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PubMed2024

Super Enhanced Purification of Denatured-Refolded Ubiquitinated Proteins by ThUBD Revealed Ubiquitinome Dysfunction in Liver Fibrosis.

Ubiquitination is crucial for maintaining protein homeostasis and plays a vital role in diverse biological processes. Ubiquitinome profiling and quantification are of great scientific significance. Artificial ubiquitin-binding domains (UBDs) have been widely employed to capture ubiquitinated proteins. The success of this enrichment relies on recognizing native spatial structures of ubiquitin and ubiquitin chains by UBDs under native conditions. However, the use of native lysis conditions presents significant challenges, including insufficient protein extraction, heightened activity of deubiquitinating enzymes and proteasomes in removing the ubiquitin signal, and purification of a substantial number of contaminant proteins, all of which undermine the robustness and reproducibility of ubiquitinomics. In this study, we introduced a novel approach that combines denatured-refolded ubiquitinated sample preparation (DRUSP) with a tandem hybrid UBD for ubiquitinomic analysis. The samples were effectively extracted using strongly denatured buffers and subsequently refolded using filters. DRUSP yielded a significantly stronger ubiquitin signal, nearly three times greater than that of the Control method. Then, eight types of ubiquitin chains were quickly and accurately restored; therefore, they were recognized and enriched by tandem hybrid UBD with high efficiency and no biases. Compared with the Control method, DRUSP showed extremely high efficiency in enriching ubiquitinated proteins, improving overall ubiquitin signal enrichment by approximately 10-fold. Moreover, when combined with ubiquitin chain-specific UBDs, DRUSP had also been proven to be a versatile approach. This new method significantly enhanced the stability and reproducibility of ubiquitinomics research. Finally, DRUSP was successfully applied to deep ubiquitinome profiling of early mouse liver fibrosis with increased accuracy, revealing novel insights for liver fibrosis research.

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PubMed2006

Studying the protein organization of the postsynaptic density by a novel solid phase- and chemical cross-linking-based technology.

Agarose beads carrying a cleavable, fluorescent, and photoreactive cross-linking reagent on the surface were synthesized and used to selectively pull out the proteins lining the surface of supramolecules. A quantitative comparison of the abundances of various proteins in the sample pulled out by the beads from supramolecules with their original abundances could provide information on the spatial arrangement of these proteins in the supramolecule. The usefulness of these synthetic beads was successfully verified by trials using a synthetic protein complex consisting of three layers of different proteins on glass coverslips. By using these beads, we determined the interior or superficial locations of five major and 19 minor constituent proteins in the postsynaptic density (PSD), a large protein complex and the landmark structure of asymmetric synapses in the mammalian central nervous system. The results indicate that alpha,beta-tubulins, dynein heavy chain, microtubule-associated protein 2, spectrin, neurofilament H and M subunits, an hsp70 protein, alpha-internexin, dynamin, and PSD-95 protein reside in the interior of the PSD. Dynein intermediate chain, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors, kainate receptors, N-cadherin, beta-catenin, N-ethylmaleimide-sensitive factor, an hsc70 protein, and actin reside on the surface of the PSD. The results further suggest that the N-methyl-d-aspartate receptors and the alpha-subunits of calcium/calmodulin-dependent protein kinase II are likely to reside on the surface of the PSD although with unique local protein organizations. Based on our results and the known interactions between various PSD proteins from data mining, a model for the molecular organization of the PSD is proposed.

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PubMed2004

Novel endogenous small molecules as the checkpoint controllers in inflammation and resolution: entrée for resoleomics.

