Downregulation of LINC00515 in high-grade serous ovarian cancer and its relationship with platinum resistance.

Aim: To investigate the expression of long intergenic noncoding RNA 00515 (LINC00515) in high-grade serous ovarian cancer (HGSOC) and its potential correlation with platinum resistance. Patients & methods: Expression of LINC00515 in HGSOC (n = 115) and normal (n = 19) tissues was detected via quantitative real-time PCR (qRT-PCR). We further explored the statistical significance of the relationship between LINC00515 expression and platinum resistance in HGSOC. Results: LINC00515 was gradually downregulated in the order of normal > platinum-sensitive > platinum-resistant tissue (p < 0.05). Results demonstrated that LINC00515 downregulation was correlated with platinum resistance and relapse-free survival (RFS) of HGSOC (p < 0.05). Conclusion: LINC00515 downregulation is correlated with HGSOC development, platinum resistance and RFS, supporting its utility as a potential biomarker to predict platinum resistance and prognosis of RFS.

The structural and biological significance of the EAAEAE insert in the alpha-domain of human neuronal growth inhibitory factor.

Human neuronal growth inhibitory factor (hGIF) is able to inhibit the outgrowth of neurons. As compared with the amino acid sequences of metallothionein 1/2, hGIF contains two insertions: a Thr at position 5 and an acidic hexapeptide EAAEAE(55-60) close to the C-terminus. Moreover, all mammalian growth inhibitory factor sequences contain a conserved CPCP(6-9) motif. Previous studies have demonstrated that the TCPCP(5-9) motif is pivotal to its bioactivity, but no specific role has been assigned to the unique EAAEAE(55-60) insert. To investigate the potential structural and biological significance of the EAAEAE(55-60) insert, several mutants were constructed and investigated in detail. Notably, deletion of the acidic insert (the Delta55-60 mutant) reduced the inhibitory activity, whereas the bioactivities of other mutants did not change much. Then, spectroscopic characterization and molecular dynamics simulation were performed to investigate the potential causes of the reduced bioactivity of the Delta55-60 mutant. It was found that the domain-domain interaction mechanism of hGIF was different from that of metallothionein 2. It was also shown that the acidic insert could regulate the interdomain interactions in hGIF, leading to the structural change in the beta-domain, which resulted in the alteration of the solvent accessibility and metal release ability, thus playing an important role in the biological activity of hGIF. Our studies provided useful information on the domain-domain interaction at the molecular level for the first time, and shed new light on the mechanism of the bioactivity of hGIF.

Important roles of the conserved linker-KKS in human neuronal growth inhibitory factor.

Metallothinein-3 (MT3), also named neuronal growth inhibitory factor (GIF), is attractive by its distinct neuronal growth inhibitory activity, which is not shared by other MT isoforms. The polypeptide chain of GIF is folded into two individual domains, which are connected by a highly conserved linker, KKS. In order to figure out the significance of the conserved segment, we constructed several mutants of human GIF (hGIF), including the K31/32A mutant, the K31/32E mutant and the KKS-SP mutant by site-directed mutagenesis. pH titration and DTNB reaction exhibited that all the three mutations made the beta-domain lower in stability and looser. More significantly, change of KKS to SP also altered the general backbone conformation and metal-thiolate cluster geometry. Notably, bioassay results showed that the bioactivity of the K31/32A mutant and the K31/32E mutant decreased obviously, while the KKS-SP mutant lost inhibitory activity completely. Based on these results, we proposed that the KKS linker was a crucial factor in modulating the stability and the solvent accessibility of the Cd(3)S(9) cluster in the beta-domain through domain-domain interactions, thus was indispensable to the biological activity of hGIF.

Incorporation of a glycine within the conserved TCPCP motif of human neuronal growth inhibitory factor significantly reduces its bioactivity.

