Exploring Sources, Biological Functions, and Potential Applications of the Ubiquitous Marine Cyclic Dipeptide: A Concise Review of Cyclic Glycine-Proline.

Cyclic glycine-proline (cGP), a prevalent marine cyclic dipeptide, possesses a distinct pyrrolidine-2,5-dione scaffold, which contributes to the chemical diversity and broad bioactivities of cGP. The diverse sources from marine-related, endogenous biological, and synthetic pathways and the in vitro and in vivo activities of cGP are reviewed. The potential applications for cGP are also explored. In particular, the pivotal roles of cGP in regulating insulin-like growth factor-1 homeostasis, enhancing neuroprotective effects, and improving neurotrophic function in central nervous system diseases are described. The potential roles of this endogenous cyclic peptide in drug development and healthcare initiatives are also highlighted. This review underscores the significance of cGP as a fundamental building block in drug discovery with exceptional drug-like properties and safety. By elucidating the considerable value of cGP, this review aims to reignite interest in cGP-related research within marine medicinal chemistry and synthetic biology.

Streptomyces-Fungus Co-Culture Enhances the Production of Borrelidin and Analogs: A Genomic and Metabolomic Approach.

The marine Streptomyces harbor numerous biosynthetic gene clusters (BGCs) with exploitable potential. However, many secondary metabolites cannot be produced under laboratory conditions. Co-culture strategies of marine microorganisms have yielded novel natural products with diverse biological activities. In this study, we explored the metabolic profiles of co-cultures involving Streptomyces sp. 2-85 and Cladosporium sp. 3-22-derived from marine sponges. Combining Global Natural Products Social (GNPS) Molecular Networking analysis with natural product database mining, 35 potential antimicrobial metabolites annotated were detected, 19 of which were exclusive to the co-culture, with a significant increase in production. Notably, the Streptomyces-Fungus interaction led to the increased production of borrelidin and the discovery of several analogs via molecular networking. In this study, borrelidin was first applied to combat Saprolegnia parasitica, which caused saprolegniosis in aquaculture. We noted its superior inhibitory effects on mycelial growth with an EC50 of 0.004 mg/mL and on spore germination with an EC50 of 0.005 mg/mL compared to the commercial fungicide, preliminarily identifying threonyl-tRNA synthetase as its target. Further analysis of the associated gene clusters revealed an incomplete synthesis pathway with missing malonyl-CoA units for condensation within this strain, hinting at the presence of potential compensatory pathways. In conclusion, our findings shed light on the metabolic changes of marine Streptomyces and fungi in co-culture, propose the potential of borrelidin in the control of aquatic diseases, and present new prospects for antifungal applications.

DPPH Radical Scavenging Activity of New Phenolics from the Fermentation Broth of Mushroom Morehella importuna.

In recent years, wild morel mushroom species have begun to be widely cultivated in China due to their high edible and medicinal values. To parse the medicinal ingredients, we employed the technique of liquid-submerged fermentation to investigate the secondary metabolites of Morehella importuna. Two new natural isobenzofuranone derivatives (1-2) and one new orsellinaldehyde derivative (3), together with seven known compounds, including one o-orsellinaldehyde (4), phenylacetic acid (5), benzoic acid (6), 4-hydroxy-phenylacetic acid (7), 3,5-dihydroxybenzoic acid (8), N,N'-pentane-1,5-diyldiacetamide (9), and 1H-pyrrole-2-carboxylic acid (10), were obtained from the fermented broth of M. importuna. Their structures were determined according to the data of NMR, HR Q-TOF MS, IR, UV, optical activity, and single-crystal X-ray crystallography. TLC-bioautography displayed that these compounds possess significant antioxidant activity with the half DPPH free radical scavenging concentration of 1.79 (1), 4.10 (2), 4.28 (4), 2.45 (5), 4.40 (7), 1.73 (8), and 6.00 (10) mM. The experimental results would shed light on the medicinal value of M. importuna for its abundant antioxidants.

