Biomedical subjects

Lukas A Huber

Publications and source records attributed to Lukas A Huber.

At least 19 recordsLinked to original sources

PubMed2007

Identification and dynamics of proteins adhering to the surface of medical silicones in vivo and in vitro.

Silicone has been used in medical practice as a paradigmatic implant material for decades despite significant detrimental side effects. Our targeted proteomics approach was aimed at identification of the proteins adsorbed to the surface of silicone because they have been characterized as key components in the onset and perpetuation of local immune reactions to silicone. The composition of the proteinacious film, the dynamics of protein deposition, and protein modifications after adsorption were analyzed both in vivo and in vitro. Differential analysis of protein deposition was performed, followed by protein identification with mass spectrometry, database matching, and Western blots. Thus far, we have identified the 30 most abundant proteins deposited on the surface of silicone, the largest known inventory of such proteins so far. Structural and extracellular matrix proteins predominated, followed by mediators of host defense, metabolism, transport, and stress related proteins. In addition, several biochemical modifications of fibronectin, vitronectin, and heat shock protein 60 were detected. Our analyses also revealed previously undetected proteins deposited on the surface of silicone. As tentative initiators and/or modulators of the response to silicone, they are therefore valuable candidates for prognosis and therapy.

Adsorption↗

PubMed2006

A novel human primary immunodeficiency syndrome caused by deficiency of the endosomal adaptor protein p14.

Lysosome-related organelles have versatile functions, including protein and lipid degradation, signal transduction and protein secretion. The molecular elucidation of rare congenital diseases affecting endosomal-lysosomal biogenesis has given insights into physiological functions of the innate and adaptive immune system. Here, we describe a previously unknown human primary immunodeficiency disorder and provide evidence that the endosomal adaptor protein p14, previously characterized as confining mitogen-activated protein kinase (MAPK) signaling to late endosomes, is crucial for the function of neutrophils, B cells, cytotoxic T cells and melanocytes. Combining genetic linkage studies and transcriptional profiling analysis, we identified a homozygous point mutation in the 3' untranslated region (UTR) of p14 (also known as MAPBPIP), resulting in decreased protein expression. In p14-deficient cells, the distribution of late endosomes was severely perturbed, suggesting a previously unknown role for p14 in endosomal biogenesis. These findings have implications for understanding endosomal membrane dynamics, compartmentalization of cell signal cascades, and their role in immunity.

Adaptor Protein Complex 4↗

PubMed2006

p14-MP1-MEK1 signaling regulates endosomal traffic and cellular proliferation during tissue homeostasis.

The extracellular signal-regulated kinase (ERK) cascade regulates proliferation, differentiation, and survival in multicellular organisms. Scaffold proteins regulate intracellular signaling by providing critical spatial and temporal specificity. The scaffold protein MEK1 (mitogen-activated protein kinase and ERK kinase 1) partner (MP1) is localized to late endosomes by the adaptor protein p14. Using conditional gene disruption of p14 in mice, we now demonstrate that the p14-MP1-MEK1 signaling complex regulates late endosomal traffic and cellular proliferation. This function its essential for early embryogenesis and during tissue homeostasis, as revealed by epidermis-specific deletion of p14. These findings show that endosomal p14-MP1-MEK1 signaling has a specific and essential function in vivo and, therefore, indicate that regulation of late endosomal traffic by extracellular signals is required to maintain tissue homeostasis.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Biomarker discovery in breast cancer serum using 2-D differential gel electrophoresis/ MALDI-TOF/TOF and data validation by routine clinical assays.

In the present study, we used 2-D differential gel electrophoresis (2-D DIGE) and MS to screen biomarker candidates in serum samples obtained from 39 patients with breast cancer and 35 controls. First, we pooled the serum samples matched with age and menopausal status. Then, we depleted the two most abundant proteins albumin and IgG by immunoaffinity chromatography under partly denaturing conditions in order to enrich low-abundance proteins and proteins with low molecular weight. Concentrated and desalted samples were labeled with three different CyDyes including one internal standard, pooled from all the samples, and separated with 2-D DIGE in triplicate experiments. Biological variations of the protein expression level were analyzed with DeCyder software and evaluated for reproducibility and statistical significance. The profile of differentially expressed protein spots between patients and controls revealed proapolipoprotein A-I, transferrin, and hemoglobin as up-regulated and three spots, apolipoprotein A-I, apolipoprotein C-III, and haptoglobin alpha2 as down-regulated in patients. Finally, routine clinical immunochemical reactions were used to validate selected candidate biomarkers by quantitative determination of specific proteins in all individual serum samples. The serum level of transferrin correlated well with the 2-D-DIGE results. However, the serum levels of apolipoprotein A-I and haptoglobin could not be detected with the clinical routine diagnostic tests. This demonstrated an advantage 2-D DIGE still has over other techniques. 2-D DIGE can distinguish between isoforms of proteins, where the overall immunochemical quantification does fail due to a lack of isoform-special antibodies.

