Chemical Tools for the Study of Intramembrane Proteases.

Intramembrane proteases (IMPs) reside inside lipid bilayers and perform peptide hydrolysis in transmembrane or juxtamembrane regions of their substrates. Many IMPs are involved in crucial regulatory pathways and human diseases, including Alzheimer's disease, Parkinson's disease, and diabetes. In the past, chemical tools have been instrumental in the study of soluble proteases, enabling biochemical and biomedical research in complex environments such as tissue lysates or living cells.

Label-free biosensors based on in situ formed and functionalized microwires in microfluidic devices.

Label-free biosensors based on in situ formed and functionalized TTF-Au wires were developed using an integrated microfluidic system. By applying different modification protocols, TTF-Au wires were successfully used for sensitive label-free detection of catecholamines and human IgG by Raman spectroscopy.

Inhibitor Fingerprinting of Rhomboid Proteases by Activity-Based Protein Profiling Reveals Inhibitor Selectivity and Rhomboid Autoprocessing.

Rhomboid proteases were discovered almost 15 years ago and are structurally the best characterized intramembrane proteases. Apart from the general serine protease inhibitor 3,4-dichloro-isocoumarin (DCI) and a few crystal structures of the Escherichia coli rhomboid GlpG with other inhibitors, there is surprisingly little information about inhibitors of rhomboids from other species, probably because of a lack of general methods to measure inhibition against different rhomboid species. We here present activity-based protein profiling (ABPP) as a general method to screen rhomboids for their activity and inhibition.

Inhibitors of rhomboid proteases.

Rhomboid proteases form one of the most widespread families of intramembrane proteases. They utilize a catalytic serine-histidine dyad located several Å below the surface of the membrane for substrate hydrolysis. Multiple studies have implicated rhomboid proteases in biologically and medically relevant processes.

Application of surface plasmon resonance imaging technique for the detection of single spherical biological submicrometer particles.

Recent proof-of-principle studies demonstrated the suitability of the surface plasmon resonance imaging (SPRi) technique for the detection of individual submicrometer and nanoparticles in solutions. In the current study, we used the SPRi technique for visualization of the binding of round-shaped viruses (inactivated influenza A virus) and virus-like particles (human immunodeficiency virus (HIV)-based virus-like particles) to the functionalized sensor surface. We show the applicability of the SPRi technique for the detection of individual virus-like particles in buffers without serum as well as in buffers containing different concentrations of serum.

Detection of protease activity in cells and animals.

Proteases are involved in a wide variety of biologically and medically important events. They are entangled in a complex network of processes that regulate their activity, which makes their study intriguing, but challenging. For comprehensive understanding of protease biology and effective drug discovery, it is therefore essential to study proteases in models that are close to their complex native environments such as live cells or whole organisms.

Analysis of disease-associated protein expression using quantitative proteomics—fibulin-5 is expressed in association with hepatic fibrosis.

Hepatic fibrosis and cirrhosis are major health problems worldwide. Until now, highly invasive biopsy remains the diagnostic gold standard despite many disadvantages. To develop noninvasive diagnostic assays for the assessment of liver fibrosis, it is urgently necessary to identify molecules that are robustly expressed in association with the disease.

Antibody induction directed against the tumor-associated MUC4 glycoprotein.

Mucin glycoproteins are important diagnostic and therapeutic targets for cancer treatment. Although several strategies have been developed to explore anti-tumor vaccines based on MUC1 glycopeptides, only few studies have focused on vaccines directed against the tumor-associated MUC4 glycoprotein. MUC4 is an important tumor marker overexpressed in lung cancer and uniquely expressed in pancreatic ductual adenocarcinoma.

Identification of novel biomarker candidates for the immunohistochemical diagnosis of cholangiocellular carcinoma.

The aim of this study was the identification of novel biomarker candidates for the diagnosis of cholangiocellular carcinoma (CCC) and its immunohistochemical differentiation from benign liver and bile duct cells. CCC is a primary cancer that arises from the epithelial cells of bile ducts and is characterized by high mortality rates due to its late clinical presentation and limited treatment options. Tumorous tissue and adjacent non-tumorous liver tissue from eight CCC patients were analyzed by means of two-dimensional differential in-gel electrophoresis and mass-spectrometry-based label-free proteomics.

Symmetric exchange of multi-protein building blocks between stationary focal adhesions and the cytosol.

