Off-target inhibition of NGLY1 by the polycaspase inhibitor Z-VAD-fmk induces cellular autophagy.
Off-target inhibition of NGLY1 by the polycaspase inhibitor Z-VAD-fmk induces cellular autophagy.
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DOI：
10.1111/febs.16345
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发表时间：
2022-06
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影响因子：
--
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文献类型：
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多肽半胱氨酸酶抑制剂Z‐VAD‐fmk作为肽：N -聚糖酶（NGLY1）的抑制剂，NGLY1是一种内糖苷酶，在内质网相关降解（ERAD）过程中从内质网（ER）输出的糖蛋白中切割N -连接的聚糖。Z‐VAD‐fmk抑制N‐甘聚糖酶和siRNA介导的NGLY1敲低（KD）均可诱导HEK 293细胞中GFP‐LC3阳性点。内质网应激标志物的激活或活性氧（ROS）的诱导在两种条件下都没有观察到。此外，Ca2+处理是不受影响的，当观察释放细胞内的储存。在药理学抑制NGLY1或NGLY1 KD的条件下，观察到自噬体形成上调而不损害自噬通量。通过免疫沉淀（IP）和基于质谱的蛋白质组学分析富集自噬体揭示了相似的自噬体蛋白质含量。对自噬小体IPs中富集蛋白的基因本体分析显示，参与蛋白质翻译、定位和靶向、RNA降解和蛋白质复合物分解的因子过多。自噬的上调代表了细胞对NGLY1抑制或KD的适应，而ATG13‐缺陷小鼠胚胎成纤维细胞（mef）在这些条件下表现出活性降低。相反，用pan - caspase抑制剂Q - VD - OPh治疗不会诱导细胞自噬。因此，使用Z‐VAD‐fmk的实验由于NGLY1抑制的影响而变得复杂，包括诱导自噬，而Q‐VD‐OPh代表了一种不受这种限制的替代caspase抑制剂。肽：N -甘聚糖酶1，肽- N(4) - （N -乙酰- β -氨基葡萄糖）天冬酰胺酰胺酶[EC:3.5.1.52]。在HEK293细胞中，多caspase抑制剂Z‐VAD‐fmk抑制NGLY1可诱导自噬，增加自噬体的形成，而不是破坏自噬通量。同样，NGLY1 siRNA敲低也能诱导自噬。免疫沉淀和蛋白质组学分析显示，在Z‐VAD‐fmk治疗和NGLY1敲除中，自噬体蛋白含量相当。另一种广谱caspase抑制剂Q‐VD‐OPh不抑制NGLY1，也未观察到诱导自噬。
The polycaspase inhibitor Z‐VAD‐fmk acts as an inhibitor of peptide: N‐glycanase (NGLY1), an endoglycosidase which cleaves N‐linked glycans from glycoproteins exported from the endoplasmic reticulum (ER) during ER‐associated degradation (ERAD). Both pharmacological N‐glycanase inhibition by Z‐VAD‐fmk and siRNA‐mediated knockdown (KD) of NGLY1 induce GFP‐LC3‐positive puncta in HEK 293 cells. The activation of ER stress markers or induction of reactive oxygen species (ROS) is not observed under either condition. Moreover, Ca2+ handling is unaffected when observing release from intracellular stores. Under conditions of pharmacological NGLY1 inhibition or NGLY1 KD, upregulation of autophagosome formation without impairment of autophagic flux is observed. Enrichment of autophagosomes by immunoprecipitation (IP) and mass spectrometry‐based proteomic analysis reveals comparable autophagosomal protein content. Gene ontology analysis of proteins enriched in autophagosome IPs shows overrepresentation of factors involved in protein translation, localization and targeting, RNA degradation and protein complex disassembly. Upregulation of autophagy represents a cellular adaptation to NGLY1 inhibition or KD, and ATG13‐deficient mouse embryonic fibroblasts (MEFs) show reduced viability under these conditions. In contrast, treatment with pan‐caspase inhibitor, Q‐VD‐OPh, does not induce cellular autophagy. Therefore, experiments with Z‐VAD‐fmk are complicated by the effects of NGLY1 inhibition, including induction of autophagy, and Q‐VD‐OPh represents an alternative caspase inhibitor free from this limitation. Peptide:N‐glycanase1, Peptide‐N(4)‐(N‐acetyl‐beta‐glucosaminyl)asparagine amidase [EC:3.5.1.52]. Inhibition of NGLY1 by polycaspase inhibitor Z‐VAD‐fmk in HEK293 cells leads to induction of autophagy and increase in autophagosome formation rather than disruption of autophagic flux. Similarly, autophagy induction is observed with NGLY1 siRNA knockdown. Immunoprecipitation and proteomic analysis reveal comparable autophagosomal protein content in both Z‐VAD‐fmk treatment and NGLY1 knockdown. The alternative broad‐spectrum caspase inhibitor Q‐VD‐OPh does not inhibit NGLY1, and no induction of autophagy is observed.
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影响因子：
3.5
作者：
Deszcz, L;Selpelt, J;Kuechler, E
通讯作者：
Kuechler, E
影响因子：
4.8
作者：
Garcia-Calvo, M;Peterson, EP;Thornberry, NA
通讯作者：
Thornberry, NA
DOI：
10.1074/mcp.m114.041012
发表时间：
2015-01
期刊：
Molecular & cellular proteomics : MCP
影响因子：
--
作者：
Keilhauer EC;Hein MY;Mann M
通讯作者：
Mann M
DOI：
10.1016/j.bbamcr.2017.02.013
发表时间：
2017-06-01
影响因子：
5.1
作者：
Luyten, Tomas;Welkenhuyzen, Kirsten;Bultynck, Geert
通讯作者：
Bultynck, Geert
影响因子：
5.3
作者：
Kaizuka, Takeshi;Mizushima, Noboru
通讯作者：
Mizushima, Noboru