Publications by authors named "Qiuhong Xiong"

20 Publications

  • Page 1 of 1

RNA and quantitative proteomic analysis of Dictyostelium knock-out cells lacking the core autophagy proteins ATG9 and/or ATG16.

BMC Genomics 2021 Jun 15;22(1):444. Epub 2021 Jun 15.

Center for Biochemistry, Medical Faculty, University of Cologne, Joseph-Stelzmann-Str. 52, 50931, Cologne, Germany.

Background: Autophagy is an evolutionary ancient mechanism that sequesters substrates for degradation within autolysosomes. The process is driven by many autophagy-related (ATG) proteins, including the core members ATG9 and ATG16. However, the functions of these two core ATG proteins still need further elucidation. Here, we applied RNA and tandem mass tag (TMT) proteomic approaches to identify differentially expressed genes (DEGs) and proteins (DEPs) in Dictyostelium discoideum ATG9‾, ATG16‾ and ATG9‾/16‾ strains in comparison to AX2 wild-type cells.

Result: In total, we identified 332 (279 up and 53 down), 639 (487 up and 152 down) and 260 (114 up and 146 down) DEGs and 124 (83 up and 41 down), 431 (238 up and 193 down) and 677 (347 up and 330 down) DEPs in ATG9‾, ATG16‾ and ATG9‾/16‾ strains, respectively. Thus, in the single knock-out strains, the number of DEGs was higher than the number of DEPs while in the double knock-out strain the number of DEPs was higher. Comparison of RNA and proteomic data further revealed, that only a small proportion of the transcriptional changes were reflected on the protein level. Gene ontology (GO) analysis revealed an enrichment of DEPs involved in lipid metabolism and oxidative phosphorylation. Furthermore, we found increased expression of the anti-oxidant enzymes glutathione reductase (gsr) and catalase A (catA) in ATG16‾ and ATG9‾/16‾ cells, respectively, indicating adaptation to excess reactive oxygen species (ROS).

Conclusions: Our study provides the first combined transcriptome and proteome analysis of ATG9‾, ATG16‾ and ATG9‾/16‾ cells. Our results suggest, that most changes in protein abundance were not caused by transcriptional changes, but were rather due to changes in protein homeostasis. In particular, knock-out of atg9 and/or atg16 appears to cause dysregulation of lipid metabolism and oxidative phosphorylation.
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http://dx.doi.org/10.1186/s12864-021-07756-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8204557PMC
June 2021

Identification of Two Novel Compound Heterozygous Mutations Underlying Wolcott-Rallison Syndrome in a Chinese Family.

Front Pediatr 2021 26;9:679646. Epub 2021 May 26.

Institutes of Biomedical Sciences, Key Laboratory of Chemical Biology and Molecular Engineering of National Ministry of Education, Shanxi University, Taiyuan, China.

Wolcott-Rallison syndrome is a rare autosomal recessive inheritance disorder caused by the defectiveness of eukaryotic translation initiation factor 2 alpha kinase 3 (), which encodes the PKR-like endoplasmic reticulum kinase (PERK). Defect in results in a permanent diabetes in early infancy or newborn period, a tendency to develop skeletal fractures and other associated disorders such as severe liver and renal dysfunction, and central hypothyroidism. Two patients with Wolcott-Rallison syndrome-like manifestations in a Chinese family and family members were genetically analyzed to identify if any variations that occurred in , which may cause Wolcott-Rallison syndrome. Whole-exome sequencing (WES) was performed to identify genetic variations, and Sanger sequencing was conducted to verify the identified variations in the family members with Wolcott-Rallison syndrome (WRS) clinical manifestations. Several bioinformatics tools were employed to predict the effect of variations on the protein function. The impact on PERK protein was analyzed by sequential analysis and evolution conservation study. Two novel heterozygous single base variations (c.2818C>T and c.2980G>C) were detected in the proband. PERK has two functional domains: one is regulatory domain (aa 1-576), and the other is catalytic domain (aa 577-1,115). Both variations are missense mutations and locate in catalytic domain of PERK; c.2818C>T resulted in a residue substitution of proline for serine at amino acid site 940 (p.Pro940Ser), and variation c.2980G>C caused an amino acid change at position 994 from glutamic acid to glutamine (p.Glu994Gln). These novel missense variations may affect the physiological functions of PERK protein. Two novel compound heterozygous variations (c.2818C>T, p.Pro940Ser and c.2980G>C, p.Glu994Gln) were found in a Chinese family. The identification of the variations and verification of their pathogenicity extended the variation spectrum of variations causing Wolcott-Rallison syndrome and enriched valuable information for precise medical intervention for Wolcott-Rallison syndrome in China.
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http://dx.doi.org/10.3389/fped.2021.679646DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8187601PMC
May 2021

Mutation Impairs the Autophagic Degradation of Transferrin Receptor and Promotes Ferroptosis.

