Dorothy Loo

Differential impact of caveolae and caveolin-1 scaffolds on the membrane raft proteome

Caveolae, a class of cholesterol-rich lipid rafts, are smooth invaginations of the plasma membrane whose formation in nonmuscle cells requires caveolin-1 (Cav1). The recent demonstration that Cav1-associated cavin proteins, in particular PTRF/cavin-1, are also required for caveolae formation supports a functional role for Cav1 independently of caveolae. In tumor cells deficient for Golgi β-1,6N-acetylglucosaminyltransferase V (Mgat5), reduced Cav1 expression is associated not with caveolae but with oligomerized Cav1 domains, or scaffolds, that functionally regulate receptor signaling and raft-dependent endocytosis. Using subdiffraction-limit microscopy, we show that Cav1 scaffolds are homogenous subdiffraction-limit sized structures whose size distribution differs from that of Cav1 in caveolae expressing cells. These cell lines displaying differing Cav1/caveolae phenotypes are effective tools for probing the structure and composition of caveolae. Using stable isotope labeling by amino acids in cell culture, we are able to quantitatively distinguish the composition of caveolae from the background of detergent-resistant membrane proteins and show that the presence of caveolae enriches the protein composition of detergent-resistant membrane, including the recruitment of multiple heterotrimeric G-protein subunits. These data were further supported by analysis of immuno-isolated Cav1 domains and of methyl-β-cyclodextrin-disrupted detergent-resistant membrane. Our data show that loss of caveolae results in a dramatic change to the membrane raft proteome and that this change is independent of Cav1 expression. The proteomics data, in combination with subdiffraction-limit microscopy, indicates that noncaveolar Cav1 domains, or scaffolds are structurally and functionally distinct from caveolae and differentially impact on the molecular composition of lipid rafts.

Nucleophosmin and Nucleolin Regulate K-Ras Plasma Membrane Interactions and MAPK Signal Transduction

The spatial organization of Ras proteins into nanoclusters on the inner leaflet of the plasma membrane is essential for high fidelity signaling through the MAPK pathway. Here we identify two selective regulators of K-Ras nanoclustering from a proteomic screen for K-Ras interacting proteins. Nucleophosmin (NPM) and nucleolin are predominantly localized to the nucleolus but also have extranuclear functions. We show that a subset of NPM and nucleolin localizes to the inner leaflet of plasma membrane and forms specific complexes with K-Ras but not other Ras isoforms. Active GTP-loaded and inactive GDP-loaded K-Ras both interact with NPM, although NPM-K-Ras binding is increased by growth factor receptor activation. NPM and nucleolin both stabilize K-Ras levels on the plasma membrane, but NPM concurrently increases the clustered fraction of GTP-K-Ras. The increase in nanoclustered GTP-K-Ras in turn enhances signal gain in the MAPK pathway. In summary these results reveal novel extranucleolar functions for NPM and nucleolin as regulators of K-Ras nanocluster formation and activation of the MAPK pathway. The study also identifies a new class of K-Ras nanocluster regulator that operates independently of the structural scaffold galectin-3.

Expression of PTRF in PC-3 Cells Modulates Cholesterol Dynamics and the Actin Cytoskeleton Impacting Secretion Pathways

Expression of caveolin-1 is up-regulated in prostate cancer metastasis and is associated with aggressive recurrence of the disease. Intriguingly, caveolin-1 is also secreted from prostate cancer cell lines and has been identified in secreted prostasomes. Caveolin-1 is the major structural component of the plasma membrane invaginations called caveolae. Co-expression of the coat protein Polymerase I and transcript release factor (PTRF) is required for caveolae formation. We recently found that expression of caveolin-1 in the aggressive prostate cancer cell line PC-3 is not accompanied by PTRF, leading to noncaveolar caveolin-1 lipid rafts. Moreover, ectopic expression of PTRF in PC-3 cells sequesters caveolin-1 into caveolae. Here we quantitatively analyzed the effect of PTRF expression on the PC-3 proteome using stable isotope labeling by amino acids in culture and subcellular proteomics. We show that PTRF reduced the secretion of a subset of proteins including secreted proteases, cytokines, and growth regulatory proteins, partly via a reduction in prostasome secretion. To determine the cellular mechanism accounting for the observed reduction in secreted proteins we analyzed total membrane and the detergent-resistant membrane fractions. Our data show that PTRF expression selectively impaired the recruitment of actin cytoskeletal proteins to the detergent-resistant membrane, which correlated with altered cholesterol distribution in PC-3 cells expressing PTRF. Consistent with this, modulating cellular cholesterol altered the actin cytoskeleton and protein secretion in PC-3 cells. Intriguingly, several proteins that function in ER to Golgi trafficking were reduced by PTRF expression. Taken together, these results suggest that the noncaveolar caveolin-1 found in prostate cancer cells generates a lipid raft microenvironment that accentuates secretion pathways, possibly at the step of ER sorting/exit. Importantly, these effects could be modulated by PTRF expression.

