Human Growth Inhibitory Factor Structure, The Nature And Function

Human neuronal growth inhibitory factor (hGIF), also named as metallothionein-3 (MT3), is a member of metallothionein (MT) family. This protein consists of 68 amino acid residues, including 20 conserved cysteine residues similar to the other members of MT family. Moreover, human GIF can bind 7 divalent metal ions such as Zn²⁺ and Cd²⁺, and forms two relatively independent domains, each wrapping around a metal-thiolate cluster. There is a 3-amino-acid linker between these two domains, and the whole molecule presents a dumbbell-like shape. Human GIF is mainly expressed in the central nerves system. In the brain cortex, the distribution of zinc-rich neurons is in accordance with that of human GIF protein, suggesting that human GIF may participate in transport and homeostasis of cellular zinc ions. Alzheimer's disease (AD) is a neuron degenerative disease. It was reported that the level of human GIF in the brain extract of patients with AD is significantly lower than that of healthy people, which can stimulate overgrowth of neuron cells and finally lead to neuron death because of exhaustion of neurotrophic factor. Moreover, animal models show that the expression level of GIF obviously alters after brain injury, indicating that human GIF may take part in the process of neuron growth and regeneration. Although the mechanism of neuron inhibitory activity of human GIF is unclear and the target of human GIF is uncertain, more and more evidences indicate that the bioactivity of human GIF is regulated by several factors cooperatively.This lab has studied the structural-functional relationship of MTs for many years: An MT expression system in E coli with high yield has been established, and the purification process has been optimized; a series of human GIF mutants was generated by molecular biology method, and their structure, stability and reactivity were investigated in detail. To further clarify the connections between protein structure, biochemical properties and bioactivity, we have conducted a systematical study on the effects of mutations at different sites of human GIF on the inhibitory activity toward neurons. These mutants include: 1) Thr5 insert mutants in the β-domain, i.e. ΔT5, T5S, T5A; 2) mutant focuses on the conserved CPCP motif in theβ-domain, i.e. P7S/P9A; 3) Glu4 mutants in the β-domain, including E4W, ΔE4 and P3S/E4R; 4) mutant focuses on the acid-base catalytic site of nitrosylation in the β-domain, i.e. E23K; 5) the hexapeptide insert mutants in the α-domain of hGIF, including Δ55-60, E58K, E55/58/60Q and G53/54A etc.; 6) the linker mutants, including Δ31-34, Δ32-33 and KKP-SP; 7) domain hybridized mutants, i.e. Δ34-36, βGIF-βGIF, and βGIF-α-MT1; 8) single domain, i.e. β-domain and α-domain of human GIF.Results of the neuron culture assay proved that 1) the β-domain is indispensable for the neuronal inhibitory activity of human GIF, and the conserved CPCP motif in the β-domain is particularly important. 2) We also found out that the hydroxyl group in Thr5 is critical for the bioactivity. Deleting the hydroxyl group leads to almost completely loss of bioactivity of the T5A mutant. Based on the structure and reactivity information, we conclude that the hydroxyl group in Thr5 may stabilize the solution structure of human GIF by forming intra-molecular hydrogen bond. On the other hand, Thr5 can also serve as a potential phosphorylation site. 3) The mutant E23K loses its bioactivity, although it keeps the critical TCPCP motif. It was pointed out that except the increasing solvent accessibility of the metal-thiolate clusters, the spectroscopic properties and the reactivity of E23K are similar to those of human GIF. It can be inferred that besides the TCPCP motif, other residues in the β-domain also contribute to the bioactivity of human GIF. 4) As we observed, changing charge distribution or increasing α-helix inclination of the EAAEAE insert in the α-domain has little effect on the bioactivity of human GIF. However, deleting this hexapeptide will impair seriously the bioactivity of human GIF. Structural analysis of the α-domain of human GIF reveals that the EAAEAE insert in the α-domain lies on an extremely flexible loop with no constraint by the metal-thiolate cluster, leading to enhancement of dynamics in the α-domain of human GIF. The increased dynamics of the α-domain will affect the properties of the β-domain through inter-domain interaction, and therefore the bioactivity of human GIF β-domain is regulated by the α-domain. 5) Finally, it is observed that the absence of linker between two domains does not significantly alter the bioactivity of mutants (in the Δ31-34 and Δ32-33 mutants ofhGIF). However, replacement of KKS (mammalian MT linker) with SP (crustacean MT linker) causes total loss of the bioactivity. The mechanism for this phenomenon is not sure, but from the point of view of evolution, this may provide a reason for why mammalian MTs choose KKS as the linker.It was reported that human GIF can interact with Rab3A in the two-hybrid-yeast screen. Moreover, human GIF binds to GDP/Rab3A specifically (K_d - 2.6 μM). Rab3A belongs to Ras family. It can bind GTP and has GTP hydrolyzing activity. When Rab3A forms GTP/Rab3A, it can interact with downward targets to regulate trafficking of synaptic vesicle. After GTP is hydrolyzed to GDP, the GDP/Rab3A is inactive. The GDP/GTP exchange rate in Rab3A is controlled by several proteins, including guanine nucleotide exchange proteins (GEPs), GTPase-activating proteins (GAPs), and GDP dissociation inhibitors (GDIs). Till now little is known about the interaction between Rab3A and human GIF. To study the mechanism of their interaction in vitro, we cloned Rab3A cDNA into GST fusion protein expression plasmid, and the GST-Rab3A fusion protein was expressed in E coli. After purification the molecular weight of GST-Rab3A is verified by mass spectroscopy, and the interaction between human GIF and Rab3A was investigated.GIF, MT1, and MT4 all belong to MT family. They exhibit high sequence identity, while only GIF has the unique neuronal growth inhibitory activity. Is the bioactivity of human GIF related to the structure and properties of its metal-thiolate clusters? Therefore we investigated the structure and properties of metal-thiolate clusters in human GIF, and compared with those of clusters in human MT1 and mouse MT4. The results show that human MT1 and mouse MT4 have similar cluster structure and stability, while the clusters in human GIF have different structure and lower stability. The ability of human GIF clusters to interact with thiol regents is also affected by the alteration of cluster structure and stability. Furthermore, the metal transfer ability of the three proteins follows human GIF > human MT1 > mouse MT4. These results indicate that the solution structure of human GIF is much looser than those of the other two MTs. Several cooperative factors may be attributed to the effect: the overall more negative charge of human GIF, the EAAEAE insert, the CPCP motif, and theinter-domain interaction et al.In summary, we have studied the structure-reactivity-function relationship of human GIF using chemical and biological tools, including protein engineering, spectroscopic characterization, and primary neuron culture assay and so on. The neuronal growth inhibitory activity of human GIF is regulated by multiple factors cooperatively, including the hydroxyl group in Thr5, the conservative CPCP motif, the inter-domain interaction, the linker, and the metal-thiolate clusters etc.