Front Mol Biosci 2021 3;8:645831. Epub 2021 May 3.

Institutes of Biomedical Sciences, Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Shanxi University, Taiyuan, China.

WDR45 is an autophagy-related protein that involves in the formation of autophagosome. Mutations in lead to the impairment of autophagy which is associated with the human β-propeller protein-associated neurodegeneration (BPAN). However, the relationship between autophagy and brain iron accumulation in patients with BPAN remains unclear. Here, we demonstrated that transferrin receptor (TfRC) which is critical for the iron import of cells was degraded autophagy. TfRC was accumulated after the inhibition of autophagy by treatment with autophagic inhibitor chloroquine or knockdown of . The intracellular iron content was increased in cells overexpressing TfRC or mutant WDR45, however, ferritin H (FTH) chain was decreased. Increased TfRC and simultaneously decreased FTH consequently resulted in an elevated level of ferrous iron (Fe) which further promoted cell ferroptosis, demonstrated by the increased lipid peroxidation and reactive oxygen species (ROS) and the decreased glutathione peroxidase 4 (GPX4) and cell viability. Taken together, these findings provide a piece of important evidence that WDR45 deficiency impairs autophagic degradation of TfRC, therefore leading to iron accumulation, and the elevated iron promotes ferroptosis which may contribute to the progression of BPAN.
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http://dx.doi.org/10.3389/fmolb.2021.645831DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8126626PMC
May 2021

Polydatin has anti-inflammatory and antioxidant effects in LPS-induced macrophages and improves DSS-induced mice colitis.

Immun Inflamm Dis 2021 09 19;9(3):959-970. Epub 2021 May 19.

Central Laboratory, Shanxi Provincial People's Hospital, Affiliate of Shanxi Medical University, Taiyuan, Shanxi, China.

Polydatin (PD), a monocrystalline compound isolated from the root and rhizome of Polygonum cuspidatum, is widely used in inhibiting the inflammatory response and oxidative stress. PD has an anti-inflammatory effect on colitis mice; however, information regulating the mechanism by which maintains the intestinal epithelium barrier is currently scarce. Here, we assessed the anti-inflammatory and antioxidant of PD in lipopolysaccharide (LPS)-induced macrophages in vitro, and explored its effects on inhibiting intestinal inflammation and maintaining the intestinal epithelium barrier in dextran sodium sulfate (DSS)-induced colitis mice. Results showed that PD reduced the level of proinflammatory cytokines and enzymes, including tumor necrosis factor-α, interleukin-4 (IL-4), IL-6, cyclooxygenase-2, and inducible nitric oxide synthase, in LPS-induced macrophages, and improved the expression level of IL-10. PD maintained the expression of tight junction proteins in medium (LPS-induced macrophages medium)-induced MCEC cells. Additionally, PD inhibited the phosphorylation of nuclear factor-κB (NF-κB), p65, extracellular signal-regulated kinase-1/2, c-Jun N-terminal kinase, and p38 signaling pathways in LPS-induced macrophages and facilitated the phosphorylation of AKT and the nuclear translocation of Nrf2, improving the expression of HO-1 and NQO1. Furthermore, PD ameliorated the intestinal inflammatory response and improved the dysfunction of the colon epithelium barrier in DSS-induced colitis mice. Taken together, our results indicated that PD inhibited inflammation and oxidative stress, maintained the intestinal epithelium barrier, and the protective role of PD was associated with the NF-κB p65, itogen-activated protein kinases, and AKT/Nrf2/HO-1/NQO1 signaling pathway.
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http://dx.doi.org/10.1002/iid3.455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8342204PMC
September 2021

The negative charge of the 343 site is essential for maintaining physiological functions of CXCR4.

BMC Mol Cell Biol 2021 Jan 23;22(1). Epub 2021 Jan 23.