Mass Fabrication of Capillary Columns Based on Centrifugal Packing.

Packed capillary columns have become the standard front-end separation device for mass spectrometry-based proteomics. The development of simple, fast, and robust capillary column technology, especially that with mass-fabrication capacity, can greatly improve analytical throughput and reproducibility in omics research. In this technical note, we report a centrifugal packing technology, which has the capability to mass fabricate high quality capillary columns with a 2886 columns/day fabrication throughput. The centrifugally packed columns presented significantly improved efficiency (reduced plate height hmin = 1.6, 37%-40% improvement compared with slurry packed columns), advanced kinetic performance limit, and excellent column-to-column reproducibility (2.0% RSD for retention time, 50 columns). Such columns enabled ∼5300 HeLa proteins identified in single-shot proteomic analysis, displaying both intercolumn and inter-run retention time stability (retention time RSD = 0.94% between nine replicates on three columns for probing peptide sequence). The mass-fabrication technology reported in this technical note may support disposable use of high quality chromatographic columns in large-scale bioanalysis.

Diversification of phenolic glucosides by two UDP-glucosyltransferases featuring complementary regioselectivity.

BACKGROUND: Glucoside natural products have been showing great medicinal values and potentials. However, the production of glucosides by plant extraction, chemical synthesis, and traditional biotransformation is insufficient to meet the fast-growing pharmaceutical demands. Microbial synthetic biology offers promising strategies for synthesis and diversification of plant glycosides. RESULTS: In this study, the two efficient UDP-glucosyltransferases (UGTs) (UGT85A1 and RrUGT3) of plant origin, that are capable of recognizing phenolic aglycons, are characterized in vitro. The two UGTs show complementary regioselectivity towards the alcoholic and phenolic hydroxyl groups on phenolic substrates. By combining a developed alkylphenol bio-oxidation system and these UGTs, twenty-four phenolic glucosides are enzymatically synthesized from readily accessible alkylphenol substrates. Based on the bio-oxidation and glycosylation systems, a number of microbial cell factories are constructed and applied to biotransformation, giving rise to a variety of plant and plant-like O-glucosides. Remarkably, several unnatural O-glucosides prepared by the two UGTs demonstrate better prolyl endopeptidase inhibitory and/or anti-inflammatory activities than those of the clinically used glucosidic drugs including gastrodin, salidroside and helicid. Furthermore, the two UGTs are also able to catalyze the formation of N- and S-glucosidic bonds to produce N- and S-glucosides. CONCLUSIONS: Two highly efficient UGTs, UGT85A1 and RrUGT3, with distinct regioselectivity were characterized in this study. A group of plant and plant-like glucosides were efficiently synthesized by cell-based biotransformation using a developed alkylphenol bio-oxidation system and these two UGTs. Many of the O-glucosides exhibited better PEP inhibitory or anti-inflammatory activities than plant-origin glucoside drugs, showing significant potentials for new glucosidic drug development.

Subergorgines A-E, five new suberosanone-purine hybrids from the South China Sea gorgonian Subergorgia suberosa.

Five new suberosanone-purine hybrids, namely subergorgines A-E (1-5), were isolated from the South China Sea gorgonian Subergorgia suberosa. Their structures were elucidated on the basis of extensive spectroscopic data and the absolute configurations were clarified by the theoretical ECD calculation. Compounds 1-5 were rare purine alkaloids merged with the same suberosanone moiety via different C (6)-N bridges. Cytotoxic activities of the isolates were tested. Compound 4 was found to be the most active against the HL-60 cancer cell line with an IC50 value of 14.3 µM. A plausible biosynthetic pathway for suberosanone-purine hybrids was also discussed.

Chemical Constituents of the Deep-Sea-Derived Penicillium citreonigrum MCCC 3A00169 and Their Antiproliferative Effects.