How can the integrin adhesome get self-assembled locally, rapidly, and correctly as diverse cell-matrix adhesion sites? Here, we investigate this question by exploring the cytosolic state of integrin-adhesome components and their dynamic exchange between adhesion sites and cytosol. Using fluorescence cross-correlation spectroscopy (FCCS) and fluorescence recovery after photobleaching (FRAP) we found that the integrin adhesome is extensively pre-assembled already in the cytosol as multi-protein building blocks for adhesion sites. Stationary focal adhesions release symmetrically the same types of protein complexes that they recruit, thereby keeping the cytosolic pool of building blocks spatiotemporally uniform.

A convergent strategy for the synthesis of type-1 elongated mucin cores 1-3 and the corresponding glycopeptides.

Mucins are a class of highly O-glycosylated proteins found on the surface of cells in epithelial tissues. O-Glycosylation is crucial for the functionality of mucins and changes therein can have severe consequences for an organism. With that in mind, the elucidation of interactions of carbohydrate binding proteins with mucins, whether in morbidly altered or unaltered conditions, continue to shed light on mechanisms involved in diseases like chronic inflammations and cancer.

Synthesis of a glycopeptide vaccine conjugate for induction of antibodies recognizing O-mannosyl glycopeptides.

In spite of the clear importance of protein O-mannosylation in brain glycobiology, tools are lacking for specific detection, enrichment, and identification of proteins containing these modifycations. We envisioned inducing antibodies that specifically recognize O-mannose glycans on proteins and peptides. With this in mind, we prepared a glycopeptide vaccine construct containing the N-acetyllactosamine-extended mannose motif Galβ1-4GlcNAcβ1-2ManαThr, found as a common core structure on almost all mammalian O-mannosyl glycoproteins identified.

The protein import machinery of mitochondria-a regulatory hub in metabolism, stress, and disease.

Mitochondria fulfill central functions in bioenergetics, metabolism, and apoptosis. They import more than 1,000 different proteins from the cytosol. It had been assumed that the protein import machinery is constitutively active and not subject to detailed regulation.

Arraying the post-translational glycoproteome (PTG).

Glycosylation is chemically the most complex post-translational modification of proteins and therefore understanding the structural and biological implications of post-translational glycosylation is a major challenge. The need for rapid and reliable investigations of protein-glycan interaction events and the substantial efforts required to synthesize glycans and glycopeptides with a variety of structures has called for the development of miniaturized analytical techniques. In the last decade, glycan and glycopeptide microarrays have enabled high-throughput analysis of diverse protein-glycan interactions.

Human chitotriosidase CHIT1 cross reacts with mammalian-like substrates.

Humans do not synthesize chitin, yet they produce a number of active and inactive chitinases. One of the active enzymes is chitotriosidase whose serum levels are elevated in a number of diseases such as Gaucher's disease and upon fungal infection. Since the biological role of chitotriosidase in disease pathogenesis is not understood we screened a panel of mammalian GlcNAc-containing glycoconjugates as alternate substrates.

Gli protein activity is controlled by multisite phosphorylation in vertebrate Hedgehog signaling.

Gli proteins are transcriptional effectors of the Hedgehog (Hh) pathway in both normal development and cancer. We describe a program of multisite phosphorylation that regulates the conversion of Gli proteins into transcriptional activators. In the absence of Hh ligands, Gli activity is restrained by the direct phosphorylation of six conserved serine residues by protein kinase A (PKA), a master negative regulator of the Hh pathway.

Synthetic glycopeptides and glycoproteins with applications in biological research.

Over the past few years, synthetic methods for the preparation of complex glycopeptides have been drastically improved. The need for homogenous glycopeptides and glycoproteins with defined chemical structures to study diverse biological phenomena further enhances the development of methodologies. Selected recent advances in synthesis and applications, in which glycopeptides or glycoproteins serve as tools for biological studies, are reviewed.

Synthetic vaccines from tumor-associated glycopeptide antigens.

The aberrantly glycosylated and extensively over-expressed membrane-bound mucin MUC1 glycoprotein forms a tumor specific epitope on the surface of epithelial cells. Using defined synthetic glycopeptide structures consisting of the MUC1 tandem repeat region for immunization, antibodies selectively binding to tumor cell surface should be induced. Recent examples of synthetic vaccines directed against the O-glycosylated MUC1 tandem repeats and their immunological evaluation will be given here.

Phosphoproteome analysis of the platelet plasma membrane.

Blood platelets are key players standing at the crossroads between physiologically occurring hemostasis and pathologic thrombus formation. As these cellular particles lack a nucleus, intra- and intercellular processes involved in platelet activity and function are almost exclusively regulated on the protein level. In particular, posttranslational protein modification by phosphorylation, which allows for a quick and highly dynamic transduction of cellular signals, is discussed in this context.