It has been reported that the (6)CPCP(9) motif near the N-terminus is pivotal to the inhibitory activity of human neuronal growth inhibitory factor (hGIF). In order to better understand the biological significance of this region on the structure, property and function of hGIF, we introduced a highly flexible residue, Gly, either in front of the (6)CPCP(9) motif (the IG6 mutant, TGCPCP) or in the middle of it (the IG8 mutant, TCPGCP) and investigated their structural and metal binding properties in detail. The results showed that the overall structure and the stability of the metal-thiolate clusters of the two mutants were comparable to that of hGIF. However, the bioassay results showed that the bioactivity of the IG6 mutant decreased significantly, while the bioactivity of the IG8 mutant was almost abolished. Molecular dynamics simulation results showed that the backbone of the IG6 mutant exhibited high similarity to that of hGIF, and the two prolines could still induce structural constraints on the (6)CPCP(9) tetrapeptide and form a similar conformation with that of hGIF, however, the conformation of the first five amino acid residues in the N-terminus was quite different. In hGIF, the five residues are twisted and form a restricted conformation, while in the IG6 mutant this peptide extends more naturally and smoothly, which is similar to that of MT2. As to the IG8 mutant, the Gly insertion broke the (6)CPCP(9) motif, thus probably abolishing the interactions with other molecules and eliminating its inhibitory activity. Based on these results, we suggested that although the structure adopted by the (6)CPCP(9) motif is the determinant factor of the inhibitory bioactivity of hGIF, other residues within the N-terminal fragment (residue 1-13) may also influence the peptide conformation and contribute to the protein's bioactivity.

Effect of alpha-domain substitution on the structure, property and function of human neuronal growth inhibitory factor.

Human metallothionein-3 (hMT3), also named human neuronal growth inhibitory factor (hGIF), is attractive due to its distinct neuronal growth inhibitory activity, which is not shown by other human MT isoforms. It has been reported that the neuronal growth inhibitory activity arises from the N-terminal beta-domain rather than its C-terminal alpha-domain. However, previous bioassay results have shown that the single beta-domain is less effective at inhibiting the neuron growth than that in intact hMT3 on a molar basis, which suggests that the alpha-domain is indispensable to the neuronal growth inhibitory activity of hMT3. In order to confirm this assumption, we constructed two domain-hybrid mutants, the beta(MT3)-beta(MT3) mutant and the beta(MT3)-alpha(MT1) mutant, and investigated their structural and metal binding properties by UV-vis spectroscopy, CD spectroscopy, pH titration, DTNB reaction, EDTA reaction, etc. The results showed that stability of the Cd(3)S(9) cluster of the beta(MT3)-beta(MT3) mutant decreased significantly while the Cd(3)S(9) cluster of the beta(MT3)-alpha(MT1) mutant had a similar stability and solvent accessibility to that of hMT3. Interestingly, the bioassay results showed that the neuronal growth inhibitory activity of the beta(MT3)-beta(MT3) mutant decreased significantly, while the beta(MT3)-alpha(MT1) mutant showed similar inhibitory activity to hMT3. Based on these results, we conclude that the alpha-domain is indispensable and plays an important role in modulating the stability of the metal cluster in the beta-domain by domain-domain interactions, thus influencing the bioactivity of hMT3.

Mutation at Glu23 eliminates the neuron growth inhibitory activity of human metallothionein-3.

Human metallothionein-3 (hMT3), first isolated and identified as a neuronal growth inhibitory factor (GIF), is a metalloprotein expressed predominantly in brain. However, until now, the exact mechanism of the bioactivity of hMT3 is still unknown. In order to study the influence of acid-base catalysis on S-nitrosylation of hMT3, we constructed the E23K mutant of hMT3. During the course of bioassay, we found out unexpectedly that mutation at E23 of hMT3 eliminates the neuronal growth inhibitory activity completely. To the best of our knowledge, it is the first report that other residues, besides the TCPCP motif, in the beta-domain can alter the bioactivity of hMT3. In order to figure out the causes for the loss of bioactivity of the E23K mutant, the biochemical properties were characterized by UV-vis spectroscopy, CD spectroscopy, pH titration, DTNB reaction, EDTA reaction, and SNOC reaction. All data demonstrated that stability of the metal-thiolate cluster and overall structure of the E23K mutant were not altered too much. However, the reaction of the E23K mutant with SNOC exhibited biphasic kinetics and the mutant protein released zinc ions much faster than hMT3 in the initial step, while hMT3 exhibited single kinetic process. The 2D [1H-15N] HSQC was also employed to characterize structural changes during the reaction of hMT3 with varying mounts of nitric oxide. It was shown that the resonance of Glu23 disappeared at a molar ratio of NO to protein of 4. Based on these results, we suggest that mutation at Glu23 may alter the NO metabolism and/or affect zinc homeostasis in brain, thus altering the neuronal growth inhibitory activity.

