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Lub Protease Calpain2a txwv Innate Immunity Los ntawm Targeting TRAF6 hauv Teleost Fish

Dec 19, 2023

TNF receptor-associated factor 6 (TRAF6) plays lub luag haujlwm tseem ceeb ntawm kev hloov pauv hauv ob qho tib si tshuaj tua kab mob thiab tshuaj tiv thaiv kab mob. Txawm li cas los xij, cov txheej txheem tswj hwm ntawm TRAF6 nyob rau hauv qis vertebrates tau tshaj tawm tsawg dua. Nyob rau hauv txoj kev tshawb no, peb txheeb xyuas calpain2a, raws li ib tug tswv cuab ntawm calcium-dependent proteases tsev neeg nrog tshwj xeeb hydrolytic enzyme kev ua si, thiab ua hauj lwm raws li ib tug tseem ceeb regulator rau antibacterial thiab antiviral tiv thaiv nyob rau hauv teleost ntses. Raws li lipopolysaccharide (LPS) stimulation, knockdown ntawm calpain2a txhawb lub upregulation ntawm inflammatory cytokines. Mechanistically, calpain2a cuam tshuam nrog TRAF6 thiab txo cov protein ntawm TRAF6 los ntawm hydrolyzing. Tom qab poob ntawm enzymatic kev ua si, mutant calpain2a sib tw inhibits dimer tsim thiab nws pib-ubiquitination ntawm TRAF6. Knockdown ntawm calpain2a kuj txhawb nqa cellular antiviral teb. Mutant calpain2a tsis muaj hydrolase kev ua ub no cuam tshuam ubiquitination ntawm IFN regulatory factor (IRF) 3/7 los ntawm TRAF6. Ua ke, cov kev tshawb pom no tau faib cov calpain2a ua tus tswj xyuas qhov tsis zoo ntawm lub cev tiv thaiv kab mob los ntawm kev tsom TRAF6 hauv cov ntses teleost.

Desert ginseng-Improve immunity (2)

cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob

Raws li thawj kab ntawm kev tiv thaiv tawm tsam invading pathogens, innate immunity exerts lub luag hauj lwm tseem ceeb hauv kev tiv thaiv lub cev los ntawm cov kab mob invading thiab kab mob. Pattern-recognition receptors (PRRs) ntawm daim nyias nyias tam sim ntawd paub txog cov kab mob cuam tshuam nrog cov qauv molecular (PAMPs) 1-3. Peb qhov kev kawm ntau tshaj plaws PRRs hauv kev tiv thaiv kab mob hauv lub cev yog Tus Xov Tooj Zoo li receptors (TLRs), RIG-I-zoo li receptors (RLRs), thiab NOD-zoo li receptors (NLRs), uas paub txog PAMPs sib txawv thiab ua rau cov teeb liab cascades los ua kom muaj cov cytokines thiab antiviral factor4–6. TNF receptor-associated factor (TRAF) 6 yog ib feem tseem ceeb ntawm kev tawm tsam cov kab mob ntxeem tau thiab kis kab mob. Raws li ib qho tseem ceeb teeb liab txoj kev qhib los ntawm lipopolysaccharide (LPS), txoj kev nuclear transcription factor-κB (NF-κB) tau mapped hauv kev nthuav dav. Feem ntau ntawm cov protein ua haujlwm hauv NF-κB kev taw qhia txoj hauv kev tau kawm ntau dua 1,7. Myeloid sib txawv qhov tseem ceeb 88 (MyD88) yog thawj zaug qhib los ntawm TLRs teb rau LPS, thiab IRAK4 ua tus neeg nruab nrab los ntawm kev ua kom IRAK1 thiab IRAK2, uas txuas MyD88 rau TRAF68–11. Ubc13 thiab Uev1A catalyze K63-txuas kev sib txuas ntawm TRAF6, uas hloov K63 ubiquitin saw mus rau TAB2 thiab TAB3, ua rau kev ua kom TAK112–16. TRAF6- tswj IKK activator 2 (TRIKA2) uas yog tsim los ntawm TAK1, TAB1 thiab TAB2, activates inhibitor ntawm IκB kinase (IKK) / phosphorylation17–19. Transcription factor NF- κB nkag mus rau hauv lub nucleus thiab txhawb kev tsim cov inflammatory cytokines20. Thaum TLR7/9 paub tus kab mob no, MyD88- IRAKs-TRAF6 complex xa cov cim rau IFN regulatory factor (IRF) 7, txhawb nqa IRF7 ubiquitination, thiab ua kom nws cov phosphorylation mus rau hauv lub nucleus21,22. Nyob rau hauv RLR signaling pathway, RIG I thiab melanoma differentiation-associated gene 5 (MDA5) qhib mitochondrial antiviral signaling protein (MAVS) 23,24. Kev sib sau ntawm MAVS yog qhov tseem ceeb ntawm qhov pib ntawm RLR teeb liab kis tau tus mob23,25,26. TRAF2/3/6 yog thawj yam ua haujlwm los ntawm MAVS27,28. TRAF3 yuav qhib TBK1-IRF3 signaling, thiab MAVS-TRAF6 complex yuav qhib NF-κB signaling, uas txhawb IFN-1 expression27,29.

Desert ginseng-Improve immunity (12)

cistanche cov txiaj ntsig rau txiv neej-ua kom muaj zog tiv thaiv kab mob

Cov txheej txheem molecular ntawm TRAF6 ua kom pom tau lawm. TRAF6 nyob rau hauv daim ntawv dimer undergoes autoubiquitination nyob rau hauv lub catalysis ntawm Ubc13 thiab Uev1A, uas muaj feem xyuam rau lub nplhaib ntiv tes sau thiab ZF ntiv tes sau. Tus qauv ntawm TRAF6 / Ubc13 ~ UB lees paub lub luag haujlwm tseem ceeb ntawm TRAF6 dimer hauv kev sib sau thiab kev sib kis ntawm K63 ubiquitin chain30. Tsis tas li ntawd, nws tau raug tshaj tawm tias Ntiv nplhaib thiab ZF ntiv tes sau npe E2 ~ ub conjugates hauv zebrafish TRAF6 siv lead ua qauv, thiab nws tau pom tias TRAF6 tuaj yeem tsim ib heterodimer nrog TRAF5 los ntawm tib tsev neeg TRAF thawj zaug16. Cov deubiquitination tsev neeg cov proteins: A20, CYLD, USP2a, USP4, thiab USP20 lub hom phiaj TRAF6 kom tshem tawm K63-txuas kev sib txuas ubiquitination thiab txo qis downstream signaling31–35. Cov poov xab ob-hybrid system tshuaj xyuas qhov kev cuam tshuam ntawm UBE2O thiab TRAF6 thiab lees paub tias qhov kev cuam tshuam no cuam tshuam ncaj qha rau kev ua kom TRAF6 los ntawm MyD8836. Nyob rau hauv lub downstream signal transduction txheej txheem ntawm TRAF6, ECSIT raws li ib tug intermediate protein interacts nrog TRAF6 thiab TAK1 feem, thiab qhov kev sib tshuam no ntxiv dag zog rau kev khi ntawm TRAF6 thiab TAK137. Hauv LPS-induced TLR4 signaling, PRDXs tiv thaiv TRAF6 los ntawm kev nrhiav ECSIT kom xa K63 ubiquitin saw thiab tseem inhibit BECN1 qhib los ntawm TRAF6 hauv autophagy pathway38. MST4 qhib TRAF6 phosphorylation ntawm Thr463 thiab Thr486 thiab inhibits TRAF6 dimerization thiab ubiquitination39. Hauv cov tsiaj txhu, cov txheej txheem kev tswj hwm cov protein ntawm TRAF6 tau kawm dav hauv kev tiv thaiv kab mob thiab tiv thaiv kab mob. Txawm li cas los xij, kev tshawb fawb ntawm TRAF6 hauv cov vertebrates qis dua tau kawm. Kev tiv thaiv kab mob hauv cov ntses teleost feem ntau yog nyob ntawm TLR thiab RLR txoj hauv kev los ua kom lub cev tiv thaiv kab mob tom qab kis kab mob. Yog li ntawd, nws yog ib qho tseem ceeb rau kev tshawb nrhiav cov txheej txheem tswj hwm ntawm TRAF6- txoj kev sib kho kom haum xeeb. calpain2 (m-calpain) yog tsev neeg ntawm calcium-dependent proteases, uas yog tsim los ntawm ntau tshaj 16 tus tswv cuab hauv cov tsiaj txhu thiab ntau lwm yam kab mob 40. Qhov kev tshawb fawb ntxov tshaj plaws ntawm tsev neeg calpain protein muaj feem xyuam nrog kev txav ntawm tes41. calpain txhawb nqa cell migration thiab tau pom tias inhibit neutrophil migration thiab ua kom p38 MAPK thiab ERK MAPK42. Kev ua haujlwm ntawm hydrolase yog ib qho ntawm cov haujlwm tseem ceeb ntawm calpain. Hauv kev tswj hwm kev tiv thaiv kab mob, calpain-2 txhawb nqa kev tso tawm NF-κB los ntawm hydrolyzing IkB thiab ua kom muaj kev taw qhia txoj hauv kev43. Hauv txoj kev tshawb no, peb tau txiav txim siab tias calpain2a cuam tshuam nrog TRAF6 thiab ua qhov tsis zoo ntawm cov tshuaj tiv thaiv kab mob thiab tshuaj tua kab mob hauv cov ntses teleost, miiuy croaker (Miichthys miiuy). calpain2a txhawb kev degradation ntawm TRAF6 protein theem los ntawm nws cov dej num hydrolase. Lub caij no, mutant calpain2a tsis muaj hydrolase kev ua si inhibits qhov tsim ntawm K63 ubiquitin saw los ntawm inhibiting lub dimer ntawm TRAF6. Nws tau cuam tshuam tias ob qho tib si calpain2a thiab mutant calpain2a txiav tawm qhov ubiquitination ntawm TRAF6 rau ECSIT / BENC1 / IRF3 / IRF7. calpain2a nres NF-κB thiab IFN signaling txheej txheem los ntawm phiaj xwm TRAF6. Peb cov txiaj ntsig tau muab cov molecule rau kev kawm ntawm TRAF6-kev tswj hwm kev tiv thaiv kab mob hauv cov kab mob qis.

