Exoproteome analysis of Pseudomonas aeruginosa response to high alkane stress.

Microbial biodegradation serves as an effective approach to treat oil pollution. However, the application of such methods for the degrading long-chain alkanes still encounters significant challenges. Comparative proteomics has extensively studied the intracellular proteins of bacteria that degrade short- and medium-chain alkanes, but the role and mechanism of extracellular proteins in many microorganism remain unclear. To enhance our understanding of the roles of extracellular proteins in the adaptation to long-chain alkanes, a label-free LC-MS/MS strategy was applied for the relative quantification of extracellular proteins of Pseudomonas aeruginosa SJTD-1-M (ProteomeXchange identifier PXD014638). 444 alkane-sentitive proteins were acquired and their cell localization analysis was performed using the Pseudomonas Genome Database. Among them, 111 proteins were found to be located in extracellular or Outer Membrane Vesicles (OMVs). The alkane-induced abundance of 11 extracellular or OMV target proteins was confirmed by parallel reaction monitoring (PRM). Furthermore, we observed that the expression levels of three proteins (Pra, PA2815, and FliC) were associated with the carbon chain length of the added alkane in the culture medium. The roles of these proteins in cell mobility, alkane emulsification, assimilation, and degradation were further discussed. OMVs were found to contain a number of enzymes involved in alkane metabolism, fatty acid beta-oxidation, and the TCA cycle, suggesting their potential as sites for facilitated alkane degradation. In this sense, this exoproteome analysis contributes to a better understanding of the role of extracellular proteins in the hydrocarbon treatment process.

Glycosidase-activated H2S donorsto enhance chemotherapy efficacy.

Hydrogen sulfide (H2S) plays a critical role in cancer biology. Herein, we developed a series of glycosidase-triggered hydrogen sulfide (H2S) donors by connecting sugar moieties (including glucose, galactose and mannose) to COS donors via a self-immolative spacer. In the presence of corresponding glycosidases, H2S was gradually released from these donors in PBS buffer with releasing efficiencies from 36 to 67 %. H2S release was also detected by H2S probe WSP-1 after treatment HepG2 cells with Man1. Cytotoxicities of these glycosylated H2S donors were evaluated against HepG2 by MTT assay. Among them, Man1 and Man2 exhibited an obvious reduction of cell viability in HepG2 cells, with cell viability as 37.6 % for 80 µM of Man. Consistently, significant apoptosis was observed in HepG2 cells after treatment with Man1 and Man2. Finally, We evaluated the potential of Man1 for combination therapy with doxorubicin. A synergistic effect was observed between Man1 and Doxorubicin in HepG2 and Hela cells. All these results indicated glycosidase-activated H2S donorshave promising potential for cancer therapy.

O-glycosylation of SARS-CoV-2 spike protein by host O-glycosyltransferase strengthens its trimeric structure.

Protein O-glycosylation, also known as mucin-type O-glycosylation, is one of the most abundant glycosylation in mammalian cells. It is initially catalyzed by a family of polypeptide GalNAc transferases (ppGalNAc-Ts). The trimeric spike protein (S) of SARS-CoV-2 is highly glycosylated and facilitates the virus's entry into host cells and membrane fusion of the virus. However, the functions and relationship between host ppGalNAc-Ts and O-glycosylation on the S protein remain unclear. Herein, we identify 15 O-glycosites and 10 distinct O-glycan structures on the S protein using an HCD-product-dependent triggered ETD mass spectrometric analysis. We observe that the isoenzyme T6 of ppGalNAc-Ts (ppGalNAc-T6) exhibits high O-glycosylation activity for the S protein, as demonstrated by an on-chip catalytic assay. Overexpression of ppGalNAc-T6 in HEK293 cells significantly enhances the O-glycosylation level of the S protein, not only by adding new O-glycosites but also by increasing O-glycan heterogeneity. Molecular dynamics simulations reveal that O-glycosylation on the protomer-interface regions, modified by ppGalNAc-T6, potentially stabilizes the trimeric S protein structure by establishing hydrogen bonds and non-polar interactions between adjacent protomers. Furthermore, mutation frequency analysis indicates that most O-glycosites of the S protein are conserved during the evolution of SARS-CoV-2 variants. Taken together, our finding demonstrate that host O-glycosyltransferases dynamically regulate the O-glycosylation of the S protein, which may influence the trimeric structural stability of the protein. This work provides structural insights into the functional role of specific host O-glycosyltransferases in regulating the O-glycosylation of viral envelope proteins.