The role of Thr5 in human neuron growth inhibitory factor.

GIF, a member of the metallothionein (MT) family (assigned as MT3), is a neuron growth inhibitory factor that inhibits neuron outgrowth in Alzheimer's disease. The conserved Thr5 is one of the main differences between GIF and other members in the MT family. However, natural sheep GIF has an unusual Ala5, casting doubt on the role of common Thr5. We constructed a series of human GIF mutants at site 5, and characterized their biochemical properties by UV spectroscopy, circular dichroism spectroscopy, EDTA reaction, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) reaction, and pH titration. Their inhibitory activity toward neuron survival and neurite extension was also examined. Interestingly, the T5A mutant exhibited distinct metal thiolate activity in the EDTA and DTNB reactions, and also lost its bioactivity. Meanwhile, the T5S mutant had similar biochemical properties and biological activity as wild-type human GIF, indicating the hydroxyl group on the Thr5 was critical to the bioactivity of human GIF. We suggest the hydroxyl group in human GIF may help stabilize the biologically active conformation. On the other hand, lack of the hydroxyl group in sheep GIF may be partially compensated by its abnormal structure.

Synthesis of Human Proinsulin C-peptide and Preparation of Specific Antibody to It.

A HPLC and CE pure human proinsulin C-peptide was synthesized by solid-phase method and TSK column purification. Its amino acid sequence and MS were consistent with theoretical values. In comparison with the formly reported chemical synthesis of C-peptide, this method has the advantage of simplicity and higher overall yield (41%). To improve the immunogenicity and specificity of oligopeptide antibody, the acrylyl-C-peptides were transformed into a polymer the product had a poly-propionyl-core matrix with C-peptide branches. This treatment gave a macromolecule with a M(r) about 25 kD. By using the polymer to immunize New Zealand rabbits for 30 days, specific antiserum was obtained with titer of 2.5x10(4) (by ELISA), which did not cross react with BSA. Thus, the poly-propionyl-peptide system provided a new approach for preparing synthetic peptide antibody and therefore is promising for the preparation of synthetic peptide-based vaccine.

Synthesis and Biological Activity of Human Calcitonin Analogue.

The potency of salmon calcitonin (sCT) is higher but the structural homology between sCT and human calcition (hCT) is only 50%. Based on the comparison of the structure between sCT and hCT, we have designed and synthesized a hCT analogue (mhCT-2) by solid phase method, using air oxidation in diluted solution to obtain a peptide with intramolecular disulfide bond. Through HPLC purification, we obtained a capillary electrophoresis-homogeneous mhCT, results of analysis of its mass spectrum and N-terminal sequencing were in accordance with the theoretical values. The results of calcitonin bioassay by estimating the blood calcium levels in rats showed that the potency of mhCT-2 was around 2 000 IU/mg, one order of magnitude higher than that of hCT. In RIA assay, we have found that the immunoactivity of mhCT-2 and hCT was much different because of their different binding abilities to anti-hCT antibody. This indicated that the conformation of mhCT-2 was changed as compared with hCT. In rat osteoporosis model, the results showed that pharmacologic effects of mhCT-2 was the same as that of sCT. The synthetic mhCT-2 seems promising to be a clinically useful peptide with high potential in osteoporosis therapy, because it is similar in biological properties to, but less immunogenic than sCT.