In-depth Profiling and Quantification of the Lysine Acetylome in Hepatocellular Carcinoma with a Trapped Ion Mobility Mass Spectrometer

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related death worldwide with limited therapeutic options. Comprehensive investigation of protein posttranslational modifications in HCC is still limited. Lysine acetylation is one of the most common types of posttranslational modification involved in many cellular processes and plays crucial roles in the regulation of cancer. In this study, we analyzed the proteome and K-acetylome in eight pairs of HCC tumors and normal adjacent tissues using a timsTOF Pro instrument. As a result, we identified 9219 K-acetylation sites in 2625 proteins, of which 1003 sites exhibited differential acetylation levels between tumors and normal adjacent tissues. Interestingly, many novel tumor-specific K-acetylation sites were characterized, for example, filamin A (K865), filamin B (K697), and cofilin (K19), suggesting altered activities of these cytoskeleton-modulating molecules, which may contribute to tumor metastasis. In addition, we observed an overall suppression of protein K-acetylation in HCC tumors, especially for enzymes from various metabolic pathways, for example, glycolysis, tricarboxylic acid cycle, and fatty acid metabolism. Moreover, the expression of deacetylase sirtuin 2 (SIRT2) was upregulated in HCC tumors, and its role of deacetylation in HCC cells was further explored by examining the impact of SIRT2 overexpression on the proteome and K-acetylome in Huh7 HCC cells. SIRT2 overexpression reduced K-acetylation of proteins involved in a wide range of cellular processes, including energy metabolism. Furthermore, cellular assays showed that overexpression of SIRT2 in HCC cells inhibited both glycolysis and oxidative phosphorylation. Taken together, our findings provide valuable information to better understand the roles of K-acetylation in HCC and to treat this disease by correcting the aberrant acetylation patterns.     

Antifungal effect of CopA3 monomer peptide via membrane-active mechanism and stability to proteolysis of enantiomeric d-CopA3

In our previous study, coprisin, a 43-mer defensin-like peptide, was derived from the dung beetle, Copris tripartitus, and a 9-mer CopA3 (monomer), truncated coprisin analog peptide, was designed. However, the antifungal effects of CopA3 are not known yet. In this study, the antifungal activity and mechanism of CopA3 were investigated and to develop a more effective antimicrobial peptide under physiological conditions, the enantiomeric d-CopA3 was designed. l- and d-CopA3 had a similar antifungal activity without chiral selectivity, and their activity was more potent than that of melittin used as a positive control. Furthermore, l- and d-CopA3 did not even show any hemolysis against human erythrocytes. Membrane studies using propidium iodide and bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC₄(3)], suggested that the antifungal effect of l- and d-CopA3 was due to the membrane-active mechanism, by contrast with coprisin possessing apoptotic mechanism without membrane permeabilization. Finally, the proteolytic resistance and antifungal activity of l- and d-CopA3 against trypsin was analyzed by HPLC and colony count assay. The results showed that only d-CopA3 maintained a potent antifungal activity despite the proteolytic condition. Therefore, this study suggests that d-CopA3 has potential as a novel antimicrobial agent.     

The heterodimerization of platelet-derived chemokines

Chemokines encompass a large family of proteins that act as chemoattractants and are involved in many biological processes. In particular, chemokines guide the migration of leukocytes during normal and inflammatory conditions. Recent studies reveal that the heterophilic interactions between chemokines significantly affect their biological activity, possibly representing a novel regulatory mechanism of the chemokine activities. The co-localization of platelet-derived chemokines in vivo allows them to interact. Here, we used nano-spray ionization mass spectrometry to screen eleven different CXC and CC platelet-derived chemokines for possible interactions with the two most abundant chemokines present in platelets, CXCL4 and CXCL7. Results indicate that many screened chemokines, although not all of them, form heterodimers with CXCL4 and/or CXCL7. In particular, a strong heterodimerization was observed between CXCL12 and CXCL4 or CXCL7. Compared to other chemokines, the main structural difference of CXCL12 is in the orientation and packing of the C-terminal alpha-helix in relation to the beta-sheet. The analysis of one possible structure of the CXCL4/CXCL12 heterodimer, CXC-type structure, using molecular dynamics (MD) trajectory reveals that CXCL4 may undergo a conformational transition to alter the alpha helix orientation. In this new orientation, the alpha-helix of CXCL4 aligns in parallel with the CXCL12 alpha-helix, an energetically more favorable conformation. Further, we determined that CXCL4 and CXCL12 physically interact to form heterodimers by co-immunoprecipitations from human platelets. Overall, our results highlight that many platelet-derived chemokines are capable of heterophilic interactions and strongly support future studies of the biological impact of these interactions.     

Cyanobacterial bioactive metabolites—A review of their chemistry and biology

Cyanobacterial blooms occur when algal densities exceed baseline population concentrations. Cyanobacteria can produce a large number of secondary metabolites. Odorous metabolites affect the smell and flavor of aquatic animals, whereas bioactive metabolites cause a range of lethal and sub-lethal effects in plants, invertebrates, and vertebrates, including humans. Herein, the bioactivity, chemistry, origin, and biosynthesis of these cyanobacterial secondary metabolites were reviewed. With recent revision of cyanobacterial taxonomy by Anagnostidis and Komárek as part of the Süβwasserflora von Mitteleuropa volumes 19(1–3), names of many cyanobacteria that produce bioactive compounds have changed, thereby confusing readers. The original and new nomenclature are included in this review to clarify the origins of cyanobacterial bioactive compounds.Due to structural similarity, the 157 known bioactive classes produced by cyanobacteria have been condensed to 55 classes. This review will provide a basis for more formal procedures to adopt a logical naming system. This review is needed for efficient management of water resources to understand, identify, and manage cyanobacterial harmful algal bloom impacts.     

Heat shock proteins: A dual carrier-adjuvant for an anti-drug vaccine against heroin

Heroin is a highly abused opioid that has reached epidemic status within the United States. Yet, existing therapies to treat addiction are inadequate and frequently result into rates of high recidivism. Vaccination against heroin offers a promising alternative therapeutic option but requires further development to enhance the vaccine’s performance. Hsp70 is a conserved protein with known immunomodulatory properties and is considered an excellent immunodominant antigen. Within an antidrug vaccine context, we envisioned Hsp70 as a potential dual carrier-adjuvant, wherein immunogenicity would be increased by co-localization of adjuvant and antigenic drug hapten. Recombinant Mycobacterium tuberculosis Hsp70 was appended with heroin haptens and the resulting immunoconjugate granted anti-heroin antibody production and blunted heroin-induced antinociception. Moreover, Hsp70 as a carrier protein surpassed our benchmark Her-KLH cocktail through antibody-mediated blockade of 6-acetylmorphine, the main mediator of heroin’s psychoactivity. The work presents a new avenue for exploration in the use of hapten-Hsp70 conjugates to elicit anti-drug immune responses.     

A two-dimensional proteome map of maize endosperm

We have established a proteome reference map for maize (Zea mays L.) endosperm by means of two-dimensional gel electrophoresis and protein identification with LC-MS/MS analysis. This investigation focussed on proteins in major spots in a 4-7 pI range and 10-100 kDa M(r) range. Among the 632 protein spots processed, 496 were identified by matching against the NCBInr and ZMtuc-tus databases (using the SEQUEST software). Forty-two per cent of the proteins were identified against maize sequences, 23% against rice sequences and 21% against Arabidopsis sequences. Identified proteins were not only cytoplasmic but also nuclear, mitochondrial or amyloplastic. Metabolic processes, protein destination, protein synthesis, cell rescue, defense, cell death and ageing are the most abundant functional categories, comprising almost half of the 632 proteins analyzed in our study. This proteome map constitutes a powerful tool for physiological studies and is the first step for investigating the maize endosperm development.     

Screening combinatorial libraries of molecularly imprinted polymer films casted on membranes in single-use membrane modules

A new and fast technique for screening combinatorial libraries of molecularly imprinted polymers is presented. The procedure is based on commercially available membrane modules which are rinsed with pre-polymerization imprinting mixtures. After the in situ polymerization and generation of MIP films on the PTFE membranes within the modules, the membranes are evaluated in terms of affinity towards the target molecule (template) R-(-)-phenylbutyric acid. Therefore, after template extraction from the freshly produced membranes a solution of this target molecule is flushed through the module. By analyzing the remaining analyte concentration in the permeate, the amount of analyte adsorbed on the membrane can be calculated and related to specific interactions with the molecular imprints. By this means, optimized recipes in terms of cross-linker to template ratios could be obtained in combination with the optimal porogen, when screening p-divinylbenzene or ethylene glycol dimethacrylate as cross-linker and porogens like acetonitrile, dimethylsulfoxide and methanol.     

Isolation and characterization of fluorescence-enhancing RNA tags

Methods for the visualization of RNAs are urgently needed for studying a wide variety of cellular processes. Here we report on-bead screening of RNA libraries and its application to the isolation of specific fluorescence-enhancing RNA sequences. A one-bead-one-compound combinatorial RNA library with over one million different sequences was synthesized using the split-and-mix method. Solid-phase synthesis of 30 mer RNAs was performed on 15 ??m and 60 ??m diameter polystyrene beads bearing a non-cleavable linker. The RNA-derivatized beads were incubated with the well-established FlAsH pre-fluorophore and then screened for fluorescence enhancement, either by manually picking the brightest beads under a fluorescence microscope or by sorting with a FACS instrument. A protocol was established for sequence determination from single beads. While numerous RNA sequences showed increased fluorescence when immobilized, only few of them influenced the fluorescence properties of the FlAsH dye when detached from the beads. One of these sequences was found to induce a bathochromic shift in the excitation (from 492 to 510 nm) and emission (from 512 to 523 nm) maxima. This shift was accompanied by a 3.6-fold fluorescence enhancement of FlAsH fluorescence intensity. Mutation studies on the sequence revealed a rather robust structural motif.     

一株乙腈降解菌的鉴定及其生物学特性

【目的】明确乙腈降解菌BX2的分类地位及生物学特性,评价其处理含乙腈废水的可行性。【方法】通过形态特征、生理生化特性以及系统发育分析对菌株BX2进行鉴定。考察温度、初始pH及接种量等因素对菌株生长的影响,确定菌株的最佳生长条件及在该条件下的乙腈降解能力。测定菌株BX2对NaCl的耐受能力。【结果】乙腈降解菌BX2的形态特征及生理生化特性与紫红红球菌(Rhodococcus rhodochrous)最相近。其16S rRNA、gyrB、secA1基因序列与紫红红球菌的相似性分别为99.37%、99.29%、97.87%。最佳生长条件为35℃,初始pH 7.5,接种量1%。此条件下,菌株BX2在16 h内对浓度为800 mg/L乙腈的降解率为95.87%。菌株BX2在NaCl含量高于6%的培养基中无法生长。【结论】菌株BX2被鉴定为紫红红球菌。该菌株有较强的环境适应能力,可降解高浓度乙腈,在含氰废水的生物修复中有很好的应用前景。     

Kidney proteome responses in the teleost fish Paralichthys olivaceus indicate a putative immune response against Streptococcus parauberis

The proteomic response to bacterial infection in a teleost fish (Paralichthys olivaceus) infected with Streptococcus parauberis was analyzed using label-free protein quantitation coupled with LC-MS(E) tandem mass spectrometry. A total of 82 proteins from whole kidney, a major lymphoid organ in this fish, were found to be differentially expressed between healthy and diseased fish analyzed 6, 24, 72 and 120 h post-infection. Among the differentially expressed proteins, those involved in mediating immune responses (e.g., heat shock proteins, cathepsins, goose-type lysozyme and complement components) were most significantly up-regulated by infection. In addition, cell division cycle 48 (CDC48) and calreticulin, which are associated with cellular recovery and glycoprotein synthesis, were up-regulated in the universal protein group, whereas the other proteins in that group were down-regulated. There was continuous activation of expression of immune-associated proteins during infection, but there was also loss of expression of proteins not involved in immune function. We expect that our findings regarding immune response at the protein level would offer new insight into the systemic response to bacterial infection of a major immune organ in teleost fish.