Institutes of Biomedical Sciences, Shanxi University, Taiyuan, 030006, China.

Background: Warts, hypogammaglobulinemia, recurrent bacterial infections and myelokathexis (WHIM) syndrome is a primary immunodeficiency disease (PID) usually caused by autosomal dominant mutations in the chemokine receptor CXCR4 gene. To date, a total of nine different mutations including eight truncation mutations and one missense mutation (E343K, CXCR4) distributed in the C-terminus of CXCR4 have been identified in humans. Studies have clarified that the loss of phosphorylation sites in the C-terminus of truncated CXCR4 impairs the desensitization process, enhances the activation of G-protein, prolongs downstream signaling pathways and introduces over immune responses, thereby causing WHIM syndrome. So far, there is only one reported case of WHIM syndrome with a missense mutation, CXCR4, which has a full length of C-terminus with entire phosphorylation sites, no change in all potential phosphorylation sites. The mechanism of the missense mutation (CXCR4) causing WHIM syndrome is unknown. This study aimed to characterize the effect of mutation at the 343 site of CXCR4 causing the replacement of arginine/E with glutamic acid/K on the receptor signal transduction, and elucidate the mechanism underling CXCR4 causing WHIM in the reported family.

Results: We completed a series of mutagenesis to generate different mutations at the 343 site of CXCR4 tail, and established a series of HeLa cell lines stably expressing CXCR4 or CXCR4 (glutamic acid/E replaced with aspartic acid/D) or CXCR4 (glutamic acid/E replaced with lysine/K) or CXCR4 (glutamic acid/E replaced with arginine/R) or CXCR4 (glutamic acid/E replaced with alanine/A) and then systematically analyzed functions of the CXCR4 mutants above. Results showed that the cells overexpressing of CXCR4 had no functional changes with comparison that of wild type CXCR4. However, the cells overexpressing of CXCR4 or CXCR4 or CXCR4 had enhanced cell migration, prolonged the phosphorylation of ERK1/2, p38, JNK1/2/3, aggravated activation of PI3K/AKT/NF-κB signal pathway, introduced higher expression of TNFa and IL6, suggesting over immune response occurred in CXCR4 mutants with charge change at the 343 site of receptor tail, as a result, causing WHIM syndrome. Biochemical analysis of those mutations at the 343 site of CXCR4 above shows that CXCR4 mutants with no matter positive or neutral charge have aberrant signal pathways downstream of activated mutated CXCR4, only CXVR4 with negative charge residues at the site shows normal signal pathway post activation with stromal-derived factor (SDF1, also known as CXCL12).

Conclusion: Taken together, our results demonstrated that the negative charge at the 343 site of CXCR4 plays an essential role in regulating the down-stream signal transduction of CXCR4 for physiological events, and residue charge changes, no matter positive or neutral introduce aberrant activities and functions of CXCR4, thus consequently lead to WHIM syndrome.
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http://dx.doi.org/10.1186/s12860-021-00347-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7825245PMC
January 2021

A novel splicing pathogenic variant in COL1A1 causing osteogenesis imperfecta (OI) type I in a Chinese family.

Mol Genet Genomic Med 2020 09 25;8(9):e1366. Epub 2020 Jun 25.

The Second Hospital, Shanxi Medical University, Taiyuan, China.

Background: Osteogenesis imperfecta (OI), a rare autosomal inheritable disorder characterized by bone fragility and skeletal deformity, is caused by pathogenic variants in genes impairing the synthesis and processing of extracellular matrix protein collagen type I. With the use of next-generation sequencing and panels approaches, an increasing number of OI patients can be confirmed and new pathogenic variants can be discovered. This study sought to identify pathogenic gene variants in a Chinese family with OI I.

Methods: Whole-exome sequencing was used to identify pathogenic variants in the proband, which is confirmed by Sanger sequencing and cosegregation analysis; MES, HSF, and Spliceman were used to analyze this splicing variant;qRT-PCR was performed to identify the mRNA expression level of COL1A1 in patient peripheral blood samples; Minigene splicing assay was performed to mimic the splicing process of COL1A1 variants in vitro; Analysis of evolutionary conservation of amino acid residues and structure prediction of the mutant protein.

