Protein analysis of tissues--current views and clinical perspectives.

Proteomics raises high expectations in finding novel and reliable biomarkers for diagnosis, prognosis and therapy prediction. The goal of the 2-day workshop "Protein analysis of tissues-current views and clinical perspectives" was to bring together scientists from multiple areas of protein research interested in tissue analysis.

Tandem affinity purification of protein complexes from mammalian cells by the Strep/FLAG (SF)-TAP tag.

Isolation and dissection of native multiprotein complexes is a central theme in functional genomics. The development of the tandem affinity purification (TAP) tag has enabled efficient and large-scale purification of native protein complexes. The SF-TAP tag, a modified version of the TAP tag, allows a fast and straightforward purification of protein complexes from mammalian cells.

Approaching clinical proteomics: current state and future fields of application in fluid proteomics.

The field of clinical proteomics offers opportunities to identify new disease biomarkers in body fluids, cells and tissues. These biomarkers can be used in clinical applications for diagnosis, stratification of patients for specific treatment, or therapy monitoring. New protein array formats and improved spectrometry technologies have brought these analyses to a level with potential for use in clinical diagnostics.

The Parkinson disease-associated protein kinase LRRK2 exhibits MAPKKK activity and phosphorylates MKK3/6 and MKK4/7, in vitro.

Autosomal dominant mutations in the human Leucine-Rich Repeat Kinase 2 (LRRK2) gene represent the most common monogenetic cause of Parkinson disease (PD) and increased kinase activity observed in pathogenic mutants of LRRK2 is most likely causative for PD-associated neurotoxicity. The sequence of the LRRK2 kinase domain shows similarity to MAP kinase kinase kinases. Furthermore, LRRK2 shares highest sequence homology with mixed linage kinases which act upstream of canonical MAPKK and are involved in cellular stress responses.

PKG activity causes photoreceptor cell death in two retinitis pigmentosa models.

Photoreceptor degeneration in retinitis pigmentosa is one of the leading causes of hereditary blindness in the developed world. Although causative genetic mutations have been elucidated in many cases, the underlying neuronal degeneration mechanisms are still unknown. Here, we show that activation of cGMP-dependent protein kinase (PKG) hallmarks photoreceptor degeneration in rd1 and rd2 human homologous mouse models.

16-BAC/SDS-PAGE analysis of membrane proteins of yeast mitochondria purified by free flow electrophoresis.

Mitochondria are essential organelles in cellular metabolism. These organelles are bounded by two membranes, the outer and inner membrane. Especially the inner membrane comprises a high content of proteins, for example, the protein complexes of the respiratory chain.

Tandem affinity purification of ciliopathy-associated protein complexes.

Ciliary dysfunction has recently been recognized as a cause for a growing number of genetically inherited disorders termed ciliopathies. Ciliopathy-associated proteins are organized in cell/context-specific complexes and in shared regulatory circuits in cilia of affected tissues. Thus, the identification of protein interactions involved in ciliary function provides a valid starting point to molecularly dissect normal ciliary function in a context and tissue specific fashion, identify novel functional candidate genes for ciliopathies as well as uncover the molecular defects that cause ciliary disease on the cellular level.

Serum PEDF levels are decreased in a spontaneous animal model for human autoimmune uveitis.

Identification of biomarkers is of critical relevance toward improving diagnosis and therapy of autoimmune disorders. Serum markers are a desirable choice as sera are easily accessible and the development of assays for routine clinical detection prompts feasible. Autoimmune uveitis, a recurrent disease affecting the eye, is characterized by returning inflammatory attacks of the inner eye followed by variable periods of quiescent stages.

Native fractionation: isolation of native membrane-bound protein complexes from porcine rod outer segments using isopycnic density gradient centrifugation.

Networks of interacting protein control physiological processes in all living cells. Considerable effort has recently been invested in understanding protein interactions under normal and diseased conditions. One approach to elucidate the composition of protein complexes is native fractionation followed by immunological or MS-based identification of individual compounds.

Identification of paracrine neuroprotective candidate proteins by a functional assay-driven proteomics approach.

Glial cells support neuronal survival and function by secreting neurotrophic cytokines. Retinal Mueller glial cells (RMGs) support retinal neurons, especially photoreceptors. These highly light-sensitive sensory neurons receive vision, and their death results in blinding diseases.

Equine recurrent uveitis--a spontaneous horse model of uveitis.

Equine recurrent uveitis (ERU) is an autoimmune disease that occurs with a high prevalence (10%) in horses. ERU represents the only reliable spontaneous model for human autoimmune uveitis. We already identified and characterized novel autoantigens (malate dehydrogenase, recoverin, CRALBP) by analyzing the autoantibody-binding pattern of horses affected by spontaneous recurrent uveitis (ERU) to the retinal proteome.

Purification of Saccharomyces cerevisiae mitochondria by zone electrophoresis in a free flow device.

This chapter describes the isolation of yeast mitochondria by differential centrifugation followed by mitochondrial purification through zone electrophoresis (ZE) using a free flow device (FFE). Starting from a yeast colony, cultures are grown under respiratory conditions to logarithmic phase. Cells are collected, their cell walls enzymatically disintegrated and the resulting spheroplasts are homogenized.