Endogenously-generated small chemical mediators or autacoids play key roles in controlling inflammation and its organized resolution. Among them, lipoxins are the trihydroxy-tetraene-containing eicosanoids that are generated primarily by tight cell-cell interactions by way of transcellular biosynthesis and serve as local endogenous anti-inflammatory mediators. These "stop signals" in inflammation and other related processes may be involved in switching the cellular response from additional PMN recruitment toward monocytes (in a nonphlogistic fashion) that could lead to resolution of the inflammatory response or promotion of repair and healing. ASA impinges on this homeostatic system and evokes the endogenous biosynthesis of the carbon 15 epimers of lipoxins, namely ATLs, that mimic the bioactions of native LX in several biologic systems and, thus, can modulate in part, the beneficial actions of ASA in humans. Moreover, the temporal and spatial components in LX formation and actions are important determinants of their impact during an acute inflammatory reaction. Generation of lipid (ie, ATL) versus protein (ie, ANXA1) mediators during the host inflammatory response display different time courses. The temporal difference suggests that ALX could regulate PMN by interacting with each class of ligands within specific phases of the inflammatory response. ALX is the first cloned lipoxygenase-derived eicosanoid receptor. The signaling pathways and bioactions of ALX are cell type-specific. In agreement with in vitro results, ALX agonists, namely LXA4 and 15-epi-LXA4 and their stable analogs, regulate PMN during acute inflammation. In addition, it seems that LXs also display organ-specific actions, in addition to host defense and immune roles in the eye, kidney, lung, and oral and gastrointestinal tract and within bone marrow progenitors, possibly involving stem cells. The development of these few synthetic stable analogs has provided valuable tools to evaluate the biologic roles, significance, and pharmacologic actions of ALX and provided novel therapies for inflammatory diseases. The relationship between LX generation and current NSAID therapies is more intertwined than currently appreciated. ASA inhibits COX-1 and converts COX-2 into an ASA-triggered lipid mediator-generating system that produces an array of novel endogenous local autacoids from dietary omega-3 PUFA. Some of the local autacoids display potent anti-inflammatory or antineutrophil recruitment activity as well as impinge on the role of these compounds in resolution, and, thus, are termed "resolvins." It is not surprising that investigators recently found a protective action for COX-2 in cardiovascular disease. Together with the lipoxins and 15-epi-lipoxins, the identification of the resolvins gives us new avenues of approach in considering therapies for inflammation, cardiovascular diseases and cancer.

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PubMed2004

Glycomic profiling of developmental changes in bovine testis by lectin histochemistry and further analysis of the most prominent alteration on the level of the glycoproteome by lectin blotting and lectin affinity chromatography.

The emerging concept of the sugar code attributes functional significance to oligosaccharides of cellular glycoconjugates by protein (lectin)-carbohydrate interactions. Hence it follows that monitoring of glycan expression (glycomic profiling) is not only valuable to delineate characteristic (phenomenological) changes in the cell's glycosylation but will also come up with the localization of epitopes with potential in biorecognition. It is for this purpose that we have set up a panel of 16 markers (plant lectins and a carbohydrate-specific antibody). The selection met two criteria: a) to be able to detect the common constituents of natural glycans; and b) to place emphasis on detection of neutral carbohydrate units at the spatially accessible branch ends of glycan chains, which are known to be active as ligands for endogenous lectins in situ. Next, we incorporated recent insights into the importance of epitope clustering to turn less abundant oligosaccharides into potent ligands into our study design. To be able to focus on such high-affinity sites, we performed systematic titration studies aimed at defining the probe concentration at which carbohydrate-independent background staining is minimal while still yielding a clear signal. These requirements were met by marker concentrations of 1.25-2.5 microg/ml. Under these conditions, we defined cell-type- and differentiation-dependent changes in bovine testis. Sertoli cells lacked reactivity, whereas gonocytes were differentially reactive with the tested markers. The extent of staining intensity was subject to developmental changes, preferentially for Gal/GalNAc presentation and in this group most prominently with the galactoside-specific lectin from Viscum album L. (mistletoe). Of interest in this context, this lectin is known as a potent mitogen and signal inductor as well as haemagglutinin. The Gal/GalNAc-dependent signals decreased markedly in the course of development and staining was completely lost in the case of mistletoe lectin 12 weeks after gestation. Spermatids of adult testis presented respective glycan epitopes. In contrast to this developmental course of staining, endothelial cells either maintained a constant signal intensity or revealed a signal increase during development for Gal/GalNAc-specific lectins. Their binding of concanavalin A and the two phyto-haemagglutinins (PHA-E/L), which were not or only weakly reactive for gonocytes, served as inherent activity control. Based on lectin blot analysis with the mistletoe lectin as the marker which detected the most prominent change, the glycoprotein patterns from fetal and adult tissue specimens were qualitatively different, rendering changes in expression of the protein part of glycoproteins more likely than remodeling a glycoprotein's glycan chains. Methodologically, results of this procedure were compared to data obtained with lectin affinity chromatography and the combination of the two procedures. Differences in the profiles were discovered that can be assigned to the disparate ways to process the detergent extracts. When access to sample quantity is limited, as is possible in the case of fetal tissue, direct lectin blotting is recommended.

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