Six new citreoviridins (citreoviridins J-O, 1-6) and twenty-two known compounds (7-28) were isolated from the deep-sea-derived Penicillium citreonigrum MCCC 3A00169. The structures of the new compounds were determined by spectroscopic methods, including the HRESIMS, NMR, ECD calculations, and dimolybdenum tetraacetate-induced CD (ICD) experiments. Citreoviridins J-O (1-6) are diastereomers of 6,7-epoxycitreoviridin with different chiral centers at C-2-C-7. Pyrenocine A (7), terrein (14), and citreoviridin (20) significantly induced apoptosis for HeLa cells with IC50 values of 5.4 µM, 11.3 µM, and 0.7 µM, respectively. To be specific, pyrenocine A could induce S phase arrest, while terrein and citreoviridin could obviously induce G0-G1 phase arrest. Citreoviridin could inhibit mTOR activity in HeLa cells.

Axinellamine E, One New Pyrrololactam Alkaloid from the South China Sea Sponge Axinella sp.

Chemical investigation of the EtOH extract of the sponge Axinella sp. collected from the South China Sea resulted in the identification of one new pyrrololactam alkaloid, axinellamine E (2), along with four known analogs (1, 3-5). Compound 1 was initially separated as enantiomers and was further separated to be optically pure compounds (1 a and 1 b) by a chiral column. The planar structure of compound 2 was determined mainly by 1D-, 2D-NMR, and HR-ESI-MS data analyses. Absolute configurations of 1 a and 1 b was defined by calculated ECD spectra method. All of the compounds were evaluated for their anti-inflammatory activities against nitric oxide production in lipopolysaccharide-induced RAW 264.7 cells among which compound 1 showed weak activity at 40 µg/mL. Plausible biosynthetic pathways corresponding to aldisine analogs of 1, 2, 4, and 5 were also discussed.

Two New Cerebroside Metabolites from the Marine Fungus Hortaea werneckii.

Two new cerebroside metabolites were isolated from the fermented sponge-derived fungus extract of Hortaea werneckii. They were hortacerebroside A (1) ((2R,3E)-N-[(2S,3R,4E,8E)-1-(ß-D-glucopyranosyloxy)-3-hydroxy-9-methylhenicosa-4,8-dien-2-yl]-2-hydroxypentadec-3-enamide) and hortacerebroside B (2) ((2R)-N-[(2S,3R,4E,8E)-1-(ß-D-glucopyranosyloxy)-3-hydroxy-9-methylhenicosa-4,8-dien-2-yl]-2-hydroxypentadecanamide). Their structures were elucidated by spectroscopic analysis and by comparison of the spectroscopic data with those of related cerebroside analogs. These two compounds showed significant inhibitory effect on NO produced by lipopolysaccharide (LPS) stimulated RAW 264.7 macrophages. The IC50 values of hortacerebroside A (1) and hortacerebroside B (2) were 7 and 5 µM, respectively. These results suggested the potential application of these cerebrosides as drug leads targeting inflammatory-related disorders.

Anti-Food Allergic Compounds from Penicillium griseofulvum MCCC 3A00225, a Deep-Sea-Derived Fungus.

Ten new (1-10) and 26 known (11-36) compounds were isolated from Penicillium griseofulvum MCCC 3A00225, a deep sea-derived fungus. The structures of the new compounds were determined by detailed analysis of the NMR and HRESIMS spectroscopic data. The absolute configurations were established by X-ray crystallography, Marfey's method, and the ICD method. All isolates were tested for in vitro anti-food allergic bioactivities in immunoglobulin (Ig) E-mediated rat basophilic leukemia (RBL)-2H3 cells. Compound 13 significantly decreased the degranulation release with an IC50 value of 60.3 µM, compared to that of 91.6 µM of the positive control, loratadine.

Roseovarius spongiae sp. nov., a bacterium isolated from marine sponge.