Results: A novel splicing pathogenic variant (c.3814+1G>T) was identified in this OI family by using whole-exome sequencing, Sanger sequencing, and cosegregation analysis. Sequencing of RT-PCR products from the COL1A1 minigene variant reveals a 132-nucleotide (nt) insertion exists at the junction between exons 48 and exon 49 of the COL1A1 cDNA. Splicing assay indicates that the mutated minigene produces an alternatively spliced transcript which may cause a frameshift resulting in early termination of protein expression. The molecular analysis suggested that the altered amino acid is located at the C-terminus of type I procollagen.

Conclusion: Our study reveals the pathogenesis of a novel COL1A1 splicing pathogenic variant c.3814+1G>T in a Chinese family with OI I.
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http://dx.doi.org/10.1002/mgg3.1366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7507304PMC
September 2020

Functional Characterisation of the Autophagy ATG12~5/16 Complex in .

Cells 2020 05 9;9(5). Epub 2020 May 9.

Centre for Biochemistry, Institute of Biochemistry I, Medical Faculty, University of Cologne, 50931 Cologne, Germany.

Macroautophagy, a highly conserved and complex intracellular degradative pathway, involves more than 20 core autophagy (ATG) proteins, among them the hexameric ATG12~5/16 complex, which is part of the essential ubiquitin-like conjugation systems in autophagy. single, double, and triple gene knock-out mutant strains displayed similar defects in the conjugation of ATG8 to phosphatidylethanolamine, development, and cell viability upon nitrogen starvation. This implies that ATG5, 12 and 16 act as a functional unit in canonical autophagy. Macropinocytosis of TRITC dextran and phagocytosis of yeast were significantly decreased in ATG5¯ and ATG5¯/12¯ and even further in ATG5¯/12¯/16¯ cells. In contrast, plaque growth on was about twice as fast for ATG5¯ and ATG5¯/12¯/16¯ cells in comparison to AX2, but strongly decreased for ATG5¯/12¯ cells. Along this line, phagocytic uptake of was significantly reduced in ATG5¯/12¯ cells, while no difference in uptake, but a strong increase in membrane association of was seen for ATG5¯ and ATG5¯/12¯/16¯ cells. Proteasomal activity was also disturbed in a complex fashion, consistent with an inhibitory activity of ATG16 in the absence of ATG5 and/or ATG12. Our results confirm the essential function of the ATG12~5/16 complex in canonical autophagy, and furthermore are consistent with autophagy-independent functions of the complex and its individual components. They also strongly support the placement of autophagy upstream of the ubiquitin-proteasome system (UPS), as a fully functional UPS depends on autophagy.
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http://dx.doi.org/10.3390/cells9051179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290328PMC
May 2020

Identification of a novel compound heterozygous IDUA mutation underlies Mucopolysaccharidoses type I in a Chinese pedigree.

Mol Genet Genomic Med 2020 01 23;8(1):e1058. Epub 2019 Nov 23.

Institutes of Biomedical Sciences, Shanxi University, Taiyuan, China.

Background: Mucopolysaccharidosis type I (MPS I) is a rare autosomal storage disorder resulting from the defective alpha-L-iduronidase (encoded by IDUA) enzyme activity and accumulation of glycosaminoglycans (GAGs) in lysosomes. So far, more than 100 IDUA causative mutations have been identified leading to three MPS I phenotypic subtypes: Hurler syndrome (severe form), Hurler/Scheie syndrome (intermediate form), and Scheie syndrome (mild form).

Methods: Whole-exome sequencing (WES) was performed to identify the underlying genetic mutations. To verify the identified variations, Sanger sequencing was performed for all available family members following PCR amplification. The impact on IDUA protein was analyzed by sequential analysis and homology modeling.

Results: A novel IDUA heterozygous single base insertion (c.1815dupT, p.V606Cfs51 ) and a known missence mutation (c.T1037G, p.L346R) were detected in our patient diagnosed as congenital heart disease with heart valve abnormalities. The novel frameshift mutation results in a complete loss of 48 amino acids in the Ig-like domain and causes the formation of a putative protein product which might affect the IDUA enzyme activity.

Conclusions: A novel compound heterozygous IDUA mutation (c.1815dupT, p.V606Cfs51 ) was found in a Chinese MPS I family. The identification of the mutation facilitated accurate genetic counseling and precise medical intervention for MPS I in China.
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http://dx.doi.org/10.1002/mgg3.1058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978265PMC
January 2020

Bacteria Exploit Autophagy For Their Own Benefit.