CRALBP is a highly prevalent autoantigen for human autoimmune uveitis.

Cellular retinaldehyde binding protein (CRALBP) is an autoantigen in spontaneous equine recurrent uveitis. In order to test whether CRALBP contributes to human autoimmune uveitis, the specificity of antibodies from human uveitis patient's sera was first evaluated in two-dimensional (2D) Western blot analysis. Subsequent identification of the immunoreactive proteins by mass spectrometry resulted in the identification of CRALBP as a putative autoantigen.

Discovering novel targets for autoantibodies in dilated cardiomyopathy.

There is increasing evidence that a large proportion of dilated cardiomyopathy (DCM) cases are mediated by autoimmune processes. Since DCM is a fatal disorder with rapid aggravation and is the leading cause of heart transplantation, further insights into disease pathogenesis are needed. Recent studies have separated the pathogenic capacity of autoantibodies and initial clinical trials removing such autoantibodies via immunoadsorption have been promising.

Delta-like 1 participates in the specification of ventral midbrain progenitor derived dopaminergic neurons.

Delta-like 1 (Dlk1), a member of the Delta/Notch protein family, is expressed in the mouse ventral midbrain (VM) as early as embryonic day 11.5 (E11.5) followed by exclusive expression in tyrosine 3-monooxygenase (TH) positive neurons from E12.

A novel tandem affinity purification strategy for the efficient isolation and characterisation of native protein complexes.

Isolation and dissection of native multiprotein complexes is a central theme in functional genomics. The development of the tandem affinity purification (TAP) tag has enabled an efficient and large-scale purification of native protein complexes. However, the TAP tag features a size of 21 kDa and requires time consuming cleavage.

Excessive activation of poly(ADP-ribose) polymerase contributes to inherited photoreceptor degeneration in the retinal degeneration 1 mouse.

Retinitis pigmentosa (RP) is an inherited blinding disease for which there is no treatment available. It is characterized by a progressive and neurodegenerative loss of photoreceptors but the underlying mechanisms are poorly understood. Excessive activation of the enzyme poly(ADP-ribose) polymerase (PARP) has recently been shown to be involved in several neuropathologies.

Two-dimensional electrophoresis of membrane proteins.

One third of all genes of various organisms encode membrane proteins, emphasizing their crucial cellular role. However, due to their high hydrophobicity, membrane proteins demonstrate low solubility and a high tendency for aggregation. Indeed, conventional two-dimensional gel electrophoresis (2-DE), a powerful electrophoretic method for the separation of complex protein samples that applies isoelectric focusing (IEF) in the first dimension and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension, has a strong bias against membrane proteins.

Membrane-initiated effects of progesterone on calcium dependent signaling and activation of VEGF gene expression in retinal glial cells.

Neurosteroids, such as progesterone, influence central nervous system development and function by regulating a broad spectrum of physiological processes. Here, we investigated membrane-initiated actions of progesterone in the retina and identified the membrane-associated progesterone receptor component 1 (PGRMC1). We found PGRMC1 expressed mainly in retinal Muller glia (RMG) and retinal pigment epithelium, and localized uniquely to microsomal and plasma membrane fractions.

Mutations in LCA5, encoding the ciliary protein lebercilin, cause Leber congenital amaurosis.

Leber congenital amaurosis (LCA) causes blindness or severe visual impairment at or within a few months of birth. Here we show, using homozygosity mapping, that the LCA5 gene on chromosome 6q14, which encodes the previously unknown ciliary protein lebercilin, is associated with this disease. We detected homozygous nonsense and frameshift mutations in LCA5 in five families affected with LCA.

Major retinal autoantigens remain stably expressed during all stages of spontaneous uveitis.

Equine recurrent uveitis (ERU) is a valuable model for autoimmune diseases, since it develops frequently and occurs spontaneously. We investigated the overall expression level of three major retinal autoantigens in normal retinas and various ERU stages. Analysis of retinal proteomes of both, healthy and diseased retinas revealed an almost unaffected expression of IRBP, S-antigen and cRALBP in ERU cases.

Retinal Mueller glial cells trigger the hallmark inflammatory process in autoimmune uveitis.

Spontaneous equine recurrent uveitis (ERU) is an incurable autoimmune disease affecting the eye. Although retinal-autoantigen specific T-helper 1 cells have been demonstrated to trigger disease progression and relapses, the molecular processes leading to retinal degeneration and consequent blindness remain unknown. To elucidate such processes, we studied changes in the total retinal proteome of ERU-diseased horses compared to healthy controls.

Down-regulation of pigment epithelium-derived factor in uveitic lesion associates with focal vascular endothelial growth factor expression and breakdown of the blood-retinal barrier.

Spontaneous equine recurrent uveitis (ERU) is an incurable autoimmune disease affecting the eye. Identifying biological markers or pathways associated with this disease may allow the understanding of its pathogenesis at a molecular level. The vitreous is the body fluid closest to the disease-affected tissue and possibly also an effector of pathological processes relevant for ERU.