Adult↗

PubMed2006

Progress in capillary electrophoresis coupled to matrix-assisted laser desorption/ionization - time of flight mass spectrometry.

In this review the most important techniques developed to hyphenate CE to MALDI-TOF-MS are summarized. The principles of the different interfaces and ways to solve the hyphenation problem are explained and discussed in detail. The most important applications especially in the proteomic field are reviewed, and the advantages of CE-MALDI-TOF-MS for the analysis of these compounds compared to other techniques such as ESI-MS are exhaustingly discussed from a critical point of view. CE coupled to MALDI-TOF-MS has started to overpass traditionally used CE-coupling techniques, especially CE-ESI-MS, offering the possibility to analyze samples of interest even weeks after CE analysis and using multiplexing systems for high-sample throughput.

Electrophoresis, Capillary↗

PubMed2006

Microscopy of the Drosophila facet eye: vademecum for standardized fixation, embedding, and sectioning.

We describe here a standardized method for histological processing of the Drosophila compound eye. Primary fixation with 2.5% glutaraldehyde, obligatorily supplemented with 0.1% household detergent regularly yielded the best structural preservation, as compared with that of other, more complicated fixation protocols tested. Notably, it proved indispensable not only to cut off the fly's head to facilitate the penetration of the reagents but also to open the chitinous head capsule. For this, we locally pierced the cuticle between the eyes, leaving the head structurally almost intact, a prerequisite for precisely aligning the head for microtomy. We developed a two-step re-embedding procedure allowing for exact and reproducible orientation of the fly heads. Thus, highly comparable series of cross sections through a representative number of ommatidia were obtained. The feasibility of our embedding and sectioning approach is finally demonstrated by three-dimensional reconstructions of the middle segments of the R1, R7, and R8 photoreceptor cells. We present reconstructions from structurally modified ommatidia, as seen after RNAi-mediated depletion of the endosomal adaptor protein p14, and from normal ommatidia corresponding to the wildtype.

Animals↗

PubMed2006

Guidelines for the next 10 years of proteomics.

In the last ten years, the field of proteomics has expanded at a rapid rate. A range of exciting new technology has been developed and enthusiastically applied to an enormous variety of biological questions. However, the degree of stringency required in proteomic data generation and analysis appears to have been underestimated. As a result, there are likely to be numerous published findings that are of questionable quality, requiring further confirmation and/or validation. This manuscript outlines a number of key issues in proteomic research, including those associated with experimental design, differential display and biomarker discovery, protein identification and analytical incompleteness. In an effort to set a standard that reflects current thinking on the necessary and desirable characteristics of publishable manuscripts in the field, a minimal set of guidelines for proteomics research is then described. These guidelines will serve as a set of criteria which editors of PROTEOMICS will use for assessment of future submissions to the Journal.

Biomarkers↗

PubMed2006

Phosphoproteomics strategies for the functional analysis of signal transduction.

Protein phosphorylation is a key regulatory mechanism of cellular signalling processes. The analysis of phosphorylated proteins and the characterisation of phosphorylation sites under different biological conditions are some of the most challenging tasks in current proteomics research. Reduction of the sample complexity is one major step for the analysis of low-abundance kinase substrates, which can be achieved by various subcellular fractionation techniques. One strategy is the enrichment of phosphorylated proteins or peptides by immunoprecipitation or chromatography, e.g. immobilised metal affinity chromatography, prior to analysis. 2-DE gels are powerful tools for the analysis of phosphoproteins when combined with new multiplexing techniques like DIGE, phosphospecific stains, autoradiography or immunoblotting. In addition, several gel-free methods combining chromatography with highly sensitive MS have been successfully applied for the analysis of complex phosphoproteomes. Recently developed approaches like KESTREL or 'chemical genetics' and also protein microarrays offer new possibilities for the identification of specific kinase targets. This review summarises various strategies for the analyses of phosphoproteins with a special focus on the identification of novel kinase substrates.