Infect Drug Resist 2019 11;12:3205-3215. Epub 2019 Oct 11.

Institutes of Biomedical Sciences, Shanxi University, Taiyuan 030006, People's Republic of China.

Autophagy is a lysosomal degradation pathway to clear long-lived proteins, protein aggregates, and damaged organelles. Certain microorganisms can be eliminated by an autophagic degradation process termed xenophagy. However, many pathogens deploy highly evolved mechanisms to evade autophagic degradation. What is more, series of pathogens have developed different strategies to exploit autophagy to ensure their survival. These bacteria could induce autophagy and/or prevent autophagosomes fusion with lysosomes through secreted effector proteins or utilizing host components, thereby maintaining the localization of the bacteria within the autophagosomes where they replicate. Here, we review the current knowledge of the mechanisms developed by the bacteria to benefit from autophagy for their survival.
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http://dx.doi.org/10.2147/IDR.S220376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6792943PMC
October 2019

Functional evidence for a de novo mutation in WDR45 leading to BPAN in a Chinese girl.

Mol Genet Genomic Med 2019 09 22;7(9):e858. Epub 2019 Jul 22.

Institutes of Biomedical Science, Shanxi University, Taiyuan, China.

Background: Beta-propeller protein-associated neurodegeneration (BPAN, OMIM 300894) is an X-linked neurodegenerative disorder caused by mutations in WDR45. WDR45 is required for autophagy, defect in WDR45 impaired autophagy which contributes for the pathogenesis of BPAN. Previously, we reported a novel de novo mutation (c.1040_1041del, p.Glu347GlyfsTer7) in WDR45 (NM_007075) in a 3-year-old Chinese girl with BPAN.

Methods: The protein structure was constructed using SWISS-MODEL and the isoelectric point (pI) was predicted by the online pI/Mw tool at ExPASy. The functional effects of this mutation were predicted by two online software programs: PROVEN and MutationTaster. Stable overexpression of Flag-tagged wild-type or mutant WDR45 in HeLa cells was constructed. Protein levels of LC3 and p62 were analyzed by western blot upon treatment with/without autophagy inhibitor Bafilomycin A1, the formation of LC3 puncta were analyzed in HeLa cells transfected with mCherry-LC3 by confocal microscopy.

Results: The mutation resulted in a shift of pI from 6.74 to 8.84 and was predicted to be pathogenic. The protein levels of LC3-II and p62 were increased in cells overexpression of wild-type and mutant WDR45 while the protein levels were not increased in cells overexpression of mutant WDR45 upon treatment with autophagy inhibitor Bafilomycin A1. Results from confocal microscopy revealed that LC3-positive puncta were increased in cells expressing both wild-type and mutant WDR45 while the number of LC3-positive puncta was not increased in cells expressing mutant WDR45 upon treatment with Bafilomycin A1.

Conclusion: Our study evidenced that this novel mutation in WDR45 impaired autophagy in cells thus this mutation is the cause for BPAN in this patient.
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http://dx.doi.org/10.1002/mgg3.858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6732291PMC
September 2019

Functional evaluation of a novel GLA causative mutation in Fabry disease.

Mol Genet Genomic Med 2019 09 18;7(9):e864. Epub 2019 Jul 18.

Institutes of Biomedical Sciences, Shanxi University, Taiyuan, China.

Background: Fabry disease (FD), a rare X-linked α-galactosidase A (GLA) deficiency, resulting in progressive lysosomal accumulation of globotriaosylceramide in a variety of cell types. More and more disease-causing mutations in GLA have been identified in FD due to the advancement of molecular diagnostic tools. We found a novel mutation in a Chinese family with predominant Fabry's disease nephropathy.

Methods: All coding regions and exon-intron splice junctions of the GLA gene were sequenced to find sequence variations. We evaluated the impact on the GLA protein by analysis of the GLA mRNA, by sequential analysis and homology modeling, and by site-directed mutagenesis and in vitro expression studies.

Results: We identified a novel heterozygous missense mutation c.280T>C in our patient with variable phenotypic presentations of renal involvement. The novel GLA variant results in low expression of GLA mRNAs, impaired or loss of the disulfate bridge structure of wild-type GLA, reduced GLA activity and defected nuclear shape in the GFP-GLA-MT transfected HEK293T cells.