A taxonomic study was carried out on strain HN-E21T, which was isolated from sponge collected from Yangpu Bay, Hainan, PR China. Cells of strain HN-E21T were Gram-stain-negative, non-spore-forming, tumbling-motile, ovoid- to rod-shaped and pale-yellow-pigmented that could grow at 15-42 °C (optimum, 37 °C), at pH 6-11 (pH 7) and in 0-14 % (w/v) NaCl (2-3 %). This isolate was positive for oxidase and catalase, but negative for hydrolysis of aesculin and gelatin. The phylogenetic tree based on 16S rRNA gene sequences showed that strain HN-E21T formed a clade with Pelagivirga sediminicola BH-SD19T, Pontibaca methylaminivorans GRP21T, Roseovarius antarcticus M-S13-148T, Roseovarius aquimarinus CAU1059T and Roseovarius nanhaiticus NH52JT within the family Rhodobacteraceae. Strain HN-E21T shared the highest similarity to Pelagivirga sediminicola BH-SD19T (95.5 %), followed by Roseovarius lutimaris 112T (95.3 %), Pelagicola litorisediminis D1-W8T (95.2 %), Roseovarius gaetbuli YM-20T (95.0 %) and Roseovarius marisflavi H50T (95.0 %). The dominant fatty acids were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c) and C16:0. The major polar lipids comprised phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, three unidentified lipids and one unidentified phospholipid. The respiratory quinone was identified as Q-10. The G+C content of the genomic DNA was 66.4 mol%. Based on the phenotypic and phylogenetic data, strain HN-E21T represents a novel species of the genus Roseovarius, for which the name Roseovarius spongiae sp. nov. is proposed, with the type strain HN-E21T (=MCCC 1K03333T=LMG 30456T).

Mangrovimonas spongiae sp. nov., a novel member of the genus Mangrovimonas isolated from marine sponge.

A taxonomic study was carried out on strain HN-E26T, which was isolated from sponge collected from Yangpu Bay, Hainan, PR China. Cells of strain HN-E26T were Gram-stain-negative, motile by gliding, yellow-pigmented and rod-shaped. The strain could grow at 10-40 °C (optimum, 25 °C), at pH 6.0-9.0 (optimum, pH 7.0) and in 0.5-12 % (w/v) NaCl (optimum, 4-7 %). This isolate was positive for oxidase, catalase, and the hydrolysis of starch, xylan, aesculin and gelatin, but negative for indole production and the reduction of nitrate. Strain HN-E26T shared the highest 16S rRNA gene sequence similarity with Mangrovimonas yunxiaonensis LYYY01T (95.5 %), followed by Formosa spongicola A2T (94.4 %), Meridianimaribacter flavus NH57NT (94.3 %) and Winogradskyella exilis 022-2-26T (94.3 %). The phylogenetic tree based on 16S rRNA gene sequences revealed that strain HN-E26T formed a distinct phylogenetic lineage within the cluster comprising Mangrovimonas yunxiaonensis LYYY01T and 'Mangrovimonas xylaniphaga' ST2L12T. The dominant fatty acids were iso-C15 : 0 and iso-C15 : 1 G. The major polar lipids comprised phosphatidylethanolamine, three unidentified aminolipids and six unidentified lipids. The respiratory lipoquinone was identified as MK-6. The G+C content of the genomic DNA was 33.9 mol%. Based on the phenotypic and phylogenetic data, strain HN-E26T represents a novel species of the genus Mangrovimonas, for which the name Mangrovimonas spongiae sp. nov. is proposed, with the type strain HN-E26T (=MCCC 1K03326T=LMG 30458T).

Asperochratides A-J, Ten new polyketides from the deep-sea-derived Aspergillus ochraceus.

