SIRT5-mediated Deacetylation Of LDHB Promotes Autophagy And Tumourigenesis In Colorectal Cancer

BackgroundMalignant tumor can be considered as a metabolic disease in which cancer cells can perform metabolism reprogramme by their own to meet the need of rapid proliferation in a nutrient-limited tumor microenvironment.In the condition of nutritional starvation,autophagy,an important metabolic strategy,is to be activated.Autophagy recycles and reuses intracellular metabolites such as proteins,lipids,or organelles to meet the needs of cells for biosynthesis and energy metabolism under conditions of undernutrition.Current studies have shown that autophagy is abnormally activated in a variety of tumor cells and is closely related to tumor proliferation,metastasis,and drug resistance.Lactate dehydrogenase B(LDHB),one of the important metabolic enzymes in the glycolysis,catalyzes the conversion of lactate and NAD~+to pyruvate,NADH and H~+.Previous studies have shown that LDHB promotes lysosomal acidification,autophagy and tumor cell proliferation.However,little is known about the molecular mechanisms that regulate LDHB.Protein post-translational modification(PTM)can significantly affect the related functions of mutiple metabolic enzymes.However,PTM of LDHB is still unclear.Sirtuin5(SIRT5)is a member of the nicotinamide adenine dinucleotide(NAD~+)-dependent class III deacetylase sirtuin family.It has been reported that SIRT5regulates tumor cell glycolysis,fatty acid oxidation,tricarboxylic acid and urea cycle by its PTM functions.However,the effects of SIRT5 on autophagy are not fully elucidated.This study confirmed the biological behavior and molecular mechanism of SIRT5 on the autophagy and proliferation of tumor cells by regulating LDHB.MethodAfter constructing the Flag-SIRT5 plasmid,the SIRT5 protein was enriched using Flag-labeled agarose beads.The proteins interacting with SIRT5 were screened by mass spectrometry.We further used immunoprecipitation,GST-Pulldown and protein immunofluorescence colocalization experiments to verify the interaction of SIRT5 and LDHB.We performed intracellular and extracellular deacetylation experiments to verify whether LDHB can be deacetylated by SIRT5.We then mutated each of the four putative acetylation sites of LDHB individually to arginine(R)to find the lysine site modificated by SIRT5.Then we examined the effects of SIRT5 on LDHB enzyme activity.In colorectal cancer(CRC)cells,we tested the effects of SIRT5/LDHB axis on autophagy,apoptosis,lactate,and ATP production.We finally observed whether the post-translational modification of LDHB by SIRT5 would affect the proliferation of colorectal cancer cells in vitro and in vivo.Fifty-four CRC patients were analyzed in our study.The expression levels of LDHB K329 acetylation were analyzed by immunohistochemistry on CRC tissues and normal tissues.The relationship between LDHB K329 acetylation expression and patients'clinical parameters was investigated.We used Kaplan–Meier survival analysis to evaluate the prognosis of 54 CRC patients.ResultsMass spectrometry results analyzed that SIRT5 might interact with LDHB.The Co-IP and GST-Pulldown techniques demonstrated a direct interaction between SIRT5 and LDHB.Immunofluorescence confocal microscopy showed that SIRT5 and LDHB co-localized in the cytoplasm.Through in vivo and in vitro deacetylation analysis and subsequent immunoblot analysis,it was confirmed that LDHB K329 site could be deacetylated by SIRT5,which up-regulated LDHB enzymatic activity.By autophagy and apoptotic protein immunoblotting,lysosomal acidification and lysosomal maturation measurements,it was confirmed that SIRT5 mediatd LDHB K329 deacetylation promoted lysosomal acidification and autophagy.By detecting cellular lactate,ATP content and cellular mitochondrial oxygen consumption,we found that deacetylation of LDHB K329 promoted lactate-fueled oxidative respiration and energy production.Cell proliferation,colony formation and subcutaneous xenograft experiments confirmed that SIRT5 deacetylation of LDHB K329 could promote the proliferation of CRC cells in vitro and tumor growth in vivo.Real-time PCR,immunoprecipitation and following western blotting showed that glucose starvation could promote the expression of SIRT5,which further reduced the acetylation level of LDHB K329,and finally enhanced the activity of LDHB.In addition,clinical studies showed that LDHB K329 acetylation level in CRC tissue was significantly lower than normal colorectal tissue.LDHB-Ac-K329levels were negatively associated with tumour size(P=0.004)and histological grade(P=0.031).Finally,we found that low LDHB-Ac-K329 staining predicted poor overall patient survival(P=0.017).ConclusionSIRT5 directly interacts with LDHB and yields strong deacetylation of LDHB at the K329 site.This effect increases LDHB activity and favours lysosomal acidification and autolysosomal maturation.Enhanced autophagy contributes to colorectal tumour progression.This confirms that targeting SIRT5/LDHB pathway might be a feasible and effective strategy in the treatment of CRC.