Conclusion: A novel GLA missense mutation, c.280T>C (Cys94Arg), was found in a Chinese family with predominant renal manifestations of FD. Our study reveals the pathogenesis of c.280T>C mutation to FD and provides scientific foundation for accurate diagnosis and precise medical intervention for FD.
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http://dx.doi.org/10.1002/mgg3.864DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6732343PMC
September 2019

A Novel α-Galactosidase A Splicing Mutation Predisposes to Fabry Disease.

Front Genet 2019 11;10:60. Epub 2019 Feb 11.

Institutes of Biomedical Sciences, Shanxi University, Taiyuan, China.

Fabry disease (FD) is a rare X-linked α-galactosidase A () deficiency, resulting in progressive lysosomal accumulation of globotriaosylceramide (Gb3) in a variety of cell types. Here, we report a novel splicing mutation (c.801 + 1G > A) that results in alternative splicing in of a FD patient with variable phenotypic presentations of renal involvement. Sequencing of the RT-PCR products from the patient's blood sample reveals a 36-nucleotide (nt) insertion exists at the junction between exons 5 and 6 of the cDNA. Splicing assay indicates that the mutated minigene produces an alternatively spliced transcript which causes a frameshift resulting in an early termination of protein expression. Immunofluorescence shows puncta in cytoplasm for mutated whereas uniform staining small dots evenly distributed inside cytoplasm for wild type in transfected HeLa cells. The increased senescence and decreased GLA enzyme activity suggest that the abnormalities might be due to the altered localization which further might result from the lack of the C-terminal end of GLA. Our study reveals the pathogenesis of splicing mutation c.801 + 1G > A to FD and provides scientific foundation for accurate diagnosis and precise medical intervention for FD.
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http://dx.doi.org/10.3389/fgene.2019.00060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6396734PMC
February 2019

The Role of ATG16 in Autophagy and The Ubiquitin Proteasome System.

Cells 2018 12 20;8(1). Epub 2018 Dec 20.

Center for Biochemistry, Medical Faculty, University of Cologne, Joseph-Stelzmann-Str. 52, 50931 Cologne, Germany.

Autophagy and the ubiquitin proteasome system (UPS) are the two major cellular degradation pathways, which are critical for the maintenance of cell homeostasis. The two pathways differ in their mechanisms and clients. The evolutionary conserved ATG16 plays a key role in autophagy and appears to link autophagy with the UPS. Here, we review the role of ATG16 in different species. We summarize the current knowledge of its functions in autophagosome membrane expansion and autophagosome formation, in Crohn's disease, and in bacterial sequestration. In addition, we provide information on its autophagy-independent functions and its role in the crosstalk between autophagy and the UPS.
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http://dx.doi.org/10.3390/cells8010002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6356889PMC
December 2018

ATG16 mediates the autophagic degradation of the 19S proteasomal subunits PSMD1 and PSMD2.

Eur J Cell Biol 2018 Nov 17;97(8):523-532. Epub 2018 Sep 17.

Center for Biochemistry, Medical Faculty, University of Cologne, Joseph-Stelzmann-Str. 52, 50931 Cologne, Germany.. Electronic address:

Autophagy and the ubiquitin proteasome system are the two major cellular processes for protein and organelle recycling and clearance in eukaryotic cells. Evidence is accumulating that these two pathways are interrelated through adaptor proteins. Here, we found that PSMD1 and PSMD2, both components of the 19S regulatory particle of the proteasome, directly interact with Dictyostelium discoideum autophagy 16 (ATG16), a core autophagosomal protein. ATG16 is composed of an N-terminal domain, which is responsible for homo-dimerization and binding to ATG5 and a C-terminal β-propeller structure. Deletion analysis of ATG16 showed that the N-terminal half of ATG16 interacted directly only with PSMD1, while the C-terminal half interacted with both, PSMD1 and PSMD2. RFP-tagged PSMD1 as well as PSMD2 were enriched in large puncta, reminiscent of autophagosomes, in wild-type cells. These puncta were absent in atg16‾ and atg9‾/16‾ cells and weaker and less frequent in atg9‾ cells, showing that ATG16 was crucial and the autophagic process important for their formation. Co-expression of ATG16-GFP or GFP-ATG8a(LC3) with RFP-PSMD1 or RFP-PSMD2, respectively, in atg16‾ or wild-type cells revealed many instances of co-localization, suggesting that RFP-PSMD1 or RFP-PSMD2 positive puncta constitute autophagosomes. LysoTracker labeling and a proteolytic cleavage assay confirmed that PSMD1 and PSMD2 were present in lysosomes in wild-type cells. In vivo, ATG16 is required for their enrichment in ATG8a positive puncta, which mature into autolysosomes. We propose that ATG16 links autophagy and the ubiquitin proteasome system.
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http://dx.doi.org/10.1016/j.ejcb.2018.09.002DOI Listing
November 2018

A novel mutation in leading to spondyloepiphyseal dysplasia congenita in a Chinese family.