Lectin Magnetic Bead Array for Biomarker Discovery

Alterations in protein glycosylation play an important role in patho-physiology, and much effort has been devoted to detecting glycoprotein biomarkers. In this manuscript, we describe the development of a novel method for monitoring alterations in protein glycosylation. Lectins are used as individual affinity reagents and coupled to magnetic beads (Dynabeads) in a microplate array format for isolation of glycosylated proteins. Isolated glycoproteins are digested with trypsin in-solution followed by LC-MS/MS, allowing a liquid handler-assisted high throughput workflow. We demonstrate the specific and reproducible affinity-isolation of glycoproteins using the lectin Dynabead array technology. When used with serum, we achieved one-step purification of glycoproteins with minimal coisolation of abundant serum proteins including albumin. We further optimized the proteomics workflow to allow transfer to a liquid handler for automation. In summary, we report the development of a high throughput platform to detect alterations in protein glycosylation which will be useful in glycoproteomics studies, particularly clinical proteomics studies where large sample sizes are required to achieve statistical power.

Co-Regulation of Cell Polarization and Migration by Caveolar Proteins PTRF/Cavin-1 and Caveolin-1

Caveolin-1 and caveolae are differentially polarized in migrating cells in various models, and caveolin-1 expression has been shown to quantitatively modulate cell migration. PTRF/cavin-1 is a cytoplasmic protein now established to be also necessary for caveola formation. Here we tested the effect of PTRF expression on cell migration. Using fluorescence imaging, quantitative proteomics, and cell migration assays we show that PTRF/cavin-1 modulates cellular polarization, and the subcellular localization of Rac1 and caveolin-1 in migrating cells as well as PKCα caveola recruitment. PTRF/cavin-1 quantitatively reduced cell migration, and induced mesenchymal epithelial reversion. Similar to caveolin-1, the polarization of PTRF/cavin-1 was dependent on the migration mode. By selectively manipulating PTRF/cavin-1 and caveolin-1 expression (and therefore caveola formation) in multiple cell systems, we unveil caveola-independent functions for both proteins in cell migration.

High-throughput lectin magnetic bead array-coupled tandem mass spectrometry for glycoprotein biomarker discovery

Alterations in protein glycosylation occur during development and progression of many diseases, hence glycomics and glycoproteomics have emerged as important tools in glycobiomarker discovery. High‐throughput glycan profiling can now be achieved with the recent developments in MS‐based techniques. To enable identification and rapid monitoring of glycosylation changes in serum proteins, we developed a semi‐automated high‐throughput glycoprotein biomarker discovery platform termed lectin magnetic bead array‐coupled tandem mass spectrometry (LeMBA‐MS) which includes (i) effective single‐step serum glycoprotein isolation using a panel of 20 individual lectin‐coated magnetic beads in microplate format, (ii) on‐bead trypsin digestion, and (iii) nanoLC‐MS/MS with lectin exclusion list. With use of appropriate sequence databases, LeMBA‐MS can detect glycosylation changes regardless of the species. By spiking known amounts of titrated ovalbumin to a serum sample, we report nanomolar sensitivity, and linearity of response of LeMBA‐MS using concanavalin A‐coupled beads. Neuraminidase treatment led to reduction of binding to sialic acid‐binding lectins. Interestingly, we found that desialylation caused increased binding of haptoglobin and hemopexin to mannose‐specific lectins, pointing to the importance of identifying a signature of lectin‐binding. High‐throughput LeMBA‐MS to generate glycosylation signatures will facilitate glycobiomarker discovery. LeMBA can be coupled to down‐stream detection platforms for validation, making it a truly versatile platform.

Molecular Characterization of Caveolin-Induced Membrane Curvature

Background: Caveolin-1 (Cav1) requires the caveolin scaffolding domain for caveola formation. Results: The Cav1 scaffolding domain and oligomerization domain are tightly juxtaposed to the membrane in caveolae. Conclusion: Concerted membrane association of the oligomerization, scaffolding, and intramembrane domains are critical for caveola biogenesis and membrane deformation. Significance: Understanding the membrane association of Cav1 is critical for dissecting how the protein regulates caveola formation and achieves regulation over cellular signaling. The generation of caveolae involves insertion of the cholesterol-binding integral membrane protein caveolin-1 (Cav1) into the membrane, however, the precise molecular mechanisms are as yet unknown. We have speculated that insertion of the caveolin scaffolding domain (CSD), a conserved amphipathic region implicated in interactions with signaling proteins, is crucial for caveola formation. We now define the core membrane-juxtaposed region of Cav1 and show that the oligomerization domain and CSD are protected by tight association with the membrane in both mature mammalian caveolae and a model prokaryotic system for caveola biogenesis. Cryoelectron tomography reveals the core membrane-juxtaposed domain to be sufficient to maintain oligomerization as defined by polyhedral distortion of the caveolar membrane. Through mutagenesis we demonstrate the importance of the membrane association of the oligomerization domain/CSD for defined caveola biogenesis and furthermore, highlight the functional significance of the intramembrane domain and the CSD for defined caveolin-induced membrane deformation. Finally, we define the core structural domain of Cav1, constituting only 66 amino acids and of great potential to nanoengineering applications, which is required for caveolin-induced vesicle formation in a bacterial system. These results have significant implications for understanding the role of Cav1 in caveola formation and in regulating cellular signaling events.