Animals↗

PubMed2006

Ultra-fast mass fingerprinting by high-affinity capture of peptides and proteins on derivatized poly(glycidyl methacrylate/divinylbenzene) for the analysis of serum and cell lysates.

The development of support materials in mass fingerprinting is an important task required for diagnostic markers in conjunction with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The material-based approach, which we introduced as material-enhanced laser desorption/ionization (MELDI), focuses not only on different functionalities, but also emphasizes the morphology, i.e. porosity or particle size of the carrier material. As a result, it provides a quick and sensitive platform for effective binding of peptides and proteins out of different biofluids, e.g. serum, spinal fluid, urine or cell lysates, and to subsequently analyze them with MALDI-TOF MS. This approach includes a built-in desalting step for serum protein profiling and is sensitive enough to detect proteins and peptides down to 100 fmol/microL. Here we co-polymerized glycidyl methacrylate (GMA) with divinylbenzene (DVB) using thermal polymerization to yield a GMA/DVB polymer for further modifications. Different affinities have been created, such as immobilized metal ion affinity (IDA-Cu2+), reversed-phase (RP) and anion-exchanger (AX) chromatography. The diverse derivatizations and the dispersity of the particles created by different chemical synthetic approaches were confirmed by characteristic infrared (IR) peaks. The polymerization carried out by non-stirring yielded an average pore radius of 6.1 microm (macro-pores) that enhanced the binding capacity enormously by offering enlarged surface areas. Moreover, atomic absorption spectrometry (AAS) provided the metal content loaded on iminodiacetic acid (IDA) in the case of poly(GMA/DVB)-IDA-Cu2+. To summarize, the optimized MELDI approach is sensitive in its performance, extremely fast and can be adapted to robotic systems for routine analysis, allowing sample preparation in less than 5 min in contrast to the conventional surface-enhanced laser desorption/ionization (SELDI) methods.

Blood Proteins↗

PubMed2005

TIS7 regulation of the beta-catenin/Tcf-4 target gene osteopontin (OPN) is histone deacetylase-dependent.

12-O-Tetradecanoylphorbol-13-acetate-induced sequence 7 (TIS7) acts as a transcriptional co-repressor interacting with SIN3, the histone deacetylase-containing complex. The overexpression of TIS7 down-regulates expression of a specific set of genes. Homozygous deletion of this gene in mice delays injury-induced muscle regeneration and inhibits muscle satellite cell differentiation and fusion of myoblasts in vitro. Osteopontin (OPN), a known beta-catenin/T cell factor-4 (Tcf-4) downstream target gene, is up-regulated in tumors and in cells with increased motility such as muscle cells. OPN promoter sequence contains binding sites for Sp1, glucocorticoid receptor, E-box-binding factors, octamer motif-binding protein, c-Myc, and other transcription factors. Previously we have shown that TIS7 regulates the OPN expression through the inhibition of the Sp1-activating effects. Here we show that TIS7 has the capacity to inhibit OPN expression also through Lef-1, the second identified OPN regulatory element. TIS7 has the capacity to down-regulate beta-catenin/Tcf-4 transcriptional activity. TIS7 homologous deletion in mouse embryonic fibroblasts increased not only the TOPflash reporter gene transcriptional activity but also the expression of c-Myc and OPN. Furthermore, we show that TIS7 overexpression leads to the beta-catenin interaction with enzymatically active histone deacetylases. We propose that TIS7 down-regulates the beta-catenin/Tcf-4 transcriptional activity via its interaction with histone deacetylase-containing complex thereby inhibiting the expression of beta-catenin downstream target genes such as c-Myc and OPN. We hypothesize that TIS7 as a negative regulator of transcriptional activity represses expression of OPN and beta-catenin/Tcf-4 target genes, which are involved in myogenesis, muscle maintenance, and regeneration in a histone deacetylase dependent manner.

Amino Acid Motifs↗

PubMed2005

Quantitative detection of phosphoproteins by combination of two-dimensional difference gel electrophoresis and phosphospecific fluorescent staining.

Here we combine a standard two-dimensional difference gel electrophoresis (DIGE) protocol with subsequent post-staining of gels with phosphospecific fluorescent Pro-Q Diamond dye. The combination of these two methods for fluorescence detection of proteins allows quantitative detection of phosphoproteins in 2-DE-gels. We established this protocol within a functional proteomics experiment. Mammary epithelial cells (EpH4) were stimulated in culture by epidermal growth factor (EGF), endosomal fractions prepared after subcellular fractionation and phosphorylated proteins successfully detected on endosomes. For instance, Endo A cytokeratin, known as phosphoprotein and differentiation marker inducible by MAPK signaling, was identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). With this protocol, all steps of combined proteome and phosphoproteome profiling experiments are significantly simplified and accelerated, taking full advantage of both methods in terms of specificity, sensitivity and accuracy of quantification.