Interplay between the bacterial protein deacetylase CobB and the second messenger c-di-GMP.

As a ubiquitous bacterial secondary messenger, c-di-GMP plays key regulatory roles in processes such as bacterial motility and transcription regulation. CobB is the Sir2 family protein deacetylase that controls energy metabolism, chemotaxis, and DNA supercoiling in many bacteria. Using an Escherichia coli proteome microarray, we found that c-di-GMP strongly binds to CobB. Further, protein deacetylation assays showed that c-di-GMP inhibits the activity of CobB and thereby modulates the biogenesis of acetyl-CoA. Interestingly, we also found that one of the key enzymes directly involved in c-di-GMP production, DgcZ, is a substrate of CobB. Deacetylation of DgcZ by CobB enhances its activity and thus the production of c-di-GMP. Our work establishes a novel negative feedback loop linking c-di-GMP biogenesis and CobB-mediated protein deacetylation.

Identification of sitagliptin binding proteins by affinity purification mass spectrometry.

Type 2 diabetes mellitus (T2DM) is recognized as a serious public health concern with increasing incidence. The dipeptidyl peptidase-4 (DPP-4) inhibitor sitagliptin has been used for the treatment of T2DM worldwide. Although sitagliptin has excellent therapeutic outcome, adverse effects are observed. In addition, previous studies have suggested that sitagliptin may have pleiotropic effects other than treating T2DM. These pieces of evidence point to the importance of further investigation of the molecular mechanisms of sitagliptin, starting from the identification of sitagliptin-binding proteins. In this study, by combining affinity purification mass spectrometry (AP-MS) and stable isotope labeling by amino acids in cell culture (SILAC), we discover seven high-confidence targets that can interact with sitagliptin. Surface plasmon resonance (SPR) assay confirms the binding of sitagliptin to three proteins, i. e., LYPLAL1, TCP1, and CCAR2, with binding affinities (K D) ranging from 50.1 µM to 1490 µM. Molecular docking followed by molecular dynamic (MD) simulation reveals hydrogen binding between sitagliptin and the catalytic triad of LYPLAL1, and also between sitagliptin and the P-loop of ATP-binding pocket of TCP1. Molecular mechanics Poisson-Boltzmann Surface Area (MMPBSA) analysis indicates that sitagliptin can stably bind to LYPLAL1 and TCP1 in active sites, which may have an impact on the functions of these proteins. SPR analysis validates the binding affinity of sitagliptin to TCP1 mutant D88A is ~10 times lower than that to the wild-type TCP1. Our findings provide insights into the sitagliptin-targets interplay and demonstrate the potential of sitagliptin in regulating gluconeogenesis and in anti-tumor drug development.

Targeted delivery of nitric oxide via a 'bump-and-hole'-based enzyme-prodrug pair.

The spatiotemporal generation of nitric oxide (NO), a versatile endogenous messenger, is precisely controlled. Despite its therapeutic potential for a wide range of diseases, NO-based therapies are limited clinically due to a lack of effective strategies for precisely delivering NO to a specific site. In the present study, we developed a novel NO delivery system via modification of an enzyme-prodrug pair of galactosidase-galactosyl-NONOate using a 'bump-and-hole' strategy. Precise delivery to targeted tissues was clearly demonstrated by an in vivo near-infrared imaging assay. The therapeutic potential was evaluated in both rat hindlimb ischemia and mouse acute kidney injury models. Targeted delivery of NO clearly enhanced its therapeutic efficacy in tissue repair and function recovery and abolished side effects due to the systemic release of NO. The developed protocol holds broad applicability in the targeted delivery of important gaseous signaling molecules and offers a potent tool for the investigation of relevant molecular mechanisms.

Piperazine-Coumarin based fluorescence probe with enhanced brightness and solubility for bio-thiol detection and esophageal carcinoma diagnosis.

The development of novel fluorescent dyes for bio-thiol is of great importance in biological, clinical and pharmaceutical sciences. Given the importance of bio-thiol anticipating in numerous physiological processes, there is a great need to construct fluorescent biosensors with high quality to detect them. Fluorophores, especially those used in bio-system, usually require high-quality properties such as high brightness, good water solubility, bio-compatible and photostability. Herein, we reported a novel fluorescent probe based on piperazine-coumarin scaffold with enhanced brightness and solubility. To further demonstrate the potential clinical applications, we performed living cell fluorescence image and human esophageal carcinoma diagnosis. The result indicated that we were able to distinguish pathological tissue from normal tissue by applying this probe. Thus, we hope this design will be helpful to develop high-quality fluorophores for clinical diagnosis.

Synthesis of difluoroalkylated 2-azaspiro[4.5]decane derivatives via copper-catalyzed difluoroalkylation/dearomatization of N-benzylacrylamides.

An efficient method for the synthesis of difluoroalkylated 2-azaspiro[4.5]decanes via copper-catalyzed difluoroalkylation of N-benzylacrylamides with ethyl bromodifluoroacetate has been established. The reaction experienced a tandem radical addition and dearomatizing cyclization process. In addition, the resultant products can be smoothly converted into a difluoroalkylated quinolinone and saturated spirocyclohexanone scaffold.

Photoactive NO hybrids with pseudo-zero-order release kinetics for antimicrobial applications.

Bacterial infection is a major threat to the health and life of humans due to the development of drug resistance, which is related to biofilm formation. Nitric oxide (NO) has emerged as an important factor in regulating biofilm formation. In order to harness the potential benefits of NO and develop effective antibacterial agents, we designed and synthesized a new class of NO hybrids in which the active scaffold benzothienoazepine was tagged with a nitroso group and further conjugated with quaternary ammoniums or phosphoniums. The temporal release of NO from these hybrids can be achieved by photoactivation. Interestingly, the NO release follows a pseudo-zero-order kinetics, which is easily determined by measuring the fluorescent benzothienoazepine or NO. Compared to the positive control ciprofloxacin, the NO hybrid with triphenyl phosphonium (TPP) exhibited more effective activity against S. aureus biofilm in darkness. Irradiation of the NO hybrid led to higher inhibition against S. aureus biofilm compared to the parental NO hybrid in darkness or the corresponding NO-released product, indicating the combined effect of NO and the NO-released product. Therefore, this new class of NO hybrids includes very promising antimicrobial agents and this work provides a new way for the design of highly effective antimicrobial agents.

Synthesis and Characterization of Hydrophilic Trityl Radical TFO for Biomedical and Biophysical Applications.

Tetrathiatriarylmethyl (TAM, trityl) radicals have found wide applications as spin probes/labels for EPR spectroscopy and imaging, and as polarizing agents for dynamic nuclear polarization. The high hydrophilicity of TAM radicals is essential for their biomedical applications. However, the synthesis of hydrophilic TAM radicals (e.g., OX063) is extremely challenging and has only been reported in the patent literature, to date. Herein, an efficient synthesis of a highly water-soluble TAM radical bis(8-carboxyl-2,2,6,6-tetramethylbenzo[1,2-d:4,5-d']bis([1,3]dithiol-4-yl)-mono-(8-carboxyl-2,2,6,6-tetrakis(2-hydroxyethyl)benzo[1,2-d:4,5-d']bis([1,3]dithiol-4-yl)methyl (TFO), which contains four additional hydroxylethyl groups, relative to the Finland trityl radical CT-03, is reported. Similar to OX063, TFO exhibits excellent properties, including high water solubility in phosphate buffer, low log P, low pKa , long relaxation times, and negligible binding with bovine serum albumin. On the other hand, TFO has a sharper EPR line and higher O2 sensitivity than those of OX063. Therefore, in combination with its facile synthesis, TFO should find wide applications in magnetic resonance related fields and this synthetic approach would shed new light on the synthesis of other hydrophilic TAM radicals.