural architecture with a phox homology (PX) phosphoinositide (PI)-binding domain and additional PX structural domains, conferring to them a wide variety of vital eukaryotic cell's functions, from signal transduction to membrane deformation and cargo binding. Although SNXs are well studied in human and yeasts, they are poorly investigated in protists. Herein, is presented the characterization of the first SNX identified in Leishmania protozoan parasites encoded by the LdBPK_352470 gene. In silico secondary and tertiary structure prediction revealed a PX domain on the N-terminal half and a Bin/amphiphysin/Rvs (BAR) domain on the C-terminal half of this protein, with these features classifying it in the SNX-BAR subfamily of SNXs. We named the LdBPK_352470.1 gene product LdSNXi, as it is the first SNX identified in Leishmania (L.) donovani. Its expression was confirmed in L. donovani promastigotes under different cell cycle phases, and it was shown to be secreted in the extracellular medium. Using an in vitro lipid binding assay, it was demonstrated that recombinant (r) LdSNXi (rGST-LdSNXi) tagged with glutathione-S-transferase (GST) binds to the PtdIns3P and PtdIns4P PIs. Using a specific a-LdSNXi antibody and immunofluorescence confocal microscopy, the intracellular localization of endogenous LdSNXi was analyzed in L. donovani promastigotes and axenic amastigotes. Additionally, rLdSNXi tagged with enhanced green fluorescent protein (rLdSNXi-EGFP) was heterologously expressed in transfected HeLa cells and its localization was examined. All observed localizations suggest functions compatible with the postulated SNX identity of LdSNXi. Sequence, structure, and evolutionary analysis revealed high homology between LdSNXi and the human SNX2, while the investigation of protein-protein interactions based on STRING (v.11.5) predicted putative molecular partners of LdSNXi in Leishmania.

Asunto(s)

Leishmania , Humanos , Leishmania/genética , Células HeLa , Nexinas de Clasificación/genética , Transducción de Señal , Anticuerpos , Glutatión Transferasa

RESUMEN

Objective: The article aims to provide genetic counseling to a family with two children who were experiencing growth and developmental delays. Methods: Clinical information of the proband was collected. Peripheral blood was collected from core family members to identify the initial reason for growth and developmental delays by whole exome sequencing (WES) and Sanger sequencing. To ascertain the consequences of the newly discovered variants, details of the variants detected were analyzed by bioinformatic tools. Furthermore, we performed in vitro experimentation targeting SNX14 gene expression to confirm whether the variants could alter the expression of SNX14. Results: The proband had prenatal ultrasound findings that included flattened frontal bones, increased interocular distance, widened bilateral cerebral sulci, and shortened long bones, which resulted in subsequent postnatal developmental delays. The older sister also displayed growth developmental delays and poor muscle tone. WES identified compound heterozygous variants of c.712A>T (p.Arg238Ter) and .2744A>T (p.Gln915Leu) in the SNX14 gene in these two children. Both are novel missense variant that originates from the father and mother, respectively. Sanger sequencing confirmed this result. Following the guideline of the American College of Medical Genetics and Genomics (ACMG), the SNX14 c.712A>T (p.Arg238Ter) variant was predicted to be pathogenic (P), while the SNX14 c.2744A>T (p.Gln915Leu) variant was predicted to be a variant of uncertain significance (VUS). The structural analysis revealed that the c.2744A>T (p.Gln915Leu) variant may impact the stability of the SNX14 protein. In vitro experiments demonstrated that both variants reduced SNX14 expression. Conclusion: The SNX14 gene c.712A>T (p.Arg238Ter) and c.2744A>T (p.Gln915Leu) were identified as the genetic causes of growth and developmental delay in two affected children. This conclusion was based on the clinical presentations of the children, structural analysis of the mutant protein, and in vitro experimental validation. This discovery expands the range of SNX14 gene variants and provides a foundation for genetic counseling and guidance for future pregnancies in the affected children's families.

RESUMEN

Autophagy eliminates cytoplasmic material by engulfment in membranous vesicles targeted for lysosome degradation. Nonselective autophagy coordinates sequestration of bulk cargo with the growth of the isolation membrane (IM) in a yet-unknown manner. Here, we show that in the budding yeast Saccharomyces cerevisiae, IMs expand while maintaining a rim sufficiently wide for sequestration of large cargo but tight enough to mature in due time. An obligate complex of Atg24/Snx4 with Atg20 or Snx41 assembles locally at the rim in a spatially extended manner that specifically depends on autophagic PI(3)P. This assembly stabilizes the open rim to promote autophagic sequestration of large cargo in correlation with vesicle expansion. Moreover, constriction of the rim by the PI(3)P-dependent Atg2-Atg18 complex and clearance of PI(3)P by Ymr1 antagonize rim opening to promote autophagic maturation and consumption of small cargo. Tight regulation of membrane rim aperture by PI(3)P thus couples the mechanism and physiology of nonselective autophagy.