Hum Genome Var 2018 11;5:17059. Epub 2018 Jan 11.

Institutes of Biomedical Sciences, Key Laboratory of Chemical Biology and Molecular Engineering of National Ministry of Education, Shanxi University, Taiyuan, China.

Spondyloepiphyseal dysplasia congenita (SEDC) is an extremely rare autosomal dominant chondrodysplasia that is usually caused by substitution of glycine with another amino acid in the triple helical region of COL2A1. Herein, we describe a case of SEDC in a Chinese family with a novel mutation in the gene, c.1150G>A (p.Gly384Ser), which may impair protein stability and lead to dysfunction of type II collagen.
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http://dx.doi.org/10.1038/hgv.2017.59DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5763142PMC
January 2018

The two Dictyostelium discoideum autophagy 8 proteins have distinct autophagic functions.

Eur J Cell Biol 2017 Jun 4;96(4):312-324. Epub 2017 Apr 4.

Center for Biochemistry, Institute of Biochemistry I, Medical Faculty, University of Cologne, Joseph-Stelzmann-Str. 52, 50931 Cologne, Germany. Electronic address:

Autophagy is a highly conserved cellular degradation pathway which is crucial for various cellular processes. The autophagic process is subdivided in the initiation, autophagosome maturation and lysosomal degradation phases and involves more than forty core and accessory autophagy-related (ATG) proteins. Autophagy 8 (ATG8, in mammals LC3) is a well-established marker of autophagy and is linked to the autophagic membrane from initiation until fusion with the lysosome. We generated single and double knock-out mutants of the two Dictyostelium paralogues, ATG8a and ATG8b, as well as strains that expressed RFP-ATG8a and/or GFP-ATG8b, RFP-ATG8b, RFP-GFP-ATG8a or RFP-GFP-ATG8b in different knock-out mutants. The ATG8b¯ mutant displayed only subtle phenotypic changes in comparison to AX2 wild-type cells. In contrast, deletion of ATG8a resulted in a complex phenotype with delayed development, reduced growth, phagocytosis and cell viability, an increase in ubiquitinylated proteins and a concomitant decrease in proteasomal activity. The phenotype of the ATG8a¯/b¯ strain was, except for cell viability, in all aforementioned aspects more severe, showing that both proteins function in parallel during most analysed cellular processes. Immunofluorescence analysis of knock-out strains expressing either RFP-GFP-ATG8a or RFP-GFP-ATG8b suggests a crucial function for ATG8b in autophagosome-lysosome fusion. Quantitative analysis of strains expressing RFP-ATG8a, RFP-ATG8b, or RFP-ATG8a and GFP-ATG8b revealed that ATG8b generally localised to small and large vesicles, whereas ATG8a preferentially co-localised with ATG8b on large vesicles, indicating that ATG8b associated with nascent autophagosomes before ATG8a, which is supported by previous results (Matthias et al., 2016). Deconvoluted confocal fluorescence images showed that ATG8b localised around ATG8a and was presumably mainly present on the outer membrane of the autophagosome while ATG8a appears to be mainly associated with the inner membrane. In summary, our data show that ATG8a and ATG8b have distinct functions and are involved in canonical as well as non-canonical autophagy. The data further suggest that ATG8b predominantly acts as adapter for the autophagy machinery at the outer and ATG8a as cargo receptor at the inner membrane of the autophagosome.
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http://dx.doi.org/10.1016/j.ejcb.2017.03.014DOI Listing
June 2017

Autophagy in Dictyostelium: Mechanisms, regulation and disease in a simple biomedical model.

Autophagy 2017 Jan 7;13(1):24-40. Epub 2016 Oct 7.

a Instituto de Investigaciones Biomédicas "Alberto Sols" (CSIC-UAM) , Madrid , Spain.

