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| 3. |
- Klionsky, Daniel J., et al.
(författare)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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Ingår i: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Forskningsöversikt (refereegranskat)abstract
- In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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- 2019
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Tidskriftsartikel (refereegranskat)
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- Wang, Mei jun, et al.
(författare)
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SIRT1-dependent deacetylation of Txnip H3K9ac is critical for exenatide-improved diabetic kidney disease
- 2023
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Ingår i: Biomedicine and Pharmacotherapy. - 0753-3322. ; 167
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Tidskriftsartikel (refereegranskat)abstract
- Glucagon-like peptide 1 receptor agonist exenatide (exendin-4) has potential protective capabilities against diabetic kidney disease (DKD). However, the underlying mechanism has not been fully elucidated. The expression of thioredoxin-interacting protein (Txnip) is upregulated during DKD progression by histone acetylation. Sirtuin 1 (SIRT1) is a deacetylase and is decreased in DKD, which indicates that it may regulate Txnip in this disease. Here, we used whole-body heterozygous Sirt1 knockout (Sirt1+/-) and kidney-specific Sirt1 knockout (KSK) mice to investigate whether SIRT1 regulates Txnip via histone deacetylation in DKD and exenatide-alleviated DKD. Exenatide substantially improved renal pathological damage, decreased the albumin-to-creatinine ratio (ACR), upregulated SIRT1 expression, and downregulated Txnip expression in kidneys of high-fat diet-treated C57BL/6J mice. However, these effects diminished in Sirt1+/- and KSK mice under exenatide treatment. The downregulation of Txnip expression by exendin-4 in high-glucose-treated SV40 MES13 cells was hampered during Sirt1 knockdown. These results demonstrate that kidney SIRT1 is indispensable in exenatide-improved DKD and downregulation of Txnip expression. Exendin-4 mechanistically downregulated Txnip histone 3 lysine 9 acetylation (H3K9ac) in a SIRT1-dependent manner and decreased spliced X-box binding protein 1 (XBP1s) recruitment to the Txnip promoter. These findings provide epigenetic evidence elucidating the specific mechanism for exenatide-mediated DKD alleviation and highlight the importance of Txnip as a promising therapeutic target for DKD.
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- Bailey, Matthew H., et al.
(författare)
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Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples
- 2020
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts.
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- Su, Xiao Dong, et al.
(författare)
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A large-scale, high-efficiency and low-cost platform for structural genomics studies
- 2006
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Ingår i: Acta Crystallographica. Section D: Biological Crystallography. - 0907-4449. ; 62:Pt 8, s. 51-843
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Tidskriftsartikel (refereegranskat)abstract
- A large-scale, high-efficiency and low-cost platform based on a Beckman Coulter Biomek FX and custom-made automation systems for structural genomics has been set up at Peking University, Beijing, People's Republic of China. This platform has the capacity to process up to 2000 genes per year for structural and functional analyses. Bacillus subtilis, a model organism for Gram-positive bacteria, and Streptococcus mutans, a major pathogen of dental caries, were selected as the main targets. To date, more than 470 B. subtilis and 1200 S. mutans proteins and hundreds of proteins from other sources, including human liver proteins, have been selected as targets for this platform. The selected genes are mainly related to important metabolism pathways and/or have potential relevance for drug design. To date, 40 independent structures have been determined; of these 11 are in the category of novel structures by the criterion of having less than 30% sequence identity to known structures. More than 13 structures were determined by SAD/MAD phasing. The macromolecular crystallography beamline at the Beijing Synchrotron Radiation Facility and modern phasing programs have been crucial components of the operation of the platform. The idea and practice of the genomic approach have been successfully adopted in a moderately funded structural biology program and it is believed this adaptation will greatly improve the production of protein structures. The goal is to be able to solve a protein structure of moderate difficulty at a cost about US 10,000 dollars.
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- Alvarez Fernandez, Marcia, et al.
(författare)
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Crystal structure of human cystatin D, a cysteine peptidase inhibitor with restricted inhibition profile.
- 2005
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 280:18, s. 18221-18228
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Tidskriftsartikel (refereegranskat)abstract
- Cystatins are natural inhibitors of papain-like (family C1) and legumain-related (family C13) cysteine peptidases. Cystatin D is a type 2 cystatin, a secreted inhibitor found in human saliva and tear fluid. Compared to its homologues, cystatin D presents an unusual inhibition profile with a preferential inhibition cathepsin S > cathepsin H > cathepsin L, and no inhibition of cathepsin B or pig legumain. To elucidate the structural reasons for this specificity, we have crystallized recombinant human Arg26-cystatin D and solved its structures at room temperature and at cryo conditions to 2.5 and 1.8 Å resolution, respectively. Human cystatin D presents the typical cystatin fold, with a five-stranded anti-parallel -sheet wrapped around a five-turn -helix. The structures reveal differences in the peptidase-interacting regions when compared to other cystatins, providing plausible explanations to the restricted inhibitory specificity of cystatin D for some papain-like peptidases, and its lack of reactivity towards legumain-related enzymes. This is the final, accepted and revised manuscript of this article. Use alternative location to go to the published article. Requires subscription.
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