4 research outputs found
Multi-lectin Affinity Chromatography and Quantitative Proteomic Analysis Reveal Differential Glycoform Levels between Prostate Cancer and Benign Prostatic Hyperplasia Sera.
Currently prostate-specific antigen is used for prostate cancer (PCa) screening, however it lacks the necessary specificity for differentiating PCa from other diseases of the prostate such as benign prostatic hyperplasia (BPH), presenting a clinical need to distinguish these cases at the molecular level. Protein glycosylation plays an important role in a number of cellular processes involved in neoplastic progression and is aberrant in PCa. In this study, we systematically interrogate the alterations in the circulating levels of hundreds of serum proteins and their glycoforms in PCa and BPH samples using multi-lectin affinity chromatography and quantitative mass spectrometry-based proteomics. Specific lectins (AAL, PHA-L and PHA-E) were used to target and chromatographically separate core-fucosylated and highly-branched protein glycoforms for analysis, as differential expression of these glycan types have been previously associated with PCa. Global levels of CD5L, CFP, C8A, BST1, and C7 were significantly increased in the PCa samples. Notable glycoform-specific alterations between BPH and PCa were identified among proteins CD163, C4A, and ATRN in the PHA-L/E fraction and among C4BPB and AZGP1 glycoforms in the AAL fraction. Despite these modest differences, substantial similarities in glycoproteomic profiles were observed between PCa and BPH sera
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The physical and chemical parameters of in vitro packaging of heterologous RNA into virus-like particles
The evolved strategies of viruses are wondrous and should be investigated and understood better to exploit their gene delivery “tricks” for the development of effective therapeutics. Simple RNA viruses – those consisting of a positive-sense/ready-to-translate RNA genome inside an ordered capsid made up of multiple copies of a single protein – have been used as platforms for delivery of therapeutic cargo to cells. For example, early work by several groups established that spontaneous self-assembly of virus-like particles (VLPs) can be carried out in vitro from purified components of RNA and the capsid protein (CP) from the plant viruses tobacco mosaic virus (TMV), cowpea chlorotic mottle virus (CCMV) and brome mosaic virus (BMV). CCMV and BMV CP can encapsulate non-viral RNAs into monodisperse, RNase-resistant spherical capsids if the length of the RNA is between 2,500 and 4,500 nucleotides (nts); longer RNAs are packaged into “multiplets” – multiple 28-nm VLPs, where RNA is shared between two or more particles. Additionally, researchers have exploited the self-replicating nature of viruses, specifically nodamura virus (NOV), to amplify expression of genes of interest (GOIs). The GOI can vary widely from reporter genes to RNA encoding for cancer or viral antigens. This self-replicating RNA, or replicon, can be packaged into CCMV VLPs using an in vitro assembly method. In this study, I investigate viral strategies such as self-assembly and self-replication with the goal of delivering self-replicating functional RNAs encapsulated in spherical VLPs to mammalian cells as therapeutics. This thesis work reports on a systematic series of experiments elucidating the physical chemical properties of self-assembled VLPs as delivery platforms for mRNA replicon therapeutics. I am interested in understanding the physical chemical parameters governing successful self-assembly – solution ionic strength, pH, protein concentration, charge and size of cargo being packaged – to develop robust therapeutics. To probe the role of these in determining RNA-protein and protein-protein interactions and ensuing assembly of nucleocapsids, interferometric scattering (iSCAT) microscopy was used to follow the assembly kinetics of individual BMV VLPs around BMV RNA1 using a range of solution ionic strengths and protein concentrations. The iSCAT experiments revealed that nucleation times become longer and more broadly distributed when RNA-protein interactions are reduced (high ionic strength) and/or protein concentrations are decreased, corresponding to an increase in nucleation barriers. To further explore the role of RNA-protein and protein-protein interactions, assemblies were carried out in a variety of assembly buffers spanning ranges of pH from 5 to 7 and of ionic strengths from 0.1 to 0.5 M, and assembly products were imaged using negative-stain electron microscopy (EM). Additionally, the salt- and pH-dependence of protein-protein interactions were characterized by differential scanning fluorimetry. To quantify the dependence of GOI protein expression on the numbers of GOI replicons (derived from NOV RNA1) transfected into mammalian cells, cell lines were transfected with increasing masses of replicon RNA mixed with an inert carrier RNA, with the total mass of the transfected RNA held constant while increasing the fraction of replicon. Fluorescence microscopy, flow cytometry, and trypan-blue exclusion assays show a non-monotonic dose-dependence of protein expression – i.e., “less is better” – in cells transfected with replicon RNA. To package long replicon molecules – those with GOI > 1,000 nts – into RNase-resistant VLPs, the RNA needs to be compacted to fit into a BMV VLP. To probe the role of charge and size of cargo on self-assembly, long RNA molecules were pre-compacted with increasing concentrations of polyvalent cations and assembled into BMV VLPs. VLPs were analyzed by negative-stain EM and gel electrophoresis. Finally, the same replicon molecule was packaged into spherical and rod-like VLPs using the CP of CCMV and TMV, respectively, with the goal of comparing how the shape of these particles impacts their efficacy as a therapeutic delivery vehicle
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Protein signatures to distinguish aggressive from indolent prostate cancer.
BACKGROUND: Distinguishing men with aggressive from indolent prostate cancer is critical to decisions in the management of clinically localized prostate cancer. Molecular signatures of aggressive disease could help men overcome this major clinical challenge by reducing unnecessary treatment and allowing more appropriate treatment of aggressive disease. METHODS: We performed a mass spectrometry-based proteomic analysis of normal and malignant prostate tissues from 22 men who underwent surgery for prostate cancer. Prostate cancer samples included Grade Groups (3-5), with 8 patients experiencing recurrence and 14 without evidence of recurrence with a mean of 6.8 years of follow-up. To better understand the biological pathways underlying prostate cancer aggressiveness, we performed a systems biology analysis and gene enrichment analysis. Proteins that distinguished recurrent from nonrecurrent cancer were chosen for validation by immunohistochemical analysis on tissue microarrays containing samples from a larger cohort of patients with recurrent and nonrecurrent prostate cancer. RESULTS: In all, 24,037 unique peptides (false discovery rate < 1%) corresponding to 3,313 distinct proteins were identified with absolute abundance ranges spanning seven orders of magnitude. Of these proteins, 115 showed significantly (p < 0.01) different levels in tissues from recurrent versus nonrecurrent cancers. Analysis of all differentially expressed proteins in recurrent and nonrecurrent cases identified several protein networks, most prominently one in which approximately 24% of the proteins in the network were regulated by the YY1 transcription factor (adjusted p < 0.001). Strong immunohistochemical staining levels of three differentially expressed proteins, POSTN, CALR, and CTSD, on a tissue microarray validated their association with shorter patient survival. CONCLUSIONS: The protein signatures identified could improve understanding of the molecular drivers of aggressive prostate cancer and be used as candidate prognostic biomarkers