Roles And Mechanisms Of SUMO-2 On Regulation Of Protein Translation And Its Significance In Wound Healing

BackgroundSUMO modification has emerged as an important regulatory mechanism for protein activity, stability and localization. Most of the SUMO targets identified thus far are involved in various cellular processes, such as nucleocytoplasmic transport, transcriptional regulation, apoptosis, response to stress, and cell cycle progression. Sumoylation regulates several aspects of gene expression, including DNA transcription, m RNA splicing and m RNA polyadenylation. However, how the SUMO modification affect protein translation is poorly understood now. In eukaryotic cells, most of proteins are synthesized by cap dependent mRNA translation. It is already known that the overexpression of the sumo E2 conjugating enzyme, namely Ubc9, can significantly increase the cap dependent expression of luciferase reporter genes, and overexpression of SUMO-1 only slightly increased the expression of luciferase reporter genes. This may be the modification by SUMO-2/3 subtypes involved in the regulation of the cap dependent mRNA translation. However, more detailed roles and intrinsic mechanisms of SUMO-2/3 modification on the regulationg of cap dependent mRNA translation needs to be further studied.The mammalian target of rapamycin(m TOR) is a serine/theronine kinase which plays an important role in the regulation of metabolic processes and protein translation rates. The AKT/m TOR pathway activation involves in initiating protein translation(promoting growth factors synthesis), angiogenesis, cellular proliferation or differentiation. The mammalian TOR(mTOR) pathway is a key regulator of cell growth and proliferation and increasing evidence suggests that its deregulation is associated with human diseases, including cancer and diabetes. Diabetes is characterised by delayed and poor wound healing due to reduced cellular proliferation and dramatic reduction in protein synthesis leading to lower levels of extracellular matrix(ECM) andcollagen deposition, growth factors, decreased cellular recruitment and migration, impaired re-epithelialization, decreased angiogenesis and granulation tissue formation. However, the underlying mechanisms responsible for diabetic wounds healing are not completely understood. In particular, the role and mechanism of m TOR pathway in the regulation of protein translation in the diabetic wound remain to be further studied.ObjectiveTo understand the role and molecular mechanism of SUMO-2 modification in the regulation of protein translation;To study the role and mechanism of m TOR pathway in diabetic wound healing.Methods1. The role and molecular mechanism of SUMO-2 in the regulation of protein translationHuman colon cancer cell strains HCT116 cells were transfected with SUMO-2 gene and luciferase reporter gene,to assay effects of SUMO-2 on cap dependent protein translation; Immunoprecipitation method was imployed to concentrate and purify protein translation initiation complexes and investigate effects of SUMO-2 on eIF4 F complex formation; Western blot method was used to determine the expression level of related proteins in the pathway.2. The role of m TOR pathway in diabetic wound pathology and the molecular mechanism of GM-CSF to regulate mTOR pathway and promote the wound healing of diabetes.Full-thickness skin excisional wounds were created on the backs of normal and streptozotocin- induced diabetic rats. The expression of key proteins in the Akt/mTOR pathway was assayed using western blotting; topical effects of GM-CSF on diabetic wounds and activation of the Akt/m TOR pathway were subsequently investigated. Activation of the Akt/m TOR pathway by GM-SCF in vitro was examined in rat primary fibroblasts.Results1. The results of co-transfection of SUMO-2 and luciferase reporter gene in HCT116 cells showed that SUMO-2 can significantly promote the expression of proteins in the cell by activating the cap dependent protein translation.2. SUMO-2 coupling is a prerequisite for the activation of cap dependent protein translation; SUMO-2 promotes the formation of e IF4 F complexes by enhancing the interaction between e IF4 E and SUMO-2; SUMO-2 can rescue the inhibitory effects of 4EGI-1 on the interaction between e IF4 E and e IF4 G.3. SUMO-2 promotes the cap dependent expression of target proteins, such as c-myc, D1 cyclin, etc.; SUMO-2 conjucation is capable of promoting cell proliferation and inhibiting apoptosis.4. Western blot analysis showed that the Akt/m TOR signaling pathway is activated in the tissue of normal wounds compared to intant skin, but dramatically downregulated in diabetic wounds. Upon topical application of GM-CSF, the wound healing time of diabetic rats was normalized, followed by increase in the wound closure rate, the microvessel density, growth factors production and the synthesis of collagen, more importantly, the treatment also increased expression of key proteins of Akt/m TOR pathway, such as Akt, m TOR, p70S6 K, S6, 4E-BP1 and e IF4 E, being a reversal of defective Akt/mTOR pathway activated.ConclusionsSUMO-2 modification significantly enhanced the interaction between e IF4 E and e IF4 G, promoted the formation of e IF4 F complex, and then significantly improve the protein expression. SUMO-2 modification is one of the important regulatory mechanisms in cells, which plays an important role in the basic mechanism of cellular translation.Akt/mTOR signaling pathway,which is regulating the protein translation process, was attenuated in wounded skin of diabetic rats; GM-CSF increased the expression and activity of key proteins of Akt/m TOR pathway, thus accelerates diabetic wound healing process, improves the quality of healing. The AKT/mTOR signaling pathway play a direct pivotal role in the diabetic wound healing, representing an attractive method for treatment.