Proteomics of canine lymphoma identifies potential cancer-specific protein markers

Purpose: Early diagnosis of cancer is crucial for the success of treatment of the disease, and there is a need for markers whose differential expression between disease and normal tissue could be used as a diagnostic tool. Spontaneously occurring malignancies in pets provide a logical tool for translational research for human oncology. Lymphoma, one of the most common neoplasms in dogs, is similar to human non-Hodgkin's lymphoma and could serve as an experimental model system. Experimental Design: Thirteen lymph nodes from normal dogs and 11 lymph nodes from dogs with B-cell lymphoma were subjected to proteomic analysis using two-dimensional PAGE separation and matrix-assisted laser desorption/ionization time-of-flight analysis. Results: A total of 93 differentially expressed spots was subjected to matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry analysis, and several proteins that showed differential expression were identified. Of these, prolidase (proline dipeptidase), triosephosphate isomerase, and glutathione S-transferase were down-regulated in lymphoma samples, whereas macrophage capping protein was up-regulated in the lymphoma samples. Conclusions: These proteins represent potential markers for the diagnosis of lymphoma and should be further investigated in human samples for validation of their utility as diagnostic markers

HIV Replication Enhances Production of Free Fatty Acids, Low Density Lipoproteins and Many Key Proteins Involved in Lipid Metabolism: A Proteomics Study

BACKGROUND: HIV-infected patients develop multiple metabolic abnormalities including insulin resistance, lipodystrophy and dyslipidemia. Although progression of these disorders has been associated with the use of various protease inhibitors and other antiretroviral drugs, HIV-infected individuals who have not received these treatments also develop lipid abnormalities albeit to a lesser extent. How HIV alters lipid metabolism in an infected cell and what molecular changes are affected through protein interaction pathways are not well-understood. RESULTS: Since many genetic, epigenetic, dietary and other factors influence lipid metabolism in vivo, we have chosen to study genome-wide changes in the proteomes of a human T-cell line before and after HIV infection in order to circumvent computational problems associated with multiple variables. Four separate experiments were conducted including one that compared 14 different time points over a period of >3 months. By subtractive analyses of protein profiles overtime, several hundred differentially expressed proteins were identified in HIV-infected cells by mass spectrometry and each protein was scrutinized for its biological functions by using various bioinformatics programs. Herein, we report 18 HIV-modulated proteins and their interaction pathways that enhance fatty acid synthesis, increase low density lipoproteins (triglycerides), dysregulate lipid transport, oxidize lipids, and alter cellular lipid metabolism. CONCLUSIONS: We conclude that HIV replication alone (i.e. without any influence of antiviral drugs, or other human genetic factors), can induce novel cellular enzymes and proteins that are significantly associated with biologically relevant processes involved in lipid synthesis, transport and metabolism (p = <0.0002-0.01). Translational and clinical studies on the newly discovered proteins may now shed light on how some of these proteins may be useful for early diagnosis of individuals who might be at high risk for developing lipid-related disorders. The target proteins could then be used for future studies in the development of inhibitors for preventing lipid-metabolic anomalies. This is the first direct evidence that HIV-modulates production of proteins that are significantly involved in disrupting the normal lipid-metabolic pathways

Secretion of Novel SEL1L Endogenous Variants Is Promoted by ER Stress/UPR via Endosomes and Shed Vesicles in Human Cancer Cells

We describe here two novel endogenous variants of the human endoplasmic reticulum (ER) cargo receptor SEL1LA, designated p38 and p28. Biochemical and RNA interference studies in tumorigenic and non-tumorigenic cells indicate that p38 and p28 are N-terminal, ER-anchorless and more stable relative to the canonical transmembrane SEL1LA. P38 is expressed and constitutively secreted, with increase after ER stress, in the KMS11 myeloma line and in the breast cancer lines MCF7 and SKBr3, but not in the non-tumorigenic breast epithelial MCF10A line. P28 is detected only in the poorly differentiated SKBr3 cell line, where it is secreted after ER stress. Consistently with the presence of p38 and p28 in culture media, morphological studies of SKBr3 and KMS11 cells detect N-terminal SEL1L immunolabeling in secretory/degradative compartments and extracellularly-released membrane vesicles. Our findings suggest that the two new SEL1L variants are engaged in endosomal trafficking and secretion via vesicles, which could contribute to relieve ER stress in tumorigenic cells. P38 and p28 could therefore be relevant as diagnostic markers and/or therapeutic targets in cancer

Proteins modulated by HIV post-infection.