Online quantitative proteomics p-value calculator for permutation-based statistical testing of peptide ratios

The utility of high-throughput quantitative proteomics to identify differentially abundant proteins en-masse relies on suitable and accessible statistical methodology, which remains mostly an unmet need. We present a free web-based tool, called Quantitative Proteomics p-value Calculator (QPPC), designed for accessibility and usability by proteomics scientists and biologists. Being an online tool, there is no requirement for software installation. Furthermore, QPPC accepts generic peptide ratio data generated by any mass spectrometer and database search engine. Importantly, QPPC utilizes the permutation test that we recently found to be superior to other methods for analysis of peptide ratios because it does not assume normal distributions.1 QPPC assists the user in selecting significantly altered proteins based on numerical fold change, or standard deviation from the mean or median, together with the permutation p-value. Output is in the form of comma separated values files, along with graphical visualization using volcano plots and histograms. We evaluate the optimal parameters for use of QPPC, including the permutation level and the effect of outlier and contaminant peptides on p-value variability. The optimal parameters defined are deployed as default for the web-tool at http://qppc.di.uq.edu.au/ .

Proteomics in Molecular Diagnosis: Typing of Amyloidosis

Amyloidosis is a group of disorders caused by deposition of misfolded proteins as aggregates in the extracellular tissues of the body, leading to impairment of organ function. Correct identification of the causal amyloid protein is absolutely crucial for clinical management in order to avoid misdiagnosis and inappropriate, potentially harmful treatment, to assess prognosis and to offer genetic counselling if relevant. Current diagnostic methods, including antibody-based amyloid typing, have limited ability to detect the full range of amyloid forming proteins. Recent investigations into proteomic identification of amyloid protein have shown promise. This paper will review the current state of the art in proteomic analysis of amyloidosis, discuss the suitability of techniques based on the properties of amyloidosis, and further suggest potential areas of development. Establishment of mass spectrometry aided amyloid typing procedures in the pathology laboratory will allow accurate amyloidosis diagnosis in a timely manner and greatly facilitate clinical management of the disease.

Senescent human hepatocytes express a unique secretory phenotype and promote macrophage migration

AIM To develop a model of stress-induced senescence to study the hepatocyte senescence associated secretory phenotype (SASP). METHODS Hydrogen peroxide treatment was used to induce senescence in the human HepG2 hepatocyte cell line. Senescence was confirmed by cytochemical staining for a panel of markers including Ki67, p21, heterochromatin protein 1β, and senescence-associated-β-galactosidase activity. Senescent hepatocytes were characterised by gene expression arrays and quantitative polymerase chain reaction (qPCR), and conditioned media was used in proteomic analyses, a human chemokine protein array, and cell migration assays to characterise the composition and function of the hepatocyte SASP. RESULTS Senescent hepatocytes induced classical markers of senescence (p21, heterochromatin protein 1β, and senescence-associated-β-galactosidase activity); and downregulated the proliferation marker, Ki67. Hepatocyte senescence induced a 4.6-fold increase in total secreted protein (P = 0.06) without major alterations in the protein profile. Senescence-induced genes were identified by microarray (Benjamini Hochberg-corrected P < 0.05); and, consistent with the increase in secreted protein, gene ontology analysis revealed a significant enrichment of secreted proteins among inducible genes. The hepatocyte SASP included characteristic factors such as interleukin (IL)-8 and IL-6, as well as novel components such as SAA4, IL-32 and Fibrinogen, which were validated by qPCR and/or chemokine protein array. Senescent hepatocyte-conditioned medium elicited migration of inflammatory (granulocyte-macrophage colony stimulating factor, GM-CSF-derived), but not non-inflammatory (CSF-1-derived) human macrophages (P = 0.022), which could contribute to a pro-inflammatory microenvironment in vivo, or facilitate the clearance of senescent cells. CONCLUSION Our novel model of hepatocyte senescence provides insights into mechanisms by which senescent hepatocytes may promote chronic liver disease pathogenesis.