| Alzheimer's disease(AD),a kind of neurodegenerative disease,is defined pathologically by amyloidβ-peptide(Aβ) plaques and neurofibrillary tangles(NFTs). The relationship between NFTs and Aβdeposits,and their role in the neuropathy of AD,is unclear.In 1990s,Uchida and coworkers,while investigating the pathogenesis of AD, established that brain extracts from AD patients stimulate the neuron survival and neurite extension of rat neuronal cultures to a greater degree than normal brain extracts,resulting in neuron outgrowth and death.This activity was proposed to be associated with the loss of a human neuronal growth inhibitory factor(hGIF),which is subsequently identified as a member of the human MT gene family and renamed as hMT3.The amino acid sequence of hGIF is highly homologous to those of other mammalian MT isoforms,including the 20 cysteine residues at conserved positions. Furthermore,like MT1/2,hGIF lacks aromatic residues and histidines and contains a high number of lysine and serine residues.However,there are two inserts in hGIF compared to MT1/2:a threonine at position 5 and a glutamate rich hexapeptide (55EAAEAE60) close to the C-terminus.Additionally,all known GIF sequences contain the conserved 6CPCp9 motif,which is absent in all other members of the MT family.Our former labmate have constructed a series of mutant proteins of hGIF by molecular biology method and investigated the roles of the Thr5 and the EAAEAE on the structure-reactivity-property-function of hGIF in detail.To further clarify the relationship between structure,biochemical properties and bioactivity of hGIF,we systematically studied the effects of mutations at different sites of human GIF on the inhibitory activity toward neurons.These mutants include:1) mutants focus on the acid-base catalytic site of nitrosylation in theβ-domain,i.e.the E23K mutant et al.,2) domain-hybrid mutants,i.e.theβ(MT3)-β(MT3) andβ(MT3)-α(MT1) mutants;3) the linker mutants,including the K31/32A,K31/32E and KKP-SP mutants;4) mutants focus on the conserved 6CPCP9 motif in theβ-domain,i.e.the P7S/P9A mutant,the IG6 and IG8 mutants et al.,5) doubleβ-domain mutants:the A33-35 mutant.Neuronal culture assay results showed that:1) The E23K mutant lost its inhibitory activity completely.The results of biochemical studies showed that whole structure and stability of the metal-thiolate clusters of the E23K mutant,compared to those of hGIF,do not change too much.However,the reaction of the E23K mutant with SNOC exhibits biphasic kinetics and the mutant protein releases zinc ions much faster than hGIF in the initial step,while hGIF exhibited single kinetic process.Based on these results,we proposed that mutation at Glu23 may alter the NO metabolism and/or affect zinc homeostasis in brain,thus abolishing the neuronal growth inhibitory activity.2) The neuronal growth inhibitory activity of theβ(MT3)-β(MT3) mutant decreases significantly while theβ(MT3)-α(MT1) mutant shares similar inhibitory activity with hGIE The results of DTNB,EDTA reaction and pH titration showed that stability of Cd3S9 cluster of theβ(MT3)-β(MT3) mutant decreases significantly while the Cd3S9 cluster of theβ(MT3)-α(MT1) mutant had similar stability and solvent accessibility with that of hGIE Hence,we conclude that although the single a-domain itself does not exhibit any neuronal growth inhibitory activity,it does play an important role in modulating the stability of the metal-thiolate cluster and conformation of theβ-domain by domain-domain interactions,thus influencing the bioactivity.3) The bioactivity of the K31/32A mutant and the K31/32E mutant decreased significantly,while the bioactivity of the KKS-SP mutant was completely abolished,pH titration and DTNB reaction experiments exhibited that all the three mutations,to some extent,made theβ-domain lower in stability and looser. Furthermore,change of the KKS to SP also altered the backbone conformation and metal-thiolate cluster geometry.The observed correlation suggested that the KKS linker was a crucial factor in modulating the stability and the solvent accessibility of the Cd3S9 cluster in theβ-domain through domain-domain interactions,thus playing an important role on the biological activity of hGIF.From the aspect of evolution,we suggested that this may be a reason of why all the mammalian MTs use KKS sequence as their linkers.4) The bioactivity of the IG6 mutant decreased significantly, while the bioactivity of the IG8 mutant was almost abolished.The results of biochemical study exhibited that the overall structure and the stability of the metal-thiolate clusters of the two mutants were comparable to that of hGIF. Simulation results showed that the backbone of the IG6 mutant exhibited high similarity to that of hGIF,and the two prolines could still induce constraints on the 6CPCp9 tetrapeptide and form a similar conformation with that of hGIF.However,the structure of the first five amino acid residues in the N-terminus was quite different.In hGIF,these five residues are twisted and form a restricted conformation,while in the IG6 mutant this peptide extends naturally and smoothly,which is similar to that of MT2.For the IG8 mutant,the Gly was put in the middle of 6CPCp9 and broke the 6CPCp9 motif,thus abolishing the interaction with other molecules.Hence,it is reasonable that the inhibitory activity of the IG8 was almost eliminated.Hence,we