Ten new C9 polyketides (asperochratides A-J, 1-10) and 14 known miscellaneous compounds (11-24) were isolated from the deep-sea-derived fungus Aspergillus ochraceus. Structures of the new compounds were elucidated by extensive spectroscopic analyses, modified Mosher's method, Mo2(OAc)4 induced circular dichroism (ICD) experiments, and ECD calculations. Structurally, compounds 1-11 and 16-18 share the same polyketide origin of the skeleton and belong to aspyrone co-metabolites. All isolates were tested for cytotoxic, anti-food allergic, anti-H1N1 virus, anti-microbe, and anti-inflammatory activities in vitro. Results showed that compounds 5-8 and 13-17 exerted significant cytotoxic effects on BV-2 cell line, and compound 16 showed the potential of anti-inflammatory activities.

Cladosporactone A, a Unique Polyketide with 7-Methylisochromen-3-one Skeleton from the Deep-Sea-Derived Fungus Cladosporium cladosporioides.

A unique polyketide cladosporactone A along with eight known compounds were isolated from the deep-sea-derived Cladosporium cladosporioides. The structure of cladosporactone A was established by spectroscopic analyses, and the absolute configuration was clarified by the theoretical ECD calculation. Cladosporactone A is the first member of polyketide with the 7-methylisochromen-3-one skeleton.

Altererythrobacter spongiae sp. nov., a novel member of the genus Altererythrobacter isolated from marine sponge.

A taxonomic study was carried out on strain HN-Y73T, which was isolated from sponge collected from Yangpu Bay, Hainan, PR China. Strain HN-Y73T was Gram-stain-negative, non-motile, ovoid- to rod-shaped, yellow-pigmented and could grow at 10-42 °C (optimum, 28 °C), at pH 5.0-9.0 (pH 8.0) and in 0.5-16 % (w/v) NaCl (3-4 %). This isolate was positive for oxidase, catalase and the hydrolysis of aesculin and xylan, but negative for the hydrolysis of casein and gelatin. Strain HN-Y73T shared the highest 16S rRNA gene sequence similarity with Altererythrobacter endophyticus BR75T (96.9 %), followed by Altererythrobacter estronivorus MH-B5T (96.0 %), Altererythrobacter indicus MSSRF26T (95.9 %) and Altererythrobacter mangrovi C9-11T (95.7 %). The phylogenetic tree showed that strain HN-Y73T forms a clade with A. endophyticus BR75T, A. indicus MSSRF26T and A. estronivorus MH-B5T within the genus Altererythrobacter. The dominant fatty acids were summed feature 8 (C18 : 1ω7c/ω6c) and C19 : 0cyclo ω8c. The major polar lipids comprised phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine, sphingoglycolipid phosphatidyldimethylethanolamine, phosphatidylmonomethylethanolamine, one unidentified glycolipid, two unidentified phospholipids and four unidentified lipids. The respiratory lipoquinone was identified as Q-10. The G+C content of the genomic DNA was 57.6 mol%. Based on the phenotypic and phylogenetic data, strain HN-Y73T represents a novel species of the genus Altererythrobacter, for which the name Altererythrobacter spongiae sp. nov. is proposed. The type strain is HN-Y73T (=MCCC 1K03324T=LMG 30461T).

Erythrobacter spongiae sp. nov., isolated from marine sponge.

A taxonomic study was carried out on strain HN-E23T, which was isolated from sponge collected from Yangpu Bay, Hainan, China. Cells of strain HN-E23T were Gram-stain-negative, non-motile, orange-yellow-pigmented, short rods, that could grow at 10-40 °C (optimum, 28 °C), at pH 5-11 (optimun, pH 7) and in 0.5-12 % (w/v) NaCl (optimum, 3 %). This isolate was positive for oxidase, catalase, and the hydrolysis of aesculin, but negative for indole production and the reduction of nitrate. The phylogenetic tree based on 16S rRNA gene sequences revealed that strain HN-E23T formed a distinct phylogenetic lineage within the cluster comprising Erythrobacter strains. Strain HN-E23T shared the highest 16S rRNA gene sequence similarity to Erythrobacter aquimixticola JSSK-14T (97.2 %), followed by Erythrobacter atlanticus s21-N3T (96.6 %), Erythrobacter luteus KA37T (96.5 %) and Erythrobacter citreus RE35F/1T (96.4 %). The digital DNA-DNA hybridization (dDDH) and the average nucleotide identity (ANI) values between strain HN-E23T and JSSK-14T were 18.8 and 74.9 %, respectively. The dDDH and ANI values are below the standard cut-off criteria for delineation of bacterial species. The dominant fatty acids were summed feature 8 (C18 : 1ω7c/ω6c), C16 : 0 and summed feature 3 (C16 : 1ω7c/ω6c). The major polar lipids comprised phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, sphingoglycolipid, an unidentified glycolipid and six unidentified lipids. The respiratory lipoquinone was identified as Q-10. The G+C content of the genomic DNA was 65.5 mol%. Based on the phenotypic and phylogenetic data, strain HN-E23T represents a novel species of the genus Erythrobacter, for which the name Erythrobacter spongiae sp. nov. is proposed, with the type strain HN-E23T (=MCCC 1K03331T=LMG 30457T).

Zhouia spongiae sp. nov., isolated from a marine sponge.

A taxonomic study was carried out on strain HN-Y44T, which was isolated from sponge collected from Yangpu Bay, Hainan, China. Cells of strain HN-Y44T were Gram-stain-negative, non-motile, rod-shaped, yellow-pigmented and grew at 10-40 °C (optimum, 28 °C), at pH 6-10 (optimum, 7-8) and in 0-8 % (w/v) NaCl (optimum, 2-5 %). This isolate was positive for nitrate reduction, denitrification, oxidase, catalase and aesculin hydrolysis, but negative for indole production and urease. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain HN-Y44T belongs to the genus Zhouia and is clearly distinct from the other described species of this genus, Zhouia amylolytica, with a 16S rRNA gene sequence similarity of 96.85 %. The dominant fatty acids were iso-C15 : 0, iso-C15 : 1 G and iso-C17 : 0 3-OH. The major polar lipids comprised phosphatidylethanolamine, four unidentified aminolipids, four unidentified phospholipids and one unidentified lipid. The respiratory lipoquinone was identified as MK-6. The G+C content of the genomic DNA was 32.9 mol%. On the basis of the phenotypic and phylogenetic data, strain HN-Y44T represents a novel species of the genus Zhouia, for which the name Zhouia spongiae sp. nov. is proposed, with the type strain HN-Y44T (=MCCC 1K03329T=LMG 30460T).

Metabolites from the Paracel Islands Soft Coral Sinularia cf. molesta.

Five new oxygenated sesquiterpenes, molestins A⁻D (1, 3⁻5) and epi-gibberodione (2), three new cyclopentenone derivatives, ent-sinulolides C, D, and F ((+)-9⁻(+)-11), one new butenolide derivative, ent-sinulolide H ((+)-13), and one new cembranolide, molestin E (14), together with 14 known related metabolites (6⁻8, (⁻)-9⁻(⁻)-11, (±)-12, (⁻)-13, 15⁻19) were isolated from the Paracel Islands soft coral Sinularia cf. molesta. The structures and absolute configurations were elucidated based on comprehensive spectroscopic analyses, quantum chemical calculations, and comparison with the literature data. Compound 5 is the first example of a norsesquiterpene with a de-isopropyl guaiane skeleton isolated from the genus Sinularia. Molestin E (14) exhibited cytotoxicities against HeLa and HCT-116 cell lines with IC50 values of 5.26 and 8.37 µM, respectively. Compounds 4, 5, and 8 showed significant inhibitory activities against protein tyrosine phosphatase 1B (PTP1B) with IC50 values of 218, 344, and 1.24 µM, respectively.

Study on Chemical Profile and Neuroprotective Activity of Myrica rubra Leaf Extract.

The chemical profile of Myrica rubra (a native species in China) leaf extract was investigated by UPLC-PDA-HRMS, and the neuroprotective activity of two characteristic constituents, myricanol and myricetrin, was evaluated with N2a cells using H2O2-inducedoxidative challenge through a series of methods, e.g., MTT assay, ROS assay and [Ca2+]i assay. Among the 188 constituents detected in the extract of Myrica rubra leaf, 116 were identified definitely or tentatively by the comprehensive utilization of precise molecular weight and abundant multistage fragmentation information obtained by quadrupole orbitrap mass spectrometry. In addition, 14 potential new compounds were reported for the first time. This work established an example for the research of microconstituents in a complex analyte and revealed that suppression of H2O2-induced cytotoxicity in N2a cells was achieved by the pretreatment with myricanol. The evidence suggested myricanol may potentially serve as a remedy for prevention and therapy of neurodegenerative diseases induced by oxidative stress.

Metal accumulation and differentially expressed proteins in gill of oyster (Crassostrea hongkongensis) exposed to long-term heavy metal-contaminated estuary.

Bio-accumulation and bio-transmission of toxic metals and the toxicological responses of organisms exposed to toxic metals have been focused, due to heavy metal contaminations have critically threatened the ecosystem and food security. However, still few investigations focused on the responses of certain organisms exposed to the long term and severe heavy metal contamination in specific environments. In present investigation, the Hong Kong oyster, Crassostrea hongkongensis were obtained from 3 sites which were contaminated by different concentrations of heavy metals (such as zinc, copper, manganese and lead etc.), respectively. Heavy metal concentrations in the sea water samples collected from the 3 sites and the dissected tissues of the oysters with blue visceral mass were determinated to estimate the metal contamination levels in environments and the bio-accumulation ratios of the heavy metals in the different tissues of oysters. Moreover, Proteomic methods were employed to analyze the differentially expressed proteins in the gills of oysters exposed to long-term heavy metal contaminations. Results indicated that the Jiulong River estuary has been severely contaminated by Cu, Zn and slightly with Cr, Ni, Mn, etc, moreover, Zn and Cu were the major metals accumulated by oysters to phenomenally high concentrations (more than 3.0% of Zn and about 2.0% of Cu against what the dry weight of tissues were accumulated), and Cr, Ni, Mn, etc were also significantly accumulated. The differentially expressed proteins in the gills of oysters exposed to heavy metals participate in several cell processes, such as metal binding, transporting and saving, oxidative-reduction balance maintaining, stress response, signal transduction, etc. Significantly up-regulated expression (about 10 folds) of an important metal binding protein, metallothionein (MT) and granular cells was observed in the gills of oysters exposed to long-term and severely heavy-metal-contaminated estuary, it suggested that binding toxic metals with metallothionein-like proteins (MTLP) and storing toxic metals in metal-rich granules (MRG) with insoluble forms were the important strategies of oyster to detoxify the toxic metals and adapt to the high level of metal-contaminated environment. Most of the stress and immunity responsive proteins, such as heat shock proteins (HSP), extracellular superoxide dismutase (ECSOD) and cavortin, and the cellular redox reaction relative proteins such as 20G-Fe (II) oxygenase family oxidoreductase, aldehyde dehydrogenase and retinal dehydrogenase 2, were detected significantly down-regulated in the gills of oysters exposed to long term heavy metal contaminated environments, it indicated that long term exposure different from emergent exposure to heavy metal contamination may significantly suppress the stress and immunity response system of oysters. Moreover, Formin homology 2 domain containing protein (FH2). The only protein domain to directly nucleate actin monomers into unbranched filament polymers, by which will subsequently control gene expression and chromatin remodelling complexes, was also detected greatly up-regulated in the gills of oysters exposed to long-term heavy metal contaminations. It indicated that nuclear activity regulation may also be important for oyster to adapt to the long-term heavy-metal-contaminated environment.