Autophagy is a fast-moving field with an enormous impact on human health and disease. Understanding the complexity of the mechanism and regulation of this process often benefits from the use of simple experimental models such as the social amoeba Dictyostelium discoideum. Since the publication of the first review describing the potential of D. discoideum in autophagy, significant advances have been made that demonstrate both the experimental advantages and interest in using this model. Since our previous review, research in D. discoideum has shed light on the mechanisms that regulate autophagosome formation and contributed significantly to the study of autophagy-related pathologies. Here, we review these advances, as well as the current techniques to monitor autophagy in D. discoideum. The comprehensive bioinformatics search of autophagic proteins that was a substantial part of the previous review has not been revisited here except for those aspects that challenged previous predictions such as the composition of the Atg1 complex. In recent years our understanding of, and ability to investigate, autophagy in D. discoideum has evolved significantly and will surely enable and accelerate future research using this model.
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http://dx.doi.org/10.1080/15548627.2016.1226737DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5240833PMC
January 2017

The phenotypes of ATG9, ATG16 and ATG9/16 knock-out mutants imply autophagy-dependent and -independent functions.

Open Biol 2015 Apr;5(4):150008

Zentrum für Biochemie, Medizinische Fakultät, Universität zu Köln, Joseph-Stelzmann-Strasse 52, Köln 50931, Germany

Macroautophagy is a highly conserved intracellular bulk degradation system of all eukaryotic cells. It is governed by a large number of autophagy proteins (ATGs) and is crucial for many cellular processes. Here, we describe the phenotypes of Dictyostelium discoideum ATG16(-) and ATG9(-)/16(-) cells and compare them to the previously reported ATG9(-) mutant. ATG16 deficiency caused an increase in the expression of several core autophagy genes, among them atg9 and the two atg8 paralogues. The single and double ATG9 and ATG16 knock-out mutants had complex phenotypes and displayed severe and comparable defects in pinocytosis and phagocytosis. Uptake of Legionella pneumophila was reduced. In addition, ATG9(-) and ATG16(-) cells had dramatic defects in autophagy, development and proteasomal activity which were much more severe in the ATG9(-)/16(-) double mutant. Mutant cells showed an increase in poly-ubiquitinated proteins and contained large ubiquitin-positive protein aggregates which partially co-localized with ATG16-GFP in ATG9(-)/16(-) cells. The more severe autophagic, developmental and proteasomal phenotypes of ATG9(-)/16(-) cells imply that ATG9 and ATG16 probably function in parallel in autophagy and have in addition autophagy-independent functions in further cellular processes.
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http://dx.doi.org/10.1098/rsob.150008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4422124PMC
April 2015

Microarray analysis of differentially expressed microRNAs in non-regressed and regressed bovine corpus luteum tissue; microRNA-378 may suppress luteal cell apoptosis by targeting the interferon gamma receptor 1 gene.

J Appl Genet 2011 Nov 8;52(4):481-6. Epub 2011 Jun 8.

College of Animal Science and Veterinary Medicine, Jilin University, Changchun, China.

MicroRNAs (miRNAs) are small non-coding endogenous RNA molecules that down-regulate the expression of target genes in a sequence-dependent manner. Recent studies indicated that miRNAs are mechanistically involved in the regulation of the mammalian corpus luteum (CL). However, few studies have profiled the different miRNA expression patterns in bovine non-regressed and regressed CL. In this study, miRNA microarray was employed to investigate the different miRNA expression patterns in bovine CL. Among the 13 differentially expressed miRNAs, seven were preferentially expressed in non-regressed CL, while six miRNAs were more highly expressed in regressed CL. Real-time RT-PCR was used to validate the microarray results. Mir-378 miRNA, known to be associated with apoptosis, was 8.54-fold (P < 0.01) up-regulated in non-regressed CL, and the interferon gamma receptor 1 (IFNGR1) gene, which potentially plays a role in apoptosis of the luteal cell, was predicted to be the target of mir-378. The results of real-time RT-PCR of mir-378 and western blot analysis of the IFNGR1 protein at different stages of CL development showed that mir-378 decreased the expression of IFNGR1 protein but not IFNGR1 mRNA. Taken together, our data support a direct role for miRNA in apoptosis of bovine CL.
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http://dx.doi.org/10.1007/s13353-011-0055-zDOI Listing
November 2011
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