1. Transmembrane Protein 214 (TMEM214) Mediates Endoplasmic Reticulum Stress-induced Caspase 4 Enzyme Activation and Apoptosis 汇报人：蓝田丰

2. IMI CONFIDENTIAL www.xianimi.com 2 Introduction Endoplasmic reticulum (ER) functions in the folding of secretory and membrane proteins, calcium homeostasis, and lipid biosynthesis Several cellular stress conditions, such as nutrient deprivation, alterations in glycosylation status, hypoxia, changes in calcium homeostasis, and treatment with a variety of agents, can lead to accumulation and aggregation of unfolded and/or misfolded proteins in the endoplasmic reticulum (ER) lumen and cause so-called ER stress (unfold protein response) Prolonged ER stress triggers apoptosis!!

3. IMI CONFIDENTIAL www.xianimi.com 3 The CCAAT/enhancer-binding protein homologous protein(CHOP)-, JNK-, and caspase-dependent pathways are involved in ER stress- induced apoptosis. Mouse: caspase 12---procaspase 9---procaspase 3---apoptosis Human: caspase 12----procaspase 9 ER stress----caspase 4----apoptosis X ?

4. IMI CONFIDENTIAL www.xianimi.com 4 Transmembrane protein 214(TMEM214) play roles in dengue virus replication and cellular mRNA degradation A functional screening for proteins found that TMEM214 can induce cell death

5. IMI CONFIDENTIAL TMEM214 was localized on the ER and mediated ER stress-induced apoptosis. Overexpression of TMEM214 led to apoptosis, which required the activity of caspase 4. Conversely, caspase 4-induced apoptosis was blocked by knockdown of TMEM214. Furthermore, TMEM214 recruited caspase 4 to the ER, which was essential for ER stress-induced procaspase 4 activation and apoptosis. The findings suggest that TMEM214 mediates stress-induced apoptosis by acting as an anchor for the recruitment of procaspase 4 to the ER and its subsequent activation summary

6. IMI CONFIDENTIAL www.xianimi.com 6 Results round up detachment positive control DNA fragmentation 结果一 TMEM214 Mediates ER Stress-induced Apoptosis

7. IMI CONFIDENTIAL www.xianimi.com 7 Overexpression of TMEM214 induced apoptosis in a dose-dependent manner unfolded protein response markers

8. IMI CONFIDENTIAL www.xianimi.com 8 chose 3 target sequences for human TMEM214 cDNA

9. IMI CONFIDENTIAL www.xianimi.com 9 毒胡萝卜素 布雷菲尔 德菌素 A external apoptosis inducer

10. IMI CONFIDENTIAL www.xianimi.com 10 放线菌素 D 依托泊苷 induce mitochondrion-dependent apoptosis TMEM214 specifically mediates ER stress-induced apoptosis

11. IMI CONFIDENTIAL www.xianimi.com 11 结果二 TMEM214-mediated Apoptosis Is Independent of CHOP and JNK knockdown of TMEM214 had no marked effects on TG induced JNK phosphorylation and CHOP induction

12. IMI CONFIDENTIAL www.xianimi.com 12 knockdown of CHOP had no marked effect on TMEM214-mediated apoptosis

13. IMI CONFIDENTIAL www.xianimi.com 13 结果三 TMEM214 Is Localized to the Outer Membrane of the ER 高尔基体 内质网 线粒体 内质网 TMEM214 was mostly colocalized with the ER marker Sec61 but with little overlapping with the Golgi body or mitochondrial marker

14. IMI CONFIDENTIAL www.xianimi.com 14 细胞分馏实验（ cell fraction ） 凋亡抑制蛋白（细胞质 ） 细胞凋亡诱导因子（线粒体） 分子伴侣（内质网） trypsin-protection assays （胰酶保护试验） TMEM214 and procaspase 4 are localized on the outer membrane of the ER TMEM214 existed mostly in the ER-containing membrane fraction

15. IMI CONFIDENTIAL www.xianimi.com 15 结果四 Caspase 4 Is Required for ER Stress-induced Apoptosis Z-LEVD-FMK ： caspase 4 inhibitor caspase 4 is localized at the ER and activated in ER stress-induced apoptosis

16. IMI CONFIDENTIAL www.xianimi.com 16 Z-LEVD-FMK inhibited the cleavage of procaspase 4 and the caspase substrate poly-(ADP-ribose) polymerase 1 (PARP-1)

17. IMI CONFIDENTIAL www.xianimi.com 17 结果五 Caspase 4 and TMEM214 Are Mutually Involved in ER Stress-induced Apoptosis

18. IMI CONFIDENTIAL www.xianimi.com 18 both TMEM214 and caspase 4 are required for ER stress-induced apoptosis

19. IMI CONFIDENTIAL www.xianimi.com 19 apoptotic morphological change induced by overexpression of procaspase 4 was inhibited in TMEM214 knockdown cells

20. IMI CONFIDENTIAL www.xianimi.com 20 the percentage of apoptotic cells induced by overexpression of procaspase 4 but not procaspase 8 was decreased significantly in TMEM214 knockdown cells

21. IMI CONFIDENTIAL www.xianimi.com 21 inactive procaspase 4(C258S), but not mutant of procaspase 8, procaspase 8(C360S), markedly inhibited cell death induced by overexpression of TMEM214

22. IMI CONFIDENTIAL www.xianimi.com 22 结果六 Anchorage of Procaspase 4 to the ER by TMEM214 Is Important for ER Stress-induced Apoptosis anti-TMEM214 (αTM) preimmune serum (Pre). TMEM214 was constitutively associated with procaspase 4 in the presence or absence of TG or BFA stimulation

23. IMI CONFIDENTIAL www.xianimi.com 23 ER-associated procaspase 4 in both unstimulated and TG-stimulated cells was decreased by knockdown of TMEM214 the level of total procaspase 4 in cells was not changed

24. IMI CONFIDENTIAL www.xianimi.com 24 knockdown of TMEM214 markedly inhibited the cleavage of procaspase 4 and PARP-1 induced by TG

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26. IMI CONFIDENTIAL www.xianimi.com 26 N-terminal cytoplasmic region of TMEM214, amino acids 176 –354, was required for its binding with procaspase 4

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29. IMI CONFIDENTIAL www.xianimi.com 29 结果七 Caspase 7 Plays a Minor Role in TMEM214-mediated Apoptosis knockdown of TMEM214 only had a minor inhibitory effect on TG-induced procaspase 7 cleavage

30. IMI CONFIDENTIAL www.xianimi.com 30 caspase 7(C186S) partially inhibited apoptosis induced by overexpression of TMEM214

31. IMI CONFIDENTIAL The major conclusion of this manuscript is that TMEM214 mediates one of the ER stress-induced apoptotic pathways.