Asunto(s)

Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Autofagia/fisiología , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Autofagosomas/metabolismo

RESUMEN

SNX29 is a potential functional gene associated with meat production traits. Previous studies have shown that SNX29 copy number variation (CNV) could be implicated with phenotype in goats. However, in Diannan small-ear (DSE) pigs, the genetic impact of SNX29 CNV on growth traits remains unclear. Therefore, this study investigated the associations between SNX29 CNVs (CNV10810 and CNV10811) and growth traits in 415 DSE pigs. The results revealed that the CNV10810 mutation was significantly associated with backfat thickness in DSE pigs at 12 and 15 months old (P < 0.05), while the CNV10811 mutation had significant effects on various growth traits at 6 and 12 months old, particularly for body weight, body height, back height and backfat thickness (P < 0.05 or P < 0.001). In conclusion, our results confirm that SNX29 CNV plays a role in regulating growth and development in pigs, thus suggesting its potential application for pig breeding programmes.

Asunto(s)

Variaciones en el Número de Copia de ADN , Nexinas de Clasificación , Porcinos/genética , Animales , Variaciones en el Número de Copia de ADN/genética , Nexinas de Clasificación/genética , Fenotipo , Peso Corporal/genética , Dosificación de Gen

RESUMEN

African swine fever virus (ASFV) causes a highly contagious and deadly disease in domestic pigs and European wild boars, posing a severe threat to the global pig industry. ASFV CP204L, a highly immunogenic protein, is produced during the early stages of ASFV infection. However, the impact of CP204L protein-interacting partners on the outcome of ASFV infection is poorly understood. To accomplish this, coimmunoprecipitation and mass spectrometry analysis were conducted in ASFV-infected porcine alveolar macrophages (PAMs). We have demonstrated that sorting nexin 32 (SNX32) is a CP204L-binding protein and that CP204L interacted and colocalized with SNX32 in ASFV-infected PAMs. ASFV growth and replication were promoted by silencing SNX32 and suppressed by overexpressing SNX32. SNX32 degraded CP204L by recruiting the autophagy-related protein Ras-related protein Rab-1b (RAB1B). RAB1B overexpression inhibited ASFV replication, while knockdown of RAB1B had the opposite effect. Additionally, RAB1B, SNX32, and CP204L formed a complex upon ASFV infection. Taken together, this study demonstrates that SNX32 antagonizes ASFV growth and replication by recruiting the autophagy-related protein RAB1B. This finding extends our understanding of the interaction between ASFV CP204L and its host and provides new insights into exploring the relationship between ASFV infection and autophagy.IMPORTANCEAfrican swine fever (ASF) is a highly contagious and acute hemorrhagic viral disease with a high mortality near 100% in domestic pigs. ASF virus (ASFV), which is the only member of the family Asfarviridae, is a dsDNA virus of great complexity and size, encoding more than 150 proteins. Currently, there are no available vaccines against ASFV. ASFV CP204L represents the most abundantly expressed viral protein early in infection and plays an important role in regulating ASFV replication. However, the mechanism by which the interaction between ASFV CP204L and host proteins affects ASFV replication remains unclear. In this study, we demonstrated that the cellular protein SNX32 interacted with CP204L and degraded CP204L by upregulating the autophagy-related protein RAB1B. In summary, this study will help us understand the interaction mechanism between CP204L and its host upon infection and provide new insights for the development of vaccines and antiviral drugs.

Asunto(s)

Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Factores de Restricción Antivirales , Autofagia , Nexinas de Clasificación , Proteínas de Unión al GTP rab1 , Animales , Proteínas Relacionadas con la Autofagia/metabolismo , Sus scrofa/virología , Porcinos/virología , Nexinas de Clasificación/metabolismo , Factores de Restricción Antivirales/metabolismo , Proteínas de Unión al GTP rab1/metabolismo , Macrófagos/virología , Replicación Viral