Cov txiaj ntsig

calpain2a cuam tshuam nrog TRAF6 thiab tsis zoo tswj NF- κB signaling.

Txhawm rau kom nkag siab txog TRAF6-cov proteins sib txuas hauv kev tiv thaiv kab mob hauv lub cev hauv cov ntses teleost, peb qhia txog miiuy croaker TRAF6 interactome siv affinity purification thiab mass spectrometry. Soj ntsuam los ntawm kev siv tsis muaj daim ntawv lo (LFQ), peb cuam tshuam ib feem ntawm cov ntaub ntawv thiab tso tawm nws (Daim duab 1a). Ntawm TRAF6-cov proteins koom nrog, calpain2a tej zaum yuav tswj hwm TRAF6 kev ua haujlwm thiab cuam tshuam TRAF6-txoj kev taw qhia kev sib kho. Ua ntej, kev sib raug zoo ntawm calpain2a thiab TRAF6 ntawm qib protein yuav tsum tau tshawb xyuas, peb tau soj ntsuam kev sib cuam tshuam ntawm calpain2a nrog TRAF6 hauv Miiuy croaker raum cell kab (MKC). Endogenous coimmunoprecipitation thwmsim qhia tias calpain2a cuam tshuam nrog TRAF6 (Fig. 1b, c). Raws li pom los ntawm kev hloov pauv hloov pauv thiab kev sim coimmunoprecipitation, overexpressed calpain2a thiab TRAF6 cuam tshuam nrog ib leeg (Daim duab 1d, e). Tag nrho cov no qhia tias calpain2a cuam tshuam nrog TRAF6 thiab xav tau kev sim ntxiv los tshawb xyuas cov txheej txheem ntawm lawv qhov kev ua. Los ntawm ntau qhov sib txawv ntawm tib neeg, nas, zebrafish, thiab tej zaum yuav croaker calpain2a proteins, calpain2a yog evolutionarily conserved ntawm ntses thiab tsiaj txhu (Fig. 1f). Txhawm rau txiav txim siab txog kev ua haujlwm ntawm calpain2a hauv kev tiv thaiv kab mob hauv lub cev, peb tau tshuaj xyuas seb calpain2a puas cuam tshuam nrog kev ua kom NF-κB ntxias los ntawm LPS stimulation. Peb kis tau cov epithelioma papulosum cyprinid (EPC) hlwb nrog NF-κB luciferase reporter, tswj sab hauv renilla luciferase reporter, thiab vector encoding calpain2a los yog khoob vector. calpain2a txo qis NF-κB tus neeg sau xov xwm ua haujlwm raws li LPS stimulation nyob rau hauv ib koob tshuaj (Daim duab 1g). Ntxiv mus, proinflammatory cytokines xws li IL-1 thiab IL-8 txhawb nqa teb rau LPS stimulation tau txo qis hauv calpain2a-overexpressed EPC hlwb (Daim duab 1h, i), qhia txog lub luag hauj lwm muaj peev xwm rau calpain2a hauv inflammatory signaling. txoj kev ntxias los ntawm LPS stimulation.

Desert ginseng-Improve immunity (23)

cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob

Nyem qhov no mus saib Cistanche Enhance Immunity khoom

【Nug ntxiv】 Email: cindy.xue@wecistanche.com / Whats App: 0086 18599088692 / Wechat: 18599088692

Kev tswj tsis zoo ntawm LPS-induced NF-κB signaling los ntawm calpain2a.

Txhawm rau tshawb xyuas lub luag haujlwm muaj peev xwm ntawm calpain2a hauv NF-κB signaling, peb tau hloov pauv calpain2a hauv MKC nrog lub sijhawm gradient ntawm LPS thiab soj ntsuam cov proinflammatory cytokines teb rau LPS. Overexpression ntawm calpain2a coj mus rau downregulation ntawm kev qhia ntawm IL-1 , IL-8, thiab IL-6 (Fig. 2a). Tom ntej no, peb tau tsim ob lub calpain2a-tshwj xeeb me me cuam tshuam RNAs (siRNAs) ua tau zoo downregulated calpain2a (Fig. 2b, d). Knockdown ntawm calpain2a tau txhim kho LPS-induced proinflammatory cytokines xws li IL-1 thiab IL-8 hauv MKC thiab Miiuy croaker intestine cell kab (MIC) (Fig. 2c, e). Hauv pab pawg ntawm LPS-stimulated hlwb, knockdown ntawm calpain2a txhim kho cell proliferation. Ib yam li ntawd, thaum tsis muaj kev txhawb nqa, peb pom tias knockdown ntawm calpain2a kuj txhim kho cell proliferation (Fig. 2f). Txhawm rau txiav txim siab seb calpain2a inhibited proteins hauv LPS-induced NF-κB signaling, calpain2a markedly inhibited endogenous protein theem ntawm TRAF6 tab sis tsis MyD88, IRAK4, los yog TAK1 (Fig. 2g). Hauv cov ntsiab lus, cov kinetics ntawm calpain2a thiab proinflammatory cytokine qhia tau qhia txog kev ua haujlwm ntawm calpain2a hauv LPS-induced NF-κB signaling.

Fig. 1 calpain2a interacts with TRAF6 and inhibits the NF-κB Signaling Pathway. a List of TRAF6 interactome based on Label-free quantification intensity (section). b, c MKC cells seeded in 6 cm2 dishes. After 24 h, cell lysates were immunoprecipitated (IP) with anti-TRAF6 or anti-calpain2a affinity gels. Then the immunoprecipitates and cell lysates were analyzed by IB with the anti-TRAF6 and anti-calpain2a Abs, respectively. d, e HEK 293 cells seeded in 6 cm2 dishes were transfected with the plasmids calpain2a-Flag and TRAF6-Myc (2 μg each). After 24 h, cell lysates were immunoprecipitated (IP) with anti-Myc d or anti-Flag e affinity gels. Then the immunoprecipitates and cell lysates were analyzed by IB with the anti-Myc and anti-Flag Abs, respectively. f The same amino acids among human calpain2a (Hu-calpain2), mouse calpain2a (Mu-calpain2), zebrafish calpain2a (ZF-calpain2a) and miiuy croaker calpain2a (M-calpain2a) are highlighted with black background. g–I EPC cells were transfected with calpain2a-Flag or empty vector together with the NF-κB, IL-1β, and IL-8 luciferase reporters. At 24 h post-transfection, cells were untreated (Mock) or treated with LPS for 6 h. The luciferase activity value was achieved against the Renilla luciferase activity (n = 3 per group). Western blot analysis was used to measure the expression of the transiently transfected calpain2a Flag. The expression of Tubulin was used as a loading control. Data were analyzed by two-way ANOVA (g, h, i). **p < 0.01. All experiments were performed in at least three independent experiments.


Daim duab 1 calpain2a cuam tshuam nrog TRAF6 thiab inhibits NF-κB Signaling Pathway. Ib Daim Ntawv Teev Npe ntawm TRAF6 interactome raws li Label-free quantification intensity (section). b, c MKC cov noob taum hauv 6 cm2 tais. Tom qab 24 teev, cell lysates tau immunoprecipitated (IP) nrog anti-TRAF6 los yog anti-calpain2a affinity gels. Tom qab ntawd cov immunoprecipitates thiab cell lysates tau soj ntsuam los ntawm IB nrog cov tshuaj tiv thaiv TRAF6 thiab anti-calpain2a Abs, raws li. d, e HEK 293 hlwb noob hauv 6 cm2 tais tau hloov nrog cov plasmids calpain2a-Flag thiab TRAF6-Myc (2 ug txhua). Tom qab 24 teev, cell lysates tau immunoprecipitated (IP) nrog anti-Myc d los yog anti-Flag e affinity gels. Tom qab ntawd cov immunoprecipitates thiab cell lysates tau soj ntsuam los ntawm IB nrog anti-Myc thiab anti-Flag Abs, feem. f Tib cov amino acids ntawm tib neeg calpain2a (Hu-calpain2), nas calpain2a (Mu-calpain2), zebrafish calpain2a (ZF-calpain2a) thiab miiuy croaker calpain2a (M-calpain2a) yog highlighted nrog dub keeb kwm yav dhau. g–I EPC cov hlwb tau hloov nrog calpain2a-Flag lossis cov vector khoob ua ke nrog NF-κB, IL-1 , thiab IL-8 luciferase reporters. Thaum 24 teev tom qab hloov pauv, cov hlwb tsis kho (Mock) lossis kho nrog LPS rau 6 teev. Qhov txiaj ntsig ntawm luciferase tau ua tiav tawm tsam Renilla luciferase kev ua (n=3 ib pab pawg). Western blot tsom xam tau siv los ntsuas qhov qhia ntawm transiently transfected calpain2a Chij. Kev qhia ntawm Tubulin tau siv los ua kev tswj xyuas kev thauj khoom. Cov ntaub ntawv tau txheeb xyuas los ntawm ob txoj kev ANOVA (g, h, i). **p < 0.01. Txhua qhov kev sim tau ua nyob rau hauv tsawg kawg peb qhov kev sim ywj pheej.

Kev sib cuam tshuam ntawm calpain2a nrog TRAF6.

Kev txheeb xyuas lub npe tau ua tiav siv Conserved Domain Database (CDD) NCBI. calpain2a suav nrog peb lub npe: Calpain tsev neeg cysteine ​​protease (CysPc) domain (aa 46-339), lub hauv paus Calpain loj subunit domain III (aa 360-499), thiab C-terminal Penta-EF tes sau ( ua 500-697). Txhawm rau txiav txim siab seb lub npe twg ntawm calpain2a xav tau rau nws qhov kev cuam tshuam nrog TRAF6, peb tsim peb lub truncation mutants: calpain2a (1-339), calpain2a (340-697), thiab calpain (500-697) (Daim duab 3a). HEK293 hlwb tau hloov nrog calpain2a-Flag wild type (WT), calpain2a-Flag truncation mutants, los yog TRAF6-HA vector. Peb pom tias ob qho tag nrho-ntev calpain2a thiab peb truncation mutants tuaj yeem cuam tshuam nrog TRAF6 (Fig. 3c). TRAF6 yog tsim los ntawm C-terminal TRAF domain, coiled-coil domain, Zinc fingers domain, thiab lub nplhaib ntiv tes domain38. Txhawm rau tshuaj xyuas seb qhov twg ntawm TRAF6 xav tau rau nws qhov kev cuam tshuam nrog calpain2a, peb tau ua cov TRAF6 truncates mutants, suav nrog TRAF6 (1-139), TRAF6 (140-574), TRAF6 ({{36). }}), thiab TRAF6 (400-574). Meanwhile, peb ua tus sau mutants: TRAF6ΔRing (nyob rau hauv uas lub nplhaib ntiv tes sau yog deleted), TRAF6ΔZF (nyob rau hauv uas lub Zinc ntiv tes yog deleted), TRAF6ΔCC (nyob rau hauv uas lub coiled-coil domain yog deleted), thiab TRAF6ΔTRAF-C ( nyob rau hauv uas C-terminal TRAF sau yog deleted) (Fig. 3b). calpain2a cuam tshuam nrog tag nrho ntev TRAF6 nrog rau kev txiav tawm ntawm TRAF6 uas muaj C-terminal TRAF domain, coiled-coil domain, Zinc fingers domain (Fig. 3d, f), tab sis tsis nrog lub nplhaib ntiv tes (Fig. 3e) . Luciferase kev soj ntsuam pom tau tias overexpression ntawm calpain2a thiab cov truncation mutants ho inhibit NF-κB thiab IL-1 reporter kev ua nyob rau hauv EPC hlwb (Fig. 3h). Tsis tas li ntawd, cov subcellular colocalizations ntawm calpain2a nrog TRAF6 tau tshawb xyuas. Peb tau hloov pauv EPC hlwb nrog TRAF6-GFP thiab ib qho vector khoob lossis calpain2a-mCherry. Confocal microscopy tsom xam pom tias cov cim ntsuab ntawm TRAF6 sib tshooj nrog cov cim liab ntawm calpain2a, uas qhia tias calpain2a co-localizes nrog TRAF6 hauv EPC hlwb (Fig. 3i, j). Cov ntaub ntawv no qhia tau hais tias lub hauv paus ntawm ntau yam ntawm TRAF6 (C-terminal TRAF domain, coiled-coil domain, Zinc fingers domain) tab sis tsis yog lub nplhaib ntiv tes yog txiav txim siab rau nws txoj kev koom tes nrog calpain2a (Fig. 3g).

Fig. 2 calpain2a negative regulates LPS-induced NF-κB Signaling Pathway. an IL-1β, IL-8, and IL-6 mRNA in MKC transduced with calpain2a-Flag or empty vector and treated with saline (0) or challenged with LPS for various times (2 h, 4 h, 6 h, 8 h, 10 h) (n = 3 per group). b, d MKC cells and MIC cells were transfected with si-calpain2a #1 and si-calpain2a #2 (100 nM) or si-ctrl (100 nM). At 48 h post-transfection, calpain2a expression was measured by qRT PCR (n = 3 per group). The level of calpain2a protein was determined by Western blotting. c, e IL-1β, IL-8 mRNA in MKC and MIC stably transduced with si-calpain2a #1 or si-ctrl (control vector) and treated with saline (0) or challenged with LPS for various times (4 h, 8 h) (n = 3 per group). f MKC cells were transfected with either si-ctrl or si-calpain2a #1. At 36 h post-transfection, the cells were stimulated with LPS for 12 h, then cell proliferation assay was measured (n = 3 per group). Scale bar, 100 μm. g calpain2a-Flag was transfected into MKC cells which were challenged with LPS at various times (3 h, 6 h, 9 h). Immunoblot analysis of cell lysates with indicated Abs. Relative mRNA level was normalized to the expression of the gene encoding β-actin in each sample. All experiments were performed in at least three independent experiments. Data were analyzed by two-way ANOVA (a, c, e, f) or one-way ANOVA (b, d). *p < 0.05, **p < 0.01.

Fig. 2 calpain2a negative regulates LPS-induced NF-κB Signaling Pathway. ib IL-1 , IL-8, thiab IL-6 mRNA hauv MKC hloov nrog calpain2a-Flag lossis khoob vector thiab kho nrog saline (0) lossis sib tw nrog LPS rau ntau yam zaug (2 h, 4 h, 6 h, 8 h, 10 h) (n=3 ib pab). b, d MKC hlwb thiab MIC hlwb tau hloov nrog si-calpain2a #1 thiab si-calpain2a #2 (100} nM) lossis si-ctrl (100 nM). Thaum 48 teev tom qab hloov pauv, calpain2a qhia tau ntsuas los ntawm qRT PCR (n=3 ib pawg). Qib ntawm calpain2a protein tau txiav txim siab los ntawm Western blotting. c, e IL-1 , IL-8 mRNA hauv MKC thiab MIC stably transduced nrog si-calpain2a #1 los yog si-ctrl (tswj vector) thiab kho nrog saline (0) los yog sib tw nrog LPS rau ntau yam lub sij hawm (4 h, 8 h) (n=3 ib pab pawg). f MKC hlwb tau kis nrog si-ctrl lossis si-calpain2a #1. Tom qab 36 teev tom qab hloov pauv, cov hlwb raug txhawb nrog LPS rau 12 teev, tom qab ntawd ntsuas kev loj hlob ntawm tes (n=3 ib pab pawg). Scale bar, 100 μm. g calpain2a-Flag tau kis mus rau MKC hlwb uas tau sib tw nrog LPS ntawm ntau lub sijhawm (3 h, 6 h, 9 h). Immunoblot tsom xam ntawm cell lysates nrog qhia Abs. Cov txheeb ze mRNA qib tau normalized rau qhov kev qhia ntawm cov noob encoding -actin hauv txhua tus qauv. Txhua qhov kev sim tau ua nyob rau hauv tsawg kawg peb qhov kev sim ywj pheej. Cov ntaub ntawv tau txheeb xyuas los ntawm ob txoj kev ANOVA (a, c, e, f) lossis ib txoj kev ANOVA (b, d). *p <0.05, **p <0.01.

Fig. 3 The interaction of calpain2a with TRAF6. a, b Schematic diagrams of domain organization in miiuy croaker calpain2a, TRAF6, and mutants were used in this study.


Daim duab 3 Kev sib cuam tshuam ntawm calpain2a nrog TRAF6. a, b Schematic daim duab ntawm lub koom haum sau npe hauv miiuy croaker calpain2a, TRAF6, thiab mutants tau siv rau hauv txoj kev tshawb no. "+" sawv cev rau kev cuam tshuam nrog TRAF6 lossis calpain2a, "-" txhais tau tias tsis muaj kev cuam tshuam. c HEK293 hlwb tau hloov nrog kev thuam, calpain2a-Flag wild type (WT), thiab calpain2a-Flag truncated mutants, los yog TRAF6-HA raws li qhia. Tom qab 24 teev tom qab kev hloov pauv, cov kab mob kis tau raug muab rho tawm thiab cov cell lysates tau txais kev tiv thaiv kab mob nrog cov tshuaj tiv thaiv HA los ntawm IB siv tshuaj tiv thaiv HA lossis tshuaj tiv thaiv Flag. d-f HEK293 hlwb tau kis nrog kev thuam, TRAF6-HA hom tsiaj qus (WT), thiab TRAF6-HA truncated mutants, los yog calpain2a-Flag raws li qhia. Tom qab 24 teev tom qab kev hloov pauv, cov hlwb raug muab rho tawm thiab cov cell lysates tau txais kev tiv thaiv kab mob nrog anti-HA antibody d, f, lossis anti-Flag antibody e ua raws li IB siv anti-HA lossis anti-Flag antibody. g Qhov chaw sib cuam tshuam ntawm TRAF6 thiab calpain2a. h EPC hlwb raug hloov nrog TRAF6-HA, calpain2a-Flag, lossis calpain2a-Flag truncated mutants ua ke nrog NF-κB thiab IL-1 luciferase reporters. Tom qab 36 teev tom qab hloov pauv, qhov txiaj ntsig ntawm luciferase tau ua tiav tawm tsam renilla luciferase kev ua haujlwm (n=3 ib pawg). Kuv j EPC hlwb raug hloov nrog kev thuam, calpain2a-mCherry, thiab TRAF6-GFP. DAPI-stained nuclei tau pom hauv xiav. calpain2a tau kuaj pom nrog cov fluorescence liab, thiab TRAF6 tau kuaj pom nrog cov fluorescence ntsuab. Scale bar, 10 μm. Txhua qhov kev sim tau ua nyob rau hauv tsawg kawg peb qhov kev sim ywj pheej. Cov ntaub ntawv tau txheeb xyuas los ntawm ib-txoj kev ANOVA (h). ** p <0.01.

calpain2a inhibits TRAF6 protein qhia theem los ntawm kev ua haujlwm proteolytic.

Txhawm rau tshawb xyuas cov txheej txheem tswj hwm ntawm calpain2a ntawm TRAF6, peb thawj zaug tshuaj xyuas seb calpain2a puas cuam tshuam rau TRAF6 ntawm qib protein. calpain2a-Myc thiab TRAF6-Flag tau sib koom ua ke rau hauv EPC hlwb, qib protein ntawm TRAF6 yog ib feem degraded ntawm 30 h (Fig. 4a). Raws li pom nyob rau hauv daim duab 4b, calpain2a txhawb kev degradation ntawm TRAF6 raws li koob tshuaj. Nyob rau hauv lub xub ntiag ntawm calpain2a, nws tsis cuam tshuam rau mRNA qib ntawm TRAF6, qhia tias calpain2a tsuas cuam tshuam rau qib protein ntawm TRAF6. Lub caij no, qhov kev ntsuam xyuas inhibitor cycloheximide (CHX) tau pom tias calpain2a tuaj yeem ua kom lub neej ib nrab ntawm TRAF6 protein (Fig. 4c). Overexpression ntawm calpain2a nyob rau hauv MKC hlwb ua rau lub destabilization ntawm endogenous TRAF6 (Fig. 4d). Txhawm rau tshawb xyuas seb qhov kev puas tsuaj ntawm TRAF6 kho los ntawm calpain2a yog nyob ntawm ubiquitin-proteasome, autophagosome, lysosomal txoj hauv kev, lossis lwm yam, EPC hlwb tau kho nrog cov reagents uas inhibited txoj hauv kev no: MG132, 3-MA, thiab NH4Cl. Txawm li cas los xij, calpain2a-mediated destabilization ntawm TRAF6 tsis thim rov qab los ntawm proteasomal inhibitor MG132, autophagic inhibitor 3-MA, lossis lysosomal inhibitor NH4Cl (Fig. 4e). Tom ntej no, EPC hlwb raug kho nrog cov reagents: E-64 (ib qho calpain inhibitor uas inactivate calpain hydrolase kev ua si) thiab PMSF (nonspecific serine protease inhibitor). Peb pom calpain inhibitor E-64 tiv thaiv kev degradation ntawm TRAF6 protein ntau (Fig. 4f). Raws li pom hauv daim duab 4g, E-64 kuj thim rov qab qhov degradation ntawm endogenous TRAF6 protein ntau ntau los ntawm calpain2a.

Fig. 4 calpain2a inhibits TRAF6 protein expression. a The time gradient experiment of empty vectors or calpain2a-Myc plasmids together with TRAF6- Flag was conducted in EPC cells. The cell lysates were subjected to IB with anti-Myc, anti-Flag, and anti–Tubulin Abs. RNA was extracted from cells and reverse transcribed, and then TRAF6 and actin were amplified by PCR primers. b EPC cells were seeded in 12-well plates overnight and co-transfected with TRAF6-Flag and calpain2a-Myc (0.3, 0.6, or 0.9 μg) for 48 h. The expression of TRAF6-Flag and calpain2a-Myc proteins was detected by Western blotting. RNA was extracted from cells and reverse transcribed, then TRAF6 and actin were amplified by PCR primers. c calpain2a-Myc or empty vectors were co-transfected with TRAF6-Flag into EPC cells. At 36 h post-transfection, the transfected cells were treated with cycloheximide (CHX) for 2 or 4 h. d MKC cells were transfected with calpain2a-Flag or empty vectors. At 48 h post-transfection, the cell lysates were subjected to IB with anti-TRAF6, anti-Flag, and anti-Tubulin Abs. e, f EPC cells were transfected with the indicated plasmids in the presence or absence of MG132, 3-MA, NH4CL, E-64 (50 or 75 μM), or PMSF for 6 h before immunoblot analysis was performed. g MKC cells were transfected with calpain2a-Flag in the presence or absence of E-64 (50 or 75 μM) for 6 h before immunoblot analysis was performed. h calpain2a-Flag and calpain2a-ΔCysPc-Flag were co-transfected with TRAF6-HA into EPC cells. At 48 h post-transfection, the cell lysates were subjected to IB with indicated Abs. I calpain2a-Flag and calpain2a-ΔCysPc-Flag were cotransfected into MKC cells. At 48 h post-transfection, the cell lysates were subjected to IB with anti-TRAF6, anti-Flag, and anti-Tubulin Abs. j IL-1β, IL-8 mRNA in MKC stably transduced with calpain2a-Flag and calpain2a-ΔCysPc-Flag and treated with saline (0) or challenged with LPS for 4 h (n = 3 per group). Relative mRNA level was normalized to the expression of the gene encoding β-actin in each sample. All experiments were performed in at least three independent experiments. Data were analyzed by two-way ANOVA (j). **p < 0.01.


Fig. 4 calpain2a inhibits TRAF6 protein qhia. a Lub sij hawm gradient sim ntawm khoob vectors lossis calpain2a-Myc plasmids ua ke nrog TRAF6- Chij tau ua nyob rau hauv EPC hlwb. Cov cell lysates tau raug rau IB nrog anti-Myc, anti-Flag, thiab anti-Tubulin Abs. RNA tau muab rho tawm los ntawm cov hlwb thiab thim rov qab, thiab tom qab ntawd TRAF6 thiab actin tau nthuav dav los ntawm PCR primers. b EPC cov hlwb tau raug cog rau hauv 12- cov phiaj zoo thaum hmo ntuj thiab sib koom ua ke nrog TRAF6-Flag thiab calpain2a-Myc (0.3, 0.6, lossis {{ 18}}.9 ug) rau 48 h. Cov lus qhia ntawm TRAF6-Flag thiab calpain2a-Myc proteins tau kuaj pom los ntawm Western blotting. RNA tau muab rho tawm los ntawm cov hlwb thiab thim rov qab, tom qab ntawd TRAF6 thiab actin tau nthuav dav los ntawm PCR primers. c calpain2a-Myc lossis cov vectors khoob tau sib koom ua ke nrog TRAF6-Flag rau hauv EPC hlwb. Thaum 36 teev tom qab hloov pauv, cov hlwb hloov pauv tau kho nrog cycloheximide (CHX) rau 2 lossis 4 teev. d MKC hlwb tau hloov nrog calpain2a-Flag lossis cov vectors khoob. Thaum 48 teev tom qab hloov pauv, cov cell lysates tau raug rau IB nrog anti-TRAF6, anti-Flag, thiab anti-Tubulin Abs. e, f EPC hlwb raug hloov nrog cov plasmids qhia nyob rau hauv lub xub ntiag los yog tsis muaj MG132, 3-MA, NH4CL, E-64 (50 los yog 75 μM), los yog PMSF rau 6 h ua ntej kev soj ntsuam immunoblot tau ua. g MKC hlwb raug hloov nrog calpain2a-Flag nyob rau hauv lub xub ntiag los yog tsis muaj E{{50}} (50 los yog 75 μM) rau 6 h ua ntej immunoblot tsom xam. h calpain2a-Flag thiab calpain2a-ΔCysPc-Flag tau co-transfected nrog TRAF6-HA rau hauv EPC hlwb. Thaum 48 teev tom qab hloov pauv, cov cell lysates tau raug rau IB nrog Abs qhia. Kuv calpain2a-Flag thiab calpain2a-ΔCysPc-Flag tau cotransfected rau hauv MKC hlwb. Thaum 48 teev tom qab hloov pauv, cov cell lysates tau raug rau IB nrog anti-TRAF6, anti-Flag, thiab anti-Tubulin Abs. j IL-1 , IL-8 mRNA hauv MKC stably hloov nrog calpain2a-Flag thiab calpain2a-ΔCysPc-Flag thiab kho nrog saline (0) lossis sib tw nrog LPS rau 4 h (n=3} ib pab pawg). Cov txheeb ze mRNA qib tau normalized rau qhov kev qhia ntawm cov noob encoding -actin hauv txhua tus qauv. Txhua qhov kev sim tau ua nyob rau hauv tsawg kawg peb qhov kev sim ywj pheej. Cov ntaub ntawv tau txheeb xyuas los ntawm ob txoj kev ANOVA (j). ** p <0.01.

Txhawm rau txiav txim siab seb qhov degradation ntawm TRAF6 puas yog nyob ntawm kev ua haujlwm hydrolase ntawm calpain2a. Ua kom zoo dua ntawm cov qauv thiab biochemical kev tshawb fawb yav dhau los ntawm calpain2a hauv cov tsiaj nyeg, peb hloov cov cysteine, histidine, thiab asparagine residues ntawm calpain2a rau alanine (calpain2a C105A, H262A, N286A, thiab 3 A), uas poob cov catalytic 46-triad. Peb pom tias qhov degradation ntawm TRAF6 tsis cuam tshuam los ntawm qhov kev hloov pauv no (Cov duab ntxiv 1a). Yog li, peb hloov pauv lub npe protease, uas yog Calpain tsev neeg cysteine ​​protease (CysPc) ntawm calpain2a (tsim yog calpain2a-ΔCysPc). Tsis yog overexpressed TRAF6 los yog endogenous TRAF6 cuam tshuam los ntawm calpain2a-ΔCysPc (Fig. 4h, i). Txawm li cas los xij, proinflammatory cytokines IL-1 thiab IL-8 ib txhij cuam tshuam los ntawm hom tsiaj qus lossis hloov pauv calpain2a raws li LPS stimulation (Fig. 4j). Mutant calpain2a tsis muaj hydrolase kev ua si tsis cuam tshuam rau qib protein ntawm TRAF6, tab sis cov mutant calpain2a tsis muaj hydrolase kev ua si tseem cuam tshuam qhov kev qhia ntawm cov dej ntws tawm ntawm cov cytokines. Qhov no ua rau peb qhov kev tshawb nrhiav rau cov txheej txheem muaj peev xwm uas calpain2a tswj TRAF6. Noj ua ke, cov txiaj ntsig no qhia tau tias calpain2a tswj NF-κB teeb liab los ntawm kev txwv qhov kev qhia ntawm TRAF6 protein ntau, los ntawm nws txoj haujlwm calpain hydrolase.

calpain2a inhibits TRAF6 ubiquitin-ligase kev ua haujlwm.

Tom ntej no, peb tshawb nrhiav cov txheej txheem molecular uas calpain2a tsis zoo tswj TRAF6- qhib NF-κB signaling, thiab seb calpain2a kev cuam tshuam nrog TRAF6 thaum poob ntawm enzymatic kev ua haujlwm hloov nws txoj haujlwm ubiquitin ligase. Peb kho MKC hlwb nrog LPS thiab tom qab ntawd immunoprecipitated TRAF6. Ubiquitination assay nrog purified recombinant proteins paub tseeb tias calpain2a thiab calpain2a-ΔCysPc txo ubiquitination ntawm TRAF6 (Fig. 5a). Raws li pom hauv daim duab 5b, c, ob qho tib si calpain2a thiab calpain2a-ΔCysPc txo qis WT thiab K63 ubiquitinated proteins ntawm TRAF6. Concentration gradients ntawm calpain2a thiab calpain2a-ΔCysPc txo WT thiab K63 ubiquitinated proteins ntawm TRAF6 raws li koob tshuaj. calpain2a-ΔCysPc tseem tiv thaiv K63 ubiquitin saw aggregation ntawm TRAF6 yam tsis muaj kev cuam tshuam rau TRAF6 protein ntau (Fig. 5d, e). Tom qab ntawd peb hloov MKC hlwb los nthuav qhia Myc-tagged TRAF6 nyob rau hauv lub xub ntiag los yog tsis muaj Chij-tagged calpain2a-ΔCysPc, ces ua immunoprecipitation thwmsim nrog anti-Myc antibody thiab soj ntsuam los ntawm immunoblot nrog anti-UB. Xws li ubiquitination ntawm TRAF6 tau txo qis los ntawm calpain2a- ΔCysPc (Fig. 5f). Stimulated los ntawm LPS, TRAF6 xa K63- txuas ubiquitin saw rau TAK1, uas txhawb nqa TAK1 autophosphorylation thiab qhib NF-κB18. WT thiab K63 ubiquitin proteins ntawm TAK1 tau txhim kho tau zoo nyob rau hauv lub xub ntiag ntawm TRAF6, whereas TAK1- txuas ubiquitination tau inhibited nyob rau hauv lub xub ntiag ntawm calpain2a-ΔCysPc (Fig. 5g, h). Cov ntaub ntawv no txhawb nqa tias mutant calpain2a tsis muaj hydrolase kev ua si zoo sib xws inhibits TRAF6 ubiquitin ligase yam tsis muaj kev cuam tshuam rau TRAF6 protein ntau.

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calpain2a inhibits TRAF6 homo-oligomerization.

Lys63 ubiquitin saw synthesized los ntawm TRAF6 plays lub luag haujlwm tseem ceeb hauv kev xa cov teeb liab. Nws tau raug tshaj tawm tias qhov dimerization ntawm TRAF6 yog qhov tseem ceeb rau kev sib txuas ntawm nws cov ubiquitin chain16. Tus qauv dimerization yog ze ze rau kev sib xyaw ntawm Ubc13 los tsim K63 ubiquitination16,30. Txhawm rau txheeb xyuas seb cov ntses TRAF6 tuaj yeem tsim cov dimers, peb siv TRAF6 plasmids nrog cov cim sib txawv: Myc-tagged TRAF6 thiab HA-tagged TRAF6 rau co-IP thwmsim. IP assay tau ua tiav siv cov tshuaj tiv thaiv HA, HATRAF6 cuam tshuam nrog Myc-TRAF6 (Fig. 6a). Txhawm rau tshawb nrhiav cov nyhuv inhibitory ntawm calpain2a hauv TRAF6-dimer kev cuam tshuam, Flag-tagged calpain2a, HA-tagged TRAF6 thiab Myc-tagged TRAF6 tau hloov pauv mus rau HEK293 hlwb. Raws li kev cia siab, TRAF6-dimer kev sib cuam tshuam tau txo qis thaum muaj calpain2a (Fig. 6b). Nyob rau tib lub sijhawm, txhawm rau kom tsis txhob muaj qhov degradation ntawm TRAF6 los ntawm calpain2a. Peb kis tau tus chij-tagged calpain2a-ΔCysPc thiab pom tias TRAF6-dimer kev sib cuam tshuam tau maj mam txo qis thaum muaj calpain2a- ΔCysPc (Fig. 6c). Raws li qhia hauv daim duab 6d, e, peb pom HA-TRAF6 txhim kho WT thiab K63 ubiquitination ntawm Myc-TRAF6. Thaum HEK293 hlwb tau koom ua ke nrog calpain2a thiab calpain2a- ΔCysPc, kev txhim kho ubiquitination yuav raug txwv. Cov txiaj ntsig no qhia tias ob hom tsiaj qus calpain2a thiab mutant calpain2a tsis muaj hydrolase kev ua si inhibit TRAF6 homooligomerization thiab nws pib-ubiquitination.

calpain2a inhibits kev xa khoom ntawm TRAF6 ubiquitination rau ECSIT thiab BECN1.

Raws li tau piav qhia hauv daim duab 5d, peb pom tias calpain2a cuam tshuam nrog cov txheej txheem ubiquitylation ntawm TRAF6 mus rau TAK1. Nws tau raug tshaj tawm tias ECSIT ua raws li kev hloov pauv protein ntawm TAK1 thiab TRAF6 los txhawb NF-κB teeb liab los ntawm LPS stimulation37. Txhawm rau txheeb xyuas seb ECSIT puas muaj cov haujlwm uas tau hais hauv teleost ntses, peb tau ua ntau qhov sib txawv ntawm ECSIT cov proteins hauv tib neeg, nas, thiab tej zaum croaker (Ntxiv daim duab 1b). Peb tau hloov pauv EPC hlwb nrog NF-κB luciferase tus neeg sau xov xwm, ECSIT tau txhim kho NF-κB tus neeg tshaj xov xwm kev ua ub no tom qab LPS stimulation (Ntxiv Fig. 1c). Tshaj tawm ntawm ECSIT hauv MKC hlwb tau coj mus rau kev tswj hwm ntawm mRNA qib ntawm TNF- (Cov Lus Qhia Ntxiv 1d). Tom qab kis tau ntses ECSIT thiab TRAF6 hauv HEK293 hlwb, peb pom tias TRAF6 cuam tshuam nrog ECIST (Fig. 7a). Ib yam li ntawd, ECSIT thiab TAK1 tau sib koom ua ke, thiab ob lub proteins kuj tseem muaj kev sib cuam tshuam (Daim duab 7b). Tom qab ntawd, txhawm rau txheeb xyuas seb calpain2a-ΔCysPc cuam tshuam qhov tsim ntawm TRAF6-ECSIT complex, peb tau hloov peb plasmids rau hauv HEK293 hlwb thiab pom tias calpain2a-ΔCysPc tiv thaiv TRAF6-ECSIT complex (Fig. ). Txhawm rau txheeb xyuas cov txheej txheem sib kis ntawm ubiquitin saw hauv NF-κB signaling. Raws li pom nyob rau hauv daim duab 7d, TRAF6 tuaj yeem txhawb kev nthuav dav ntawm ECSIT; Raws li qhov xav tau, calpain2a-ΔCysPc inhibited cov txheej txheem uas TRAF6 kis tau cov saw ubiquitin rau ECSIT. BECN1 yog ib qho protein tseem ceeb hauv LPS-induced autophagy. Nws tau raug lees paub tias K63 ubiquitination tuaj yeem hloov kho thiab qhib BECN1. Deubiquitinating enzyme A20 tshem tawm ubiquitination ntawm BECN1 thiab inhibits autophagy tshwm sim los ntawm LPS, thiab TRAF6 yog E3 ligase lub luag haujlwm rau BECN1 ubiquitination hauv qhov teeb meem no47. Raws li pom nyob rau hauv daim duab 7e, TRAF6 cuam tshuam nrog BECN1 thiab txhawb nqa ubiquitination ntawm BECN1. Tom qab ntawd peb tau hloov pauv BECN1, TRAF6, calpain2a, thiab calpain2a-ΔCysPc rau hauv HEK293 hlwb, TRAF6 txhawb nqa ubiquitination ntawm BECN1. calpain2a-ΔCysPc inhibited tus txheej txheem los ntawm TRAF6 kis cov ubiquitin saw rau BECN1 (Fig. 7f). Cov txiaj ntsig no qhia tias hom tsiaj qus calpain2a thiab mutant calpain2a tsis muaj hydrolase kev ua haujlwm inhibit qhov muaj peev xwm ntawm TRAF6 hauv LPS-induced autophagy thiab NF-κB signaling.

Kev tswj tsis zoo ntawm cov tshuaj tiv thaiv kab mob los ntawm calpain2a.

Nyob rau hauv txoj kev RLR, TRAF6 ua raws li E3 ligase recruited los ntawm MAVS los qhib NF-κB, zus tau tej cov IFNs thiab cytokines. Peb tom ntej no tau tshuaj xyuas seb calpain2a tau ua lub luag haujlwm los tiv thaiv TRAF6 hauv IFN-mediated antiviral txheej txheem. Hauv EPC hlwb, IFN-1, ISRE, thiab IRF3 txhawb nqa-tsav luciferase cov neeg sau xov xwm tau qhia. Peb pom calpain2a txo qis cov kev txhawb nqa-tsav luciferase kev ua ub no nyob rau hauv koob tshuaj raws li Ploy(I: C) stimulation thiab Siniperca cheats rhabdovirus (SCRV) kab mob (Fig. 8a, b). Knockdown ntawm calpain2a tau txhim kho Ploy (I: C) thiab SCRV-triggered gene qhia nrog rau Viperin, Mx1, thiab ISG15 (Fig. 8c, d). Knockdown ntawm calpain2a ua rau muaj kev nthuav qhia ntau dua ntawm cell viability thaum SCRV kab mob hauv MKC hlwb (Fig. 8e). Raws li pom nyob rau hauv daim duab 8f, kev soj ntsuam kev loj hlob ntawm tes qhia tau hais tias knockdown ntawm calpain2a txhim kho cell proliferation raws li tus kab mob SCRV. Overexpression ntawm calpain2a sharply txhawb tus kab mob propagation, raws li xav tau los ntawm viral replication tom qab SCRV kab mob (Fig. 8g). calpain2a thiab calpain2a-ΔCysPc txo qis IFN-1, ISRE thiab IRF3 cov neeg txhawb nqa-tsav luciferase cov neeg sau xov xwm raws li Ploy(I:C) stimulation thiab SCRV kab mob (Ntxiv Fig. 2a, b). Cov txiaj ntsig no qhia tias calpain2a tuaj yeem tswj tsis tau RLR teeb liab txoj hauv kev los ntawm inhibition ntawm TRAF6, yog li inhibiting kev ua kom IFN.

Fig. 5 calpain2a inhibits TRAF6 ubiquitin-ligase activity. a Ubiquitination of endogenous TRAF6 in MKC cells transduced with calpain2a-Flag and calpain2a-ΔCysPc-Flag and unchallenged (−) or challenged with LPS (+), assessed by immunoblot analysis with anti-ubiquitin after immunoprecipitation with anti-TRAF6 and input immunoblot analysis with indicated Abs. b, c HEK293 cells were cotransfected with TRAF6-HA and WT-ubiquitin-His or K63O ubiquitin-His (in which only lysine 63 is kept) together with calpain2a-Flag, calpain2a-ΔCysPc-Flag or empty vector. After 24 h post-transfection, the cells were lysed and purified with Ni-NTA agarose. d, e HEK293 cells were cotransfected with TRAF6-HA and WT-ubiquitin-His or K63O-ubiquitin-His (in which only lysine 63 is kept) together with calpain2a-Flag (0.5 μg, 1 μg), calpain2a-ΔCysPc-Flag (0.5 μg, 1 μg) or empty vector. After 24 h post-transfection, the cells were lysed and purified with Ni-NTA agarose. f Ubiquitination of overexpressed TRAF6 in MKC cells transduced with calpain2a-ΔCysPc-Flag, assessed by immunoblot analysis with anti-ubiquitin after immunoprecipitation with anti-Myc and input immunoblot analysis with indicated Abs. g, h HEK293 cells were cotransfected with TAK1-Myc, TRAF6-HA, and WT-ubiquitin-His or K63O-ubiquitin-His (in which only lysine 63 is kept) together with calpain2a-Flag, calpain2a-ΔCysPc-Flag or empty vector. After 24 h post-transfection, the cells were lysed and purified with Ni-NTA agarose. All the immunoprecipitates and input immunoblot analysis with anti-Myc, anti-Flag, anti-HA, and anti-Tubulin Abs. All experiments were performed in at least three independent experiments.


Fig. 5 calpain2a inhibits TRAF6 ubiquitin-ligase kev ua si. Ubiquitination ntawm endogenous TRAF6 nyob rau hauv MKC hlwb transduced nrog calpain2a-Flag thiab calpain2a-ΔCysPc-Flag thiab unchallenged (−) los yog sib tw nrog LPS (+), soj ntsuam los ntawm immunoblot tsom xam nrog anti-ubiquitin tom qab immunoprecipitation nrog tshuaj tiv thaiv-TRAF6. nrog qhia Abs. b, c HEK293 hlwb tau cotransfected nrog TRAF6-HA thiab WT-ubiquitin-Nws lossis K63O ubiquitin-Nws (uas tsuas yog lysine 63 khaws cia) ua ke nrog calpain2a-Flag, calpain2a-ΔCysPc-Flag lossis vector. Tom qab 24 teev tom qab hloov pauv, cov hlwb raug lysed thiab purified nrog Ni-NTA agarose. d, e HEK293 hlwb tau cotransfected nrog TRAF{{30}}HA thiab WT-ubiquitin-Nws los yog K63O-ubiquitin-Nws (uas tsuas yog lysine 63 khaws cia) ua ke nrog calpain2a-Flag ({{45 }}.5 ug, 1 ug), calpain2a-ΔCysPc-Flag (0.5 ug, 1 ug) los yog npliag vector. Tom qab 24 teev tom qab hloov pauv, cov hlwb raug lysed thiab purified nrog Ni-NTA agarose. f Ubiquitination ntawm overexpressed TRAF6 nyob rau hauv MKC hlwb transduced nrog calpain2a-ΔCysPc-Flag, soj ntsuam los ntawm immunoblot tsom xam nrog anti-ubiquitin tom qab immunoprecipitation nrog anti-Myc thiab input immunoblot tsom xam nrog qhia Abs. g, h HEK293 hlwb tau cotransfected nrog TAK1-Myc, TRAF6-HA, thiab WT-ubiquitin-Nws lossis K63O-ubiquitin-Nws (uas tsuas yog lysine 63 khaws cia) ua ke nrog calpain2a-Flag , calpain2a-ΔCysPc-Flag lossis vector khoob. Tom qab 24 teev tom qab hloov pauv, cov hlwb raug lysed thiab purified nrog Ni-NTA agarose. Tag nrho cov immunoprecipitates thiab input immunoblot tsom xam nrog anti-Myc, anti-Flag, anti-HA, thiab anti-Tubulin Abs. Txhua qhov kev sim tau ua nyob rau hauv tsawg kawg peb qhov kev sim ywj pheej.

calpain2a suppresses ubiquitination ntawm IRF3 thiab IRF7 los ntawm TRAF6.

Nws tau tshaj tawm tias nyob rau hauv cov teeb liab tshuaj tiv thaiv tshwm sim los ntawm MAVS-TRAF3/6 complex, kev ua ntawm TRAF6 yog qhov tsim nyog rau kev ua kom IRF3 thiab NF-κB48. Raws li pom hauv daim duab 9a, b, TRAF6 txhawb nqa WT thiab K63- txuas ubiquitination ntawm IRF3, thiab qhov kev txhim kho no yog inhibited los ntawm kev qhia ntawm calpain2a- ΔCysPc. Lub caij no, thaum tus kab mob ua rau TLR-raws li lub cim qhia, TRAF6 muaj feem cuam tshuam nrog IRF7 thiab txhawb nqa kev nthuav dav ntawm IRF7 nrog rau cov tshuaj tiv thaiv kab mob tau qhib 22,49,50. TRAF6 txhawb nqa WT thiab K63- txuas ubiquitination ntawm IRF7, thiab qhov kev txhim kho no yog inhibited los ntawm kev qhia ntawm calpain2a-ΔCysPc (Fig. 9c, d). Cov ntaub ntawv no qhia tias cov tsiaj qus calpain2a thiab mutant calpain2a tsis muaj hydrolase kev ua ub no inhibit TRAF6-kev sib kho ubiquitination ntawm IRF3 thiab IRF7.

Kev sib tham

TRAF6 yog ib tug ntawm xya tus tswv cuab ntawm Tumor necrosis factor (TNF) receptor-associated factor (TRAFs) tsev neeg, uas tau raug txheeb xyuas tias yog lub teeb liab adapter thiab exerts teeb liab transduction51,52. TRAF tsev neeg muaj lub nplhaib ntiv tes, uas yog tus tswv ntawm E3 ligase kev ua. E3 ligases nrog HECT domain txhawb nqa substrate polyubiquitination, thiab E3s nrog lub nplhaib domain xav tau E2 ~ ub xa polyubiquitination53,54. Lub dimer Ub-binding enzyme Ubc13 / Uev1A tau raug lees paub los koom nrog hauv kev sib sau ntawm TRAF6 polyubiquitination16. Thaum K124 ntawm nas TRAF6 yog khob tawm, TRAF6 yuav poob qhov kev ua ntawm K63 auto-ubiquitination, thiab kev hloov pauv ntawm qhov chaw no tshem tawm cov kev sib txuas ntawm TRAF6-koom haum NEMO thiab ua kom TAK1, IKK, thiab NF-κB55 . Nws yog qhov tseeb vim tias PRDX1 khi rau TRAF6 Ntiv nplhaib ntiv tes sau muaj nyob hauv K124 thiab tshem tawm qhov kev nthuav dav ntawm TRAF638. Nws tau raug tshaj tawm txog qhov ubiquitination ntawm TRAF6 yog ze ze rau lub dimerization ntawm nws N-terminal domain (Ntiv nplhaib ntiv tes thiab ZF ntiv tes sau). Ntau qhov chaw N-terminal kev sib cuam tshuam nrog Ubc13 tau hloov pauv, thiab Ubc13 tsis tuaj yeem xa poly~ub mus rau TRAF616. Kev sib cuam tshuam ntawm calpain2a thiab TRAF6 mutants tau pom tias feem ntau ntawm cov thawj ntawm TRAF6 cuam tshuam nrog calpain2a, tshwj tsis yog rau lub nplhaib ntiv tes. Vim muaj kev cuam tshuam ntawm calpain2a thiab ZF ntiv tes sau ntawm TRAF6, peb kwv yees tias calpain2a tuaj yeem cuam tshuam nws pib-ubiquitination los ntawm kev thaiv cov dimerization ntawm TRAF6. Tom qab ntawd, peb tau txheeb xyuas qhov sib cuam tshuam ntawm cov cim sib txawv ntawm TRAF6. Raws li kev qhia ntawm mutant calpain2a tsis muaj hydrolase kev ua haujlwm tau nce, kev sib cuam tshuam ntawm cov cim sib txawv ntawm TRAF6 tsis muaj zog. HA-tagged TRAF6 txhawb nqa ubiquitination ntawm Myc-tagged TRAF6, thiab mutant calpain2a inhibits ubiquitination enhanced los ntawm HA-tagged TRAF6. Raws li qhov nruab nrab hauv cov txheej txheem ntawm TRAF6-TAK1 teeb liab kis tau tus mob, ECSIT khi rau C-terminal domain ntawm TRAF6 (CC domain thiab TRAF-C domain) thiab tau lees paub tias koom nrog hauv NF-κB signaling37. Kev nthuav qhia ntawm proin- flammatory cytokines txo qis hauv ECSITKD THP-1 hlwb. Txawm li cas los xij, tom qab tshaj tawm ntawm ECSIT hauv ECSITKD THP-1 hlwb, cov noob xws li IRF7, IL-1 , thiab CD44 tau nce ntau 37. PRDX1 khi rau lub nplhaib ntiv tes ntawm TRAF6 thiab inhibits ubiquitination ntawm ECSIT thiab BECN1 hauv NF-κB thiab autophagy signals los ntawm inhibiting qhov tsim ntawm TRAF6 polyubiquitination38. Hauv cov ntses teleost, peb tau lees paub ECSIT tau qhib NF-κB kev taw qhia txoj hauv kev, cuam tshuam nrog TRAF6 thiab TAK1 feem, thiab kis tau cov saw polyubiquitin uas tsim los ntawm TRAF6. calpain2a khi rau ZF domain, CC domain, thiab TRAF-C domain ntawm TRAF6, uas cuam tshuam kev cuam tshuam ntawm TRAF6-ECSIT. Peb tau lees paub tias mutant calpain2a tsis muaj kev ua haujlwm hydrolase tiv thaiv cov saw polyubiquitin los ntawm TRAF6 rau ECSIT thiab BECN1.

Fig. 6 calpain2a attenuates autoubiquitination of TRAF6. a HEK293 cells were transfected with TRAF6-Myc, TRAF6-HA, or empty vector. After 24 h post-transfection, the cells were lysed and IP analyzed with HA antibody. b HEK293 cells were transfected with TRAF6-Myc, TRAF6-HA, empty vector, or calpain2a-Flag. After 24 h post-transfection, the cells were lysed and IP analyzed with HA antibody. c HEK293 cells were transfected with TRAF6-Myc, TRAF6-HA, empty vector, or different concentrations of calpain2a-ΔCysPc-Flag. After 24 h post-transfection, the cells were lysed and IP analyzed with HA antibody. d, e HEK293 cells were cotransfected with TRAF6-Myc, TRAF6-HA, and WT-ubiquitin-His or K63O-ubiquitin-His (in which only lysine 63 is kept) together with calpain2a-Flag, calpain2a-ΔCysPc-Flag or empty vector. After 24 h post-transfection, the cells were lysed and purified with Ni-NTA agarose. The immunoprecipitates and input immunoblot analysis with anti-Myc, anti-Flag, anti-HA, and anti-Tubulin Abs. All experiments were performed in at least three independent experiments.

Daim duab 6 calpain2a attenuates autoubiquitination ntawm TRAF6. HEK293 hlwb tau hloov nrog TRAF6-Myc, TRAF6-HA, lossis vector khoob. Tom qab 24 teev tom qab hloov pauv, cov hlwb raug lysed thiab IP tshuaj xyuas nrog HA antibody. b HEK293 hlwb tau hloov nrog TRAF6-Myc, TRAF6-HA, khoob vector, lossis calpain2a-Flag. Tom qab 24 teev tom qab hloov pauv, cov hlwb raug lysed thiab IP tshuaj xyuas nrog HA antibody. c HEK293 hlwb tau hloov pauv nrog TRAF6-Myc, TRAF6-HA, khoob vector, lossis ntau qhov sib txawv ntawm calpain2a-ΔCysPc-Flag. Tom qab 24 teev tom qab hloov pauv, cov hlwb raug lysed thiab IP tshuaj xyuas nrog HA antibody. d, e HEK293 hlwb tau cotransfected nrog TRAF6-Myc, TRAF6-HA, thiab WT-ubiquitin-Nws los yog K63O-ubiquitin-Nws (uas tsuas yog lysine 63 khaws cia) ua ke nrog calpain2a-Flag , calpain2a-ΔCysPc-Flag lossis vector khoob. Tom qab 24 teev tom qab hloov pauv, cov hlwb raug lysed thiab purified nrog Ni-NTA agarose. Lub immunoprecipitates thiab input immunoblot tsom xam nrog anti-Myc, anti-Flag, anti-HA, thiab anti-Tubulin Abs. Txhua qhov kev sim tau ua nyob rau hauv tsawg kawg peb qhov kev sim ywj pheej.

Fig. 7 calpain2a inhibits TRAF6-mediated ubiquitination of ECSIT and BECN1. a HEK293 cells were transfected with ECSIT-Myc, TRAF6-Flag, or empty vector. After 24 h post-transfection, the cells were lysed and IP analyzed with Flag antibody. b HEK293 cells were transfected with TAK1-Flag, ECSIT-Myc, or empty vector. After 24 h post-transfection, the cells were lysed and IP analyzed with Myc antibody. c HEK293 cells were transfected with TRAF6-Myc, ECSIT-HA, empty vector, or different concentrations of calpain2a-ΔCysPc-Flag. After 24 h post-transfection, the cells were lysed and IP analyzed with Myc antibody. d HEK293 cells were cotransfected with ECSIT-Myc, TRAF6-HA, WT-ubiquitin-His together with calpain2a-Flag, calpain2a-ΔCysPc-Flag or empty vector. After 24 h post-transfection, the cells were lysed and purified with Ni-NTA agarose. e HEK293 cells were transfected with BECN1-Flag, TRAF6-Myc, or empty vector. After 24 h post-transfection, the cells were lysed and IP analyzed with Myc antibody. f HEK293 cells were cotransfected with BECN1-HA, TRAF6-Myc, WT-ubiquitin-His together with calpain2a-Flag, calpain2a-ΔCysPc-Flag or empty vector. After 24 h post-transfection, the cells were lysed and purified with Ni-NTA agarose. The immunoprecipitates and inputs with anti-Myc, anti-Flag, anti-HA, and anti-Tubulin Abs. All experiments were performed in at least three independent experiments.


Daim duab 7 calpain2a inhibits TRAF6-mediated ubiquitination ntawm ECSIT thiab BECN1. HEK293 hlwb tau hloov nrog ECSIT-Myc, TRAF6-Flag, lossis vector khoob. Tom qab 24 teev tom qab hloov pauv, cov hlwb raug lysed thiab IP tshuaj xyuas nrog Flag antibody. b HEK293 hlwb tau hloov pauv nrog TAK1-Flag, ECSIT-Myc, lossis vector khoob. Tom qab 24 teev tom qab hloov pauv, cov hlwb raug lysed thiab IP tshuaj xyuas nrog Myc antibody. c HEK293 hlwb tau hloov nrog TRAF6-Myc, ECSIT-HA, khoob vector, lossis ntau qhov sib txawv ntawm calpain2a-ΔCysPc-Flag. Tom qab 24 teev tom qab hloov pauv, cov hlwb raug lysed thiab IP tshuaj xyuas nrog Myc antibody. d HEK293 hlwb tau cotransfected nrog ECSIT-Myc, TRAF6-HA, WT-ubiquitin-Nws ua ke nrog calpain2a-Flag, calpain2a-ΔCysPc-Flag los yog khoob vector. Tom qab 24 teev tom qab hloov pauv, cov hlwb raug lysed thiab purified nrog Ni-NTA agarose. e HEK293 hlwb tau hloov nrog BECN1-Flag, TRAF6-Myc, lossis vector khoob. Tom qab 24 teev tom qab hloov pauv, cov hlwb raug lysed thiab IP tshuaj xyuas nrog Myc antibody. f HEK293 hlwb tau cotransfected nrog BECN1-HA, TRAF6-Myc, WT-ubiquitin-His ua ke nrog calpain2a-Flag, calpain2a-ΔCysPc-Flag lossis vector khoob. Tom qab 24 teev tom qab hloov pauv, cov hlwb raug lysed thiab purified nrog Ni-NTA agarose. Cov immunoprecipitates thiab inputs nrog anti-Myc, anti-Flag, anti-HA, thiab anti-Tubulin Abs. Txhua qhov kev sim tau ua nyob rau hauv tsawg kawg peb qhov kev sim ywj pheej.

Peb pom tias calpain2a muaj cov haujlwm hydrolase ua ib qho ntawm cov calcium-dependent proteases tsev neeg. m-calpain (calpain-2) ua lub luag haujlwm zoo hauv NF-κB taw qhia txoj hauv kev ntawm HepG2 lub siab hlwb. Kev ywj pheej ntawm 26 s proteasome mus kom ze, calpain2a hydrolyzes IkB kom tso tawm qhov kev hloov pauv NF-κB. Qhov tshwj xeeb inhibitor ntawm calpain protein tiv thaiv kev degradation ntawm IkB 56. Txawm li cas los xij, rau calpain, muaj qee qhov kev tshawb fawb txog ntses lub cev tiv thaiv kab mob. Hauv MKC hlwb, overexpression ntawm calpain2a inhibited NF-κB downstream proinflammatory cytokines induced los ntawm LPS. Qhov tshwm sim no tsis sib xws rau cov txiaj ntsig hauv HepG2 daim siab hlwb. Tej zaum nws yuav yog cov hom tshwj xeeb ntawm tsev neeg calpain protein ua rau muaj kev cuam tshuam sib txawv. Ib yam li ntawd, peb siv cov inhibitor tshwj xeeb ntawm calpain protein E-64 los tiv thaiv calpain2a los ntawm hydrolyzing TRAF6. Mutant calpain2a tsis muaj kev ua haujlwm hydrolase inhibits TRAF6 ubiquitin ligase kev ua haujlwm yam tsis muaj kev cuam tshuam rau nws cov qib protein. Peb tsis pom qhov chaw enzyme-active rau calpain2a. Raws li kev tshawb fawb yav dhau los hauv cov tsiaj nyeg, peb qhov chaw: yog cysteine ​​(C105A), histidine (H262A), thiab asparagine residues (N286A). Txawm li cas los xij, peb qhov kev sim tau lees paub tias peb qhov chaw nquag ntawm miiuy croaker calpain2a tsis cuam tshuam rau kev qhia ntawm TRAF6 protein ntau, tab sis mutant calpain2a tsis muaj hydrolase domain ua. Yog li ntawd, peb tau xaiv los hloov tag nrho cov enzyme-activating structural domain. Txawm hais tias cov protein calpain2a kuj tau txais txiaj ntsig zoo hauv cov tsiaj nyeg thiab cov vertebrates qis, cov chaw tshwj xeeb enzymatic yuav tsum tau tshawb xyuas ntxiv.

Desert ginseng-Improve immunity (15)

cistanche cog-nce kev tiv thaiv kab mob

Hauv cov ntsiab lus, peb tau txheeb xyuas calpain2a ua tus tswj tsis zoo ntawm LPS-induced NF-κB signaling thiab virus-induced IFN activation (Fig. 9e). Hauv huab hwm coj spectrometry, calpain2a tuaj yeem cuam tshuam nrog TRAF6 thiab peb tau pom qhov tshwm sim no los ntawm kev sim endogenous coimmunoprecipitation. Peb pom tias calpain2a koom nrog hauv kev tswj hwm LPS-induced NF-κB txoj kev taw qhia. Tsis tas li ntawd, TRAF6 kuj tseem ua lub luag haujlwm tseem ceeb hauv cov kab mob tiv thaiv kab mob. Peb siv SCRV thiab Ploy (I: C) los txhawb txoj hauv kev IFN signaling thiab pom tias calpain2a kuj tseem tuaj yeem cuam tshuam IFN signaling los ntawm kev tswj hwm qib protein thiab autoubiquitination ntawm TRAF6. Txij li thaum TLR3 tuaj yeem paub txog cov qauv ob-stranded RNA (dsRNA) tsim los ntawm cov kab mob kis. Peb xav paub ntau ntxiv txog kev nthuav qhia calpain2a kev tswj hwm ntawm TRAF6- kho IFN teeb liab txoj hauv kev tab sis tsis suav nrog kev sib raug zoo ntawm calpain2a thiab TLR3 teeb liab txoj hauv kev. calpain2a txhawb kev degradation ntawm TRAF6 nyob rau hauv ib tug hydrolytic yam thiab interacted nrog TRAF6 inhibit qhov tsim ntawm dimers thiab nws pib-ubiquitination. Qhov kev tiv thaiv no tiv thaiv kev cuam tshuam ntawm TRAF6 thiab ECSIT thiab cov txheej txheem hloov pauv ntawm TRAF6 mus rau ECSIT, BECN1, IRF3, thiab IRF7. Peb cov txiaj ntsig tam sim no yuav ua rau muaj kev nkag siab txog cov kev cai tsis zoo los ntawm kev tsom mus rau TRAF6 hauv cov lus teb hauv lub cev. Tsis tas li ntawd, qhov kev tshawb fawb no qhia txog lub luag haujlwm tseem ceeb ntawm TRAF6 homooligomerization thiab auto-ubiquitination hauv cov txheej txheem ntawm cov ntses lub cev tiv thaiv kab mob. Concomitantly, calpain2a tau npaj los ua cov txheej txheem tswj hwm cov protein kom inhibit lub cev tiv thaiv kab mob ntawm TRAF6 thiab tiv thaiv txoj hauv kev ib txwm muaj thiab teeb tsa. Cov kev pom muaj txiaj ntsig zoo rau kev nkag siab txog vertebrate immunology thiab evolution ntawm vertebrate immune system.

Fig. 8 calpain2a inhibits SCRV- or Poly(I: C)-induced IFN activation. a b EPC cells were transfected with different concentrations of calpain2a-Flag or empty vector together with the IFN-1, ISRE, and IRF3-pro luciferase reporters. At 24 h post-transfection, cells were mock-infected or infected with SCRV or Poly(I: C) for 12 h (n = 3 per group). Western blot analysis was used to measure the expression of transiently transfected calpain2a-Flag. The expression of Tubulin was used as a loading control. c, d Viperin, Mx1, ISG15 mRNA in MKC stably transduced with si-calpain2a #1 or si-ctrl (control vector) and treated with saline (0) or challenged with SCRV or Poly(I: C) for 24 h. Relative mRNA level was normalized to the expression of the gene encoding β-actin in each sample (n = 3 per group). e, f MKC cells were transfected with either si-ctrl or si-calpain2a #1. At 24 h post-transfection, the cells were stimulated with SCRV for 24 h, then cell viability assay and cell proliferation assay were measured (n = 3 per group). Scale bar, 100 μm. g MKC cells were transfected with calpain2a-Flag or pcDNA3.1, then infected with SCRV for 24 h (n = 3 per group). The qRT-PCR analysis was conducted for intracellular and supernatant SCRV RNA expression. All experiments were performed in at least three independent experiments. Data were analyzed by one-way ANOVA (a, b), two-way ANOVA (c, d, e, f), or two-tailed Student's t-test (g). *p < 0.05, **p < 0.01.


Fig. 8 calpain2a inhibits SCRV- lossis Poly(I: C)-induced IFN activation. ab EPC cov hlwb tau hloov nrog ntau qhov sib txawv ntawm calpain2a-Flag lossis khoob vector ua ke nrog IFN-1, ISRE, thiab IRF3-pro luciferase reporters. Thaum 24 teev tom qab hloov pauv, cov hlwb raug thuam los yog kis tus kab mob SCRV lossis Poly(I: C) rau 12 teev (n=3 ib pab). Western blot tsom xam tau siv los ntsuas qhov kev qhia ntawm transiently transfected calpain2a-Flag. Kev qhia ntawm Tubulin tau siv los ua kev tswj xyuas kev thauj khoom. c, d Viperin, Mx1, ISG15 mRNA hauv MKC stably transduced nrog si-calpain2a #1 los yog si-ctrl (tswj vector) thiab kho nrog saline (0) los yog sib tw nrog SCRV los yog Poly (I: C) rau 24 h ua. Cov txheeb ze mRNA qib tau normalized rau qhov kev qhia ntawm cov noob encoding -actin hauv txhua tus qauv (n=3 ib pab pawg). e, f MKC hlwb tau kis nrog si-ctrl lossis si-calpain2a #1. Thaum 24 teev tom qab kev hloov pauv, cov hlwb raug txhawb nrog SCRV rau 24 teev, tom qab ntawd kev ntsuas kev muaj peev xwm ntawm tes thiab kev ntsuam xyuas cell proliferation tau ntsuas (n=3 ib pab pawg). Scale bar, 100 μm. g MKC hlwb tau kis nrog calpain2a-Flag lossis pcDNA3.1, tom qab ntawd kis nrog SCRV rau 24 teev (n=3 ib pawg). Kev tshuaj xyuas qRT-PCR tau ua rau cov kab mob hauv nruab nrog cev thiab supernatant SCRV RNA qhia. Txhua qhov kev sim tau ua nyob rau hauv tsawg kawg peb qhov kev sim ywj pheej. Cov ntaub ntawv tau txheeb xyuas los ntawm ib-txoj kev ANOVA (a, b), ob txoj kev ANOVA (c, d, e, f), lossis ob-txoj kev Tub Ntxhais Kawm T-test (g). *p <0.05, **p <0.01.

Cov txheej txheem

Cell kab lis kev cai.

Tib neeg lub raum embryonic (HEK) 293 cell kab thiab Epithelioma papulosum cyprini (EPC) cell kab, tau yuav los ntawm American Type Culture Collection thiab khaws cia hauv peb lub chaw kuaj mob. Miiuy croaker (M. miiuy) raum cell kab (MKC) thiab Miiuy croaker intestine cell kab (MIC) tau coj los ntawm lub raum thiab cov ntaub so ntswg ntawm miiuy croaker57,58. Miiuy croaker raum cell kab thiab Miiuy croaker txoj hnyuv cell kab tau coj los ntawm 26 degree nyob rau hauv ib tug humidified incubator uas muaj 0.5% CO2 nyob rau hauv L-15 nruab nrab (HyClone, USA) supplemented nrog 15% FBS (Gibco , USA), 100 U/ml penicillin (Gibco, USA), 100 ug/ml streptomycin (Gibco, USA), thiab 20 U/ml heparin (Solarbio, China). Epithelioma papulosum cyprini hlwb tau loj hlob ntawm 26 degree nyob rau hauv ib tug humidified incubator uas muaj 5% CO2 nyob rau hauv nruab nrab 199 (Hyclone) ntxiv nrog 10% FBS, 2 mM L-glutamine, 100 U / ml penicillin, thiab 100 mg / ml streptomycin. Tib neeg lub raum embryonic 293 hlwb tau loj hlob ntawm 37 degree nyob rau hauv ib tug humidified incubator uas muaj 5% CO2 nyob rau hauv Dulbecco lub modified Eagle's nruab nrab (DMEM) (Invitrogen) ntxiv nrog 10% FBS, 2 mM L-glutamine, 100 U / ml penicillin, thiab 10. /ml streptomycin.

Fig. 9 calpain2a inhibits TRAF6-mediated ubiquitination of IRF7 and IRF3. b HEK293 cells were cotransfected with IRF3-Myc, TRAF6-HA, and WTubiquitin-His or K63O-ubiquitin-His (in which only lysine 63 is kept) together with calpain2a-Flag, calpain2a-ΔCysPc-Flag or empty vector. After 24 h posttransfection, the cells were lysed and purified with Ni-NTA agarose. c, d HEK293 cells were cotransfected with IRF7-Myc, TRAF6-HA, and WT-ubiquitinHis or K63O-ubiquitin-His (in which only lysine 63 is kept) together with calpain2a-Flag, calpain2a-ΔCysPc-Flag or empty vector. After 24 h post transfection, the cells were lysed and purified with Ni-NTA agarose. All the immunoprecipitates and input with anti-Myc, anti-Flag, anti-HA, and anti Tubulin Abs. All experiments were performed in at least three independent experiments. e Model detailing the roles of calpain2a in TRAF6-mediated signaling pathways. Upon LPS and virus, TRAF6 could be activated, homo-oligomerization and auto-ubiquitination. BECN1, ECSIT, IRF7, and IRF3 are ubiquitinated by TRAF6 and trigger the activation of downstream signals. calpain2a as a negative regulator targets TRAF6 to inhibit TRAF6-mediated signaling pathways.


Daim duab 9 calpain2a inhibits TRAF6-mediated ubiquitination ntawm IRF7 thiab IRF3. b HEK293 hlwb tau cotransfected nrog IRF3-Myc, TRAF6-HA, thiab WTubiquitin-Nws los yog K63O-ubiquitin-Nws (uas tsuas yog lysine 63 khaws cia) ua ke nrog calpain2a-Flag, calpain2a-ΔCysPc - Chij los yog khoob vector. Tom qab 24 teev posttransfection, cov hlwb raug lysed thiab purified nrog Ni-NTA agarose. c, d HEK293 hlwb tau cotransfected nrog IRF7-Myc, TRAF6-HA, thiab WT-ubiquitinHis lossis K63O-ubiquitin-Nws (uas tsuas yog lysine 63 khaws cia) ua ke nrog calpain2a-Flag, calpain2a -ΔCysPc-Flag lossis vector khoob. Tom qab 24 teev tom qab hloov pauv, cov hlwb raug lysed thiab purified nrog Ni-NTA agarose. Tag nrho cov immunoprecipitates thiab tawm tswv yim nrog anti-Myc, anti-Flag, anti-HA, thiab anti Tubulin Abs. Txhua qhov kev sim tau ua nyob rau hauv tsawg kawg peb qhov kev sim ywj pheej. e Qauv piav qhia txog lub luag haujlwm ntawm calpain2a hauv TRAF6-txoj kev taw qhia kev sib kho. Raws li LPS thiab tus kab mob, TRAF6 tuaj yeem qhib tau, homo-oligomerization thiab auto-ubiquitination. BECN1, ECSIT, IRF7, thiab IRF3 yog ubiquitinated los ntawm TRAF6 thiab ua rau kev ua kom lub suab qis qis. calpain2a raws li qhov tsis zoo regulator lub hom phiaj TRAF6 txhawm rau tiv thaiv TRAF6-txoj kev kho kom haum xeeb.

Cov ntaub ntawv

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