Animals↗

PubMed2005

Enrichment of low-abundant serum proteins by albumin/immunoglobulin G immunoaffinity depletion under partly denaturing conditions.

We present a simple protocol for affinity depletion to remove the two most abundant serum proteins, albumin and immunoglobulin G (IgG). Under native conditions, albumin/IgG were efficiently removed and several proteins were enriched as shown by two-dimensional electrophoresis (2-DE). Besides that, partly denaturing conditions were established by adding 5 or 20% acetonitrile (ACN) in order to disrupt the binding of low-molecular-weight (LMW) proteins to the carrier proteins albumin/IgG. 2-DE results showed that the total number of detected LMW proteins increased under denaturing conditions when compared to native conditions. Interestingly, the presence of 5% ACN in serum revealed better enrichment of LMW proteins when compared to 20% ACN condition. Seven randomly distributed spots in albumin/IgG depleted serum samples under 5% ACN condition were picked from the 2-DE gels and identified by mass spectrometry (MS). The intensity of five LMW protein spots increased under denaturing conditions when compared to native conditions. Three of the seven identified spots (serum amyloid P, vitamin D-binding protein, and transthyretin) belong to a group of relatively low-abundant proteins, which make up only 1% of all serum proteins. The method presented here improves the resolution of the serum proteome by increasing the number of visualized spots on 2-D gels and allowing the detection and MS identification of LMW proteins and proteins of lower abundance.

Acetonitriles↗

PubMed2005

Phosphoproteomic analysis using immobilized metal ion affinity chromatography on the basis of cellulose powder.

Detailed characterization of phosphoproteins as well as other post-translationally modified proteins such as glycoproteins, is required to fully understand protein function and regulatory events in cells and organisms. Therefore, an experimental strategy for the isolation of phosphoproteins using a new immobilized metal ion affinity chromatograph (IMAC) material on the basis of cellulose has been developed and characterized. Different approaches have been used to test the material. Recovery rates were determined by 32P labelling of a myelin basic protein fragment and by reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry using a tryptic digest of the model protein bovine beta-casein. Selectivity was demonstrated by enrichment and separation of phosphopeptides from different samples, such as from a digest of horse myoglobin as well as from a digest of in vitro phosphorylated extracellular signal regulates kinase 2 (ERK2) mixed with synthetic phosphopeptides, phosphorylated on different amino acid residues. Furthermore, simplification and optimization of sample pretreatment was achieved by combining the separating (IMAC) and desalting (C18) step during preparative high performance liquid chromatography. The comparison between our material and a commercially available IMAC system (POROS 20 MC; Perspective BioSystems) emphasizes the competitiveness of the cellulose. Confirmed by the obtained data, the cellulose material performed as well as the commercially available sorbent, however with the advantage, that it can be produced rather easily and at very low cost.

Animals↗

PubMed2005

Apoptosis resistance of senescent human fibroblasts is correlated with the absence of nuclear IGFBP-3.

Signaling through the insulin/IGF axis plays a major role in determining the rate of aging in many species. IGF-binding proteins (IGFBPs) modulate the IGF pathway in higher organisms. IGFBP-3 accumulates in conditioned medium of senescent human fibroblasts, suggesting that it may contribute to the senescent phenotype. IGFBP-3 can enhance apoptotic cell death in tumor cells due to its ability to target intracellular regulators of apoptosis, including nuclear transcription factors. Senescent fibroblasts are highly resistant to apoptosis, suggesting that IGFBP-3 fails to induce apoptosis in this cell type; however, mechanisms of apoptosis resistance in senescent cells are poorly understood. To address this question, we studied the production and intracellular localization of IGFBP-3 in senescent fibroblasts. Whereas IGFBP-3 is highly overexpressed by senescent fibroblasts, IGFBP-3 was not detectable in the nucleus of senescent fibroblasts. In tumor cells, IGFBP-3 can be internalized by endocytosis, which is considered as a prerequisite for the intracellular functions of IGFBP-3 and probably also for its transport to the nucleus; we show here that endocytotic uptake of IGFBP-3 does not occur in senescent human fibroblasts. This is correlated with a generally decreased endocytotic activity of these cells, as shown with the model substrate transferrin. The data are consistent with a model where IGFBP-3 accumulation in conditioned medium of senescent fibroblasts contributes to growth arrest of these cells, whereas the failure to endocytose IGFBP-3 and the absence of nuclear IGFBP-3 may contribute to the well-established apoptosis resistance of senescent human fibroblasts.