RESUMEN

BACKGROUND: Liver ischemia reperfusion (IR) injury is a common cause of liver dysfunction in patients post liver partial resection and liver transplantation. However, the cellular defense mechanisms underlying IR are not well understood. Macrophage mediated sterile inflammation plays critical roles in liver IR injury. Sorting nexin (SNX) 10, a member of the SNX family which functions in regulation of endosomal sorting. This study aimed to explore the role of sorting nexin 10 (SNX10) during liver IR injury with a focus on regulating macrophage function. METHODS: Both the gene and protein expression levels of SNX10 were analyzed in human specimens from 10 patients undergoing liver partial resection with ischemic insult and in a mouse model of liver IR. The in vivo effects of SNX10 in liver IR injury and sterile inflammation in mice were investigated. Bone marrow derived macrophages (BMDMs) were used to determine the role of SNX10 in modulating macrophage function in vitro. RESULTS: Increased expression of SNX10 was observed both in human specimens and mice livers post IR. SNX10 knockdown alleviated IR induced sterile inflammation and liver damage in mice. SNX10 promoted M1 polarization of macrophage treated with LPS and facilitated inflammatory response by activating NLRP3 inflammasome. CONCLUSIONS: We report for the first time that SNX10 is upregulated in IR-stressed livers. SNX10 activation aggravates liver IR injury and sterile inflammation by facilitating macrophage M1 polarization and inflammatory response suggesting SNX10 as a potential therapeutic target for liver IR injury.

Asunto(s)

Inflamasomas , Daño por Reperfusión , Humanos , Animales , Ratones , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Nexinas de Clasificación/genética , Nexinas de Clasificación/metabolismo , Hígado/metabolismo , Macrófagos/metabolismo , Inflamación/metabolismo , Daño por Reperfusión/metabolismo

RESUMEN

BACKGROUND: Sorting Nexin 27 (SNX27)-retromer complex facilitates cargo recycling from endosomes to the plasma membrane. SNX27 downregulation in neurons, as the result of Trisomy 21 (T21), has been linked with cognitive deficits due to impairment of AMPA and NMDA receptor recycling. Studies in human T cell lines likewise demonstrated that SNX27 regulates the correct delivery of cargoes to the immune synapse limiting the activation of pro-inflammatory pathways. Nevertheless, the physiological consequences of partial SNX27 loss in T cell homeostasis are still unclear. RESULTS: In this study, we have explored the consequences of T cell specific partial SNX27 downregulation in mice. T cells with partial SNX27 deficiency show a marked deficit in the CD4+ T cell pool, a hallmark of aging in mice and humans, and a well-characterized comorbidity of individuals with Down syndrome (DS). When analyzed ex vivo, CD4+ T cells with partial SNX27 deletion demonstrate enhanced proliferation but diminished IL-2 production. In contrast, the CD8+ population show enhanced expression of pro-inflammatory cytokines and lytic enzymes. CONCLUSIONS: This mouse model supports the relevance of SNX27 in the organization of the immune synapse, previously described in cell lines, as well as in the control of T cell homeostasis. Individuals with DS experiment an acceleration of the aging process, which particularly affects the immune and central nervous systems. Thus, we hypothesize that reduced SNX27 expression in DS could contribute to the dysregulation of these systems and further research in SNX27 will shed light on the molecular factors underlying the phenotypes observed in people with DS and its contribution to aging.

RESUMEN

Sorting nexin17 (SNX17) is a member of the sorting nexin family, which plays a crucial role in endosomal trafficking. Previous research has shown that SNX17 is involved in the recycling or degradation of various proteins associated with neurodevelopmental and neurological diseases in cell models. However, the significance of SNX17 in neurological function in the mouse brain has not been thoroughly investigated. In this study, we generated Snx17 knockout mice and observed that the homozygous deletion of Snx17 (Snx17-/-) resulted in lethality. On the other hand, heterozygous mutant mice (Snx17+/-) exhibited anxiety-like behavior with a reduced preference for social novelty. Furthermore, Snx17 haploinsufficiency led to impaired synaptic transmission and reduced maturation of dendritic spines. Through GST pulldown and interactome analysis, we identified the SRC kinase inhibitor, p140Cap, as a potential downstream target of SNX17. We also demonstrated that the interaction between p140Cap and SNX17 is crucial for dendritic spine maturation. Together, this study provides the first in vivo evidence highlighting the important role of SNX17 in maintaining neuronal function, as well as regulating social novelty and anxiety-like behaviors.

Asunto(s)

Espinas Dendríticas , Nexinas de Clasificación , Animales , Ratones , Espinas Dendríticas/metabolismo , Homocigoto , Transporte de Proteínas , Eliminación de Secuencia , Nexinas de Clasificación/genética , Nexinas de Clasificación/metabolismo