concluded that although the structure adopted by the 6CPCp9 motif is the determinant factor of the inhibitory bioactivity of hGIF,other residues within the N-terminus (residue 1-13) may also contribute to the bioactivity.5) Bioassay results showed that the neuronal growth inhibitory activity of the A33-35 mutant was similar to that of hGIF,while theβ(MT3)-β(MT3) mutant showed reduced bioactivity.The result of DTNB and EDTA reaction exhibited that the Cd3S9 cluster of the A33-35 mutant had similar stability and solvent accessibility with that of hGIF while the stability of Cd3S9 cluster of theβ(MT3)-β(MT3) mutant decreases significantly.Therefore,we proposed that the property of the metal-thiolate cluster in theβ-domain played an important role on the bioactivity of hGIEBased on these data,we suggested that the bioactivity of hOIF is regulated by multiple factors,including(1) the defined conformation of residues 1-13,which is pivotal to the bioactivity of hGIF,no such conformation no GIF bioactivity appears; (2) domain-domain interactions,which might play important roles in modulating the stability of the metal-thiolate cluster and the conformation of theβ-domain;(3) the solvent accessibility of the metal-thiolate cluster,which is closely associated with the structure of the protein and mutual accessibility of metal-thiolate clusters with biologically sensitive small molecules such as NO;(4) the linker and other unknown factors.In 2002,sheep GIF(sGIF) cDNA and encoded protein were isolated by Chung et al.In contrast to other mammalian GIF,sGIF appears to containing three fewer cysteine residues,owing to the apparent deletion of the sequence Ser-Cys-Cys normally found at positions 33-35 of hGIF and the replacement of a cysteine residue normally found at position 30 with serine.Additionally,the conserved 5TCPCP9 motif in other species GIF is replaced by 5ACPCP9 in sGIF.The bioassay result revealed that sGIF retains part of its inhibitory activity compared with hGIF.However, previous study focusing on the function of Thr5 of hGIF clearly showed that T5A mutant,mimicking the 5ACPCP9 motif in sGIF,almost losses its inhibitory activity completely,while the T5S mutant shares similar bioactivity with hGIF,implying that the importance of the hydroxyl group for the biological activity of hGIF.These seemed conflicting reports promote us to find out the causes responsible for the unusual bioactivity of sGIF.The ESI-Mass and Cd2+ titration results showed that sGIF can bind 7 metal ions although there are total 17 cysteine residues in sGIF.The 7 metal ions were wrapped into two separate metal-thiolate clusters:one 3-metal(M3) cluster and one 4-metal(M4) cluster,in which the M3 cluster is less stable than the M4 cluster.Result of biochemical studies showed that the whole structure and dynamic properties of sGIF,as well as the solvent accessibility and stability of the metal-thiolate clusters,changed much compared to that of hGIE Hence,we proposed that the critical role of the hydroxyl group may be partly compensated by the unusual structure and dynamic properties in sGIF.More and more evidences suggest that amyliodβ-peptide(Aβ) plays a central role in the development of Alzheimer's disease pathology,emerging from a variety of genetic,pathological and biochemical studies.However,the exact mechanism of the neurotoxicity of Aβremains elusive.Among the proposed mechanisms,Met35 is thought to be a key residue in the neurotoxicity of Aβ.In the present study,we constructed 3 mutants on Met35,including Aβ1-35M35C,Aβ1-35M35P and Aβ1-35M35V, and systematically studied their biochemical properties and tried to find out the reasons for the importance of Met35 in the neurotoxicity of Aβ.MTT cell reduction assay results showed that the toxicity of Aβ1-35 was almost rescued with Met35 substituted by Cys35(Aβ1-35M35C),the Aβ1-35M35V mutant was more toxic while the Aβ1-35M35P mutant was less toxic than wild type Aβ1-35.CD experiments showed that Aβ1-35,Aβ1-35M35P and Aβ1-35M35V exhibited similar structural transformation kinetics and formed theβ-sheet conformation within 1 h.However,Aβ1-35M35C exhibited a structural transformation from random coil toα-helix,andα-helical conformation can exist stably for more than 24 hours.ThT binding assay results showed that Aβ1-35 and Aβ1-35M35P shared similar aggregation dynamics and their fluorescence reached maximum after 48h's incubation.By contrast,Aβ1-35M35V aggregated more rapidly.Surprisingly,we found the Aβ1-35M35C mutant did not produce fluorescence even after incubation for more than 72h.Our results confirmed the correlation between the ability of structural transformation from random coil toβ-sheet(aggregation ability) and the neurotoxicity of Aβ.Based on these results,we proposed that Met35 played an important role on the structural transformation from random coil toβ-sheet,thus was crucial for the neurotoxicity of Aβ.It was reported that hGIF can antagonize both the neurotrophic and neurotoxic effects of Aβ,however,till now little is known about the interaction between hGIF and Aβ.To illustrate the exact mechanism involved in their interaction,we investigated the effect of different hGIF mutants on the neurotoxicity of Aβ.Results showed that the P7S/P9A and△55-60 did not exhibit the toxicity of Aβin rat neuronal culture. |
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