Apoptosis↗

PubMed2005

Persistent STAT3 activation in colon cancer is associated with enhanced cell proliferation and tumor growth.

Colorectal carcinoma (CRC) is a major cause of morbidity and mortality in Western countries. It has so far been molecularly defined mainly by alterations of the Wnt pathway. We show here for the first time that aberrant activities of the signal transducer and activator of transcription STAT3 actively contribute to this malignancy and, thus, are a potential therapeutic target for CRC. Constitutive STAT3 activity was found to be abundant in dedifferentiated cancer cells and infiltrating lymphocytes of CRC samples, but not in non-neoplastic colon epithelium. Cell lines derived from malignant colorectal tumors lost persistent STAT3 activity in culture. However, implantation of colon carcinoma cells into nude mice resulted in restoration of STAT3 activity, suggesting a role of an extracellular stimulus within the tumor microenvironment as a trigger for STAT activation. STAT3 activity in CRC cells triggered through interleukin-6 or through a constitutively active STAT3 mutant promoted cancer cell multiplication, whereas STAT3 inhibition through a dominant-negative variant impaired IL-6-driven proliferation. Blockade of STAT3 activation in CRC-derived xenograft tumors slowed down their development, arguing for a contribution of STAT3 to colorectal tumor growth.

Animals↗

PubMed2004

Crystal structure of the p14/MP1 scaffolding complex: how a twin couple attaches mitogen-activated protein kinase signaling to late endosomes.

Signaling pathways in eukaryotic cells are often controlled by the formation of specific signaling complexes, which are coordinated by scaffold and adaptor proteins. Elucidating their molecular architecture is essential to understand the spatial and temporal regulation of cellular signaling. p14 and MP1 form a tight (K(d) = 12.8 nM) endosomal adaptor/scaffold complex, which regulates mitogen-activated protein kinase (MAPK) signaling. Here, we present the 1.9-A crystal structure of a biologically functional p14/MP1 complex. The overall topology of the individual MP1 and p14 proteins is almost identical, having a central five-stranded beta-sheet sandwiched between a two-helix and a one-helix layer. Formation of the p14/MP1 heterodimer proceeds by beta-sheet augmentation and yields a unique, almost symmetrical, complex with several potential protein-binding sites on its surface. Mutational analysis allowed identification of the p14 endosomal adaptor motif, which seems to orient the complex relative to the endosomal membrane. Two highly conserved and hydrophobic protein-binding sites are located on the opposite "cytoplasmic" face of the p14/MP1 heterodimer and might therefore function as docking sites for the target proteins extracellular regulated kinase (ERK) 1 and MAPK/ERK kinase 1. Furthermore, detailed sequence analyses revealed that MP1/p14, together with profilins, define a protein superfamily of small subcellular adaptor proteins, named ProflAP. Taken together, the presented work provides insight into the spatial regulation of MAPK signaling, illustrating how p14 and MP1 collaborate as an endosomal adaptor/scaffold complex.

Animals↗

PubMed2004

Zooming in: fractionation strategies in proteomics.

The recent development of mass spectrometry, i.e., high sensitivity, automation of protein identification and some post-translational modifications (PTMs) significantly increased the number of large-scale proteomics projects. However, there are still considerable limitations as none of the currently available proteomics techniques allows the analysis of an entire proteome in a single step procedure. On the other hand, there are several successful studies analyzing well defined groups of proteins, e.g., proteins of purified organelles, membrane microdomains or isolated proteins with certain PTMs. Coupling of advanced separation methodologies (different prefractionation strategies, such as subcellular fractionation, affinity purification, fractionation of proteins and peptides according to their physicochemical properties) to highly sensitive mass spectrometers provides powerful means to detect and analyze dynamic changes of low abundant regulatory proteins in eukaryotic cells on the subcellular level. This review summarizes and discusses recent strategies in proteomics approaches where different fractionation strategies were successfully applied.

Animals↗

PubMed2004

Muscle regeneration and myogenic differentiation defects in mice lacking TIS7.

The tetradecanoyl phorbol acetate-induced sequence 7 gene (tis7) is regulated during cell fate processes and functions as a transcriptional coregulator. Here, we describe the generation and analysis of mice lacking the tis7 gene. Surprisingly, TIