TAT-Conjugated NDUFS8 Can Be Transduced into Mitochondria in a Membrane-Potential-Independent Manner and Rescue Complex I Deficiency.

Bo-Yu Lin,Pin-Chao Liao,Mou-Chieh Kao,Kai-Wen Teng,Chao-Chang Lee,Gui-Teng Zheng,Juan-Yu Chang

doi:10.3390/ijms22126524

Abstract

NADH dehydrogenase (ubiquinone) Fe-S protein 8 (NDUFS8) is a nuclear-encoded core subunit of human mitochondrial complex I. Defects in NDUFS8 are associated with Leigh syndrome and encephalomyopathy. Cell-penetrating peptide derived from the HIV-1 transactivator of transcription protein (TAT) has been successfully applied as a carrier to bring fusion proteins into cells without compromising the biological function of the cargoes. In this study, we developed a TAT-mediated protein transduction system to rescue complex I deficiency caused by NDUFS8 defects. Two fusion proteins (TAT-NDUFS8 and NDUFS8-TAT) were exogenously expressed and purified from Escherichia coli for transduction of human cells. In addition, similar constructs were generated and used in transfection studies for comparison. The results showed that both exogenous TAT-NDUFS8 and NDUFS8-TAT were delivered into mitochondria and correctly processed. Interestingly, the mitochondrial import of TAT-containing NDUFS8 was independent of mitochondrial membrane potential. Treatment with TAT-NDUFS8 not only significantly improved the assembly of complex I in an NDUFS8-deficient cell line, but also partially rescued complex I functions both in the in-gel activity assay and the oxygen consumption assay. Our current findings suggest the considerable potential of applying the TAT-mediated protein transduction system for treatment of complex I deficiency.

Highlights

Human complex I, called NADH-coenzyme Q oxidoreductase, is the first, largest, and most complicated respiratory complex in the oxidative phosphorylation system (OXPHOS), which is critical in catalyzing NADH oxidation to transfer two electrons to ubiquinone and in coupling proton translocation for ATP generation [1,2]
We generated a protein transduction system using transcription protein (TAT) as the PTD to rdNoirdNotnefhrefDsriDtsteiacthhcaUhulUeueeleceFxIecFnoetpStotrSxrmth8eytmhy8ph-erp-ppeTeipiTsmdslsrAdAieliienensmexeTTfxtnfiidudIcecctI.ciidnioa.ogieAgelAutyuneeanc,slslsccldcedwcttycostiiyoeooteeercinrcnntdnnagdtattiuigteteniuenee,nsrnrggsssewgmeetmt,dtssdrewto.i.aiobttpIbItooeoynneoyrcucpdeuhtaharthsrrdohdaoeerednreednsnpedesdidtidtsrutneyirruoiyodiltositatleasnfnets;afdu;s,iu,t,tnnbbnhbahnbycooiyticosntsrtvtiihtaoifnoifiehnoinvncnlencnsleemdxludodxluooidifuoinmdnfgtitgcgongiethintehctngwiewonghoeoacNooanauNahnusnsnDssoDsdfulyfnuyUluurnUysdrinrpFtcatFtperochSrlhimSoruoae8peo8uldrprrsduppusolcuceuuoslrtcobreeiocnbennurioetdfunfigendifonrirTifiOnmTTtmmiAtOAXAiieneipmTXTPndTdo-tHP-pNathbrNbhHotsOyeyDeDrpOtSmtthtUahmU,hpSteieFFeht,FiaotCPSrFwtSroech8TeCC8cshawsDhauPCayoun,oalnnPlttiidnytdsnno,swhich the translocation of these two TAT fusion proteins across the inner mitochondrial membrane was independent of ∆Ψm
Using TAT-NDUFS8 as an example, we showed that after treating exogenously produced TAT-NDUFS8 for 48 h, the level of complex I assembly increased from 11.4% to 105.2% in the NDUFS8 knockdown cell line, short-hairpin RNA (shRNA)-C3 compared to that of normal cells

Summary

Introduction

Human complex I, called NADH-coenzyme Q oxidoreductase, is the first, largest, and most complicated respiratory complex in the oxidative phosphorylation system (OXPHOS), which is critical in catalyzing NADH oxidation to transfer two electrons to ubiquinone and in coupling proton translocation for ATP generation [1,2] It is the major source of reactive oxygen species (ROS) production [2,3,4]. This protein complex has an L-shaped structure containing a peripheral arm protruding in the matrix and a membrane arm embedded in the inner membrane of mitochondria [5]. Due to its dual origins, structural complexity, and important roles in OXPHOS, complex I deficiency has been linked to a large variety of pathological conditions such as fatal infantile lactic acidosis (FILA), Leigh syndrome [9,10], Leber hereditary optic neuropathy (LHON), and mitochondrial encephalomyopathy with lactic acidosis (MELAS) [8], and is associated with the pathogenesis of many devastating neurodegenerative disorders including Parkinson’s disease [11,12]

Methods

Results

Conclusion