The Study Of Interaction Between HSPB8 And NEFL, And The Influence Of HSPB8 On Cell Relative Viability

Charcot-Marie-Tooth disease (CMT), also named hereditary motor and sensory neuropathy (HMSN), is the most common hereditary peripheral neuropathy with highly clinical and genetic heterogeneity.CMT is characterized by progressive and symmetric distal muscle atrophy, hyporeflexia and hypoesthesia in distal limbs. The incidence of CMT is about 40/10,0000. According to the electrophysiological and histopathological criteria, CMT can be divided into two forms: the demyelinating form (CMT1) and the axonal form (CMT2). According to different modes of inheritance, CMT can be divided into autosomal dominant (AD), autosomal recessive (AR) and X-linked dominant or recessive (XD/XR). So far 29 gene loci have been identified and 21 distinct genes have been cloned. Recent researches showed that some subtypes of CMT2 were caused by the mutations in genes encoding small heat shock proteins (sHSPs) and neurofilament light chain (NEFL). In 2000, Mersiyanova et al found the Q333P mutation in NEFL gene in a large Russian CMT2E family and confirmed NEFL as the disease causative gene of CMT2E. In 2004, Evgrafov OVet al found S135F and R127W mutation in gene encoding Heat shock protein B1 (HSPB 1) in a large Russian and a large Belgian CMT2F family respectively. In consequence, they confirmed HSPB1 as the disease causative gene of CMT2F. In 2004, we found a large Chinese CMT2 family in Hunan and Hubei provinces which was proved to be a novel genotype designated as CMT2L. After mapping the locus to chromosome I2q24.2-12q24.3, we identified a novel 423G→T mutation of HSPB8 and confirmed amino-acid change K141N as the causative gene defect in CMT2L.HSPB8 and HSPB 1 belong to sHSPs family. Recent studies revealed that HSPB8 is a molecular chaperone which can stabilize cytoskeletons and assist their correct assembly. NEFL, as an important structure that can maintain cytoskeleton, is a main intermediate filament. Recent studies showed that both wild type HSPB8 and K141N mutant HSPB8 can interact with wild type HSPB 1. Furthermore, S 135F mutant HSPB 1 may have interaction with wild type NEFL. However, there were no reports concerning with the interaction between wild type HSPB8 and wild type NEFL, nor the interaction between K141N mutant HSPB8 and wild type NEFL. In former studies, we have successfully constructed the wild type HSPB8 and K141N mutant type HSPB8 eukaryotic expression vectors. In this study, we co-transfected wild type HSPB8/K141N mutant HSPB8 and wild type NEFL eukaryotic expression vector into Hela cell line. We analyzed the interactions between HSPB8 and NEFL by using indirect immunofluorescence technique. Our results indicated that both wild type and K141N mutant HSPB8 co-locate with NEFL, which suggests the interactions between HSPB8 and NEFL. We further confirmed the interactions between HSPB8 and NEFL by co-immunoprecipitation technique. Our results suggested that both wild type HSPB8 and K141N mutant type HSPB8 interact with wild type NEFL.To investigate the influence of HSPB8 mutation on cell relative viability in heat shock stress, we transfected wild type HSPBS/K141N mutant HSPB8 eukaryotic expression vector into SHSY-SY cell line and investigated the cell relative viability by using MTT. We confirmed that SHSY-SY cell that overexpressed K141N mutant HSPB8 is less viable than the wild type. In a word, this study has set up a foundation for our following researches on HSPB8 functions.