<p>Up-regulated proteins are APOB and LRP1 and downregulated proteins are ACBP, STIP1, LRP2, and PDIA3. X-axis = protein names (abbreviations according to SwissPROT). Each protein was detected in multiple gels. Y-Axis = average of normalized quantities and standard deviations for each protein detected in multiple gels. The line limits are +/− one standard deviation for the range of data points for each protein. Full protein names and Accession #s of each protein are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003003#pone-0003003-t001" target="_blank">Tables 1</a> & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003003#pone-0003003-t002" target="_blank">2</a>.</p

Proteins detected exclusively in HIV-infected cells by MALDI-TOF mass spectrometry from multiple gels.

<p>These proteins were not detected in counterpart uninfected cells tested at multiple time points and various stages of cell growth. X-axis = protein names (abbreviations) are according to SwissPROT: Y-axis = average of normalized quantity and standard deviations for each protein expressed in multiple gels. The line limits are +/− one standard deviation for the range of data points for each protein. Full protein names and Accession #s of each protein are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003003#pone-0003003-t001" target="_blank">Table 1</a>.</p

Figure 5: Two-dimensional gel electrophoresis patterns of HIV-infected and counterpart uninfected cells T-cells.

<p>Protein profiles were evaluated 48 hours after treatment with azidothymidine (AZT), or mock treatment with phosphate buffered saline. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003003#pone-0003003-g005" target="_blank">Figure 5A</a> represents protein profile of uninfected T-cells and shows theexpression of Apolipoprotein B-100 (APOB) and the absence of HIV p24 antigen. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003003#pone-0003003-g005" target="_blank">Figure 5B</a> represents the protein profile of HIV-infected T-cells and displays the presence of both APOB and p24 antigen. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003003#pone-0003003-g005" target="_blank">Figure 5C</a> indicates that APOB is NOT inhibited by treatment of HIV-infected cells by AZT while the viral proteins are inhibited by the antiviral drug.</p

Protein-interaction pathways of proteins associated with various aspects of lipid metabolism.

<p>The functional significance of each protein was defined using Ingenuity bioinformatics platforms designed for Systems Analyses of genes and protein expressed in human health and disease. All proteins were uploaded into the Strategene Pathway Architect (SPA) program and protein-interaction pathways were constructed according to the manufacturer's instructions. Proteins identified by our present proteomics studies are shown with blue outlines; purple-colored proteins are enzymes and kinases and pink-colored (with blue outlines) are other significant proteins involved in the lipid metabolism. Lines connecting proteins indicate molecular networks of interactions. The greater the number of lines originating from a protein, the more significant the molecular interactions of that protein with other protein/s. Note the extensive networks of interactions between all proteins identified in the present study (blue circles), particularly those of APOB and APOA-1. Full protein names, abbreviations and accession numbers of each protein are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003003#pone-0003003-t001" target="_blank">Tables 1</a>&<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003003#pone-0003003-t002" target="_blank">2</a>.</p

Diagram showing putative biological processes involved in lipid metabolism.

<p>Each circle represents proteins associated with the respective function/s. Proteins in red were downregulated post-HIV infection (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003003#pone-0003003-g003" target="_blank">Figure 3</a>). All other proteins were either expressed exclusively in HIV infected cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003003#pone-0003003-g002" target="_blank">Figure 2</a>) or upregulated after virus infection (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003003#pone-0003003-g003" target="_blank">Figure 3</a>). Full names, abbreviations and accession numbers of each protein are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003003#pone-0003003-t001" target="_blank">Tables 1</a>&<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003003#pone-0003003-t002" target="_blank">2</a>.</p

HIV- Modulated Proteins Associated with Lipid Metabolism

<p>The three downregulated proteins are marked with (^*). All other (n = 8) proteins were either expressed exclusively in HIV-infected cells or were upregulated compared to those detected in the uninfected cells. Protein names, abbreviations, locations and accession numbers are according to SwissProt. Putative functions and significance scores in relation to lipid metabolism were calculated by Fisher Exact Test using software from Ingenuity Systems Inc. A p-value of <0.05 represents a statistically significant non-random association of a specific protein with a lipid-related function/s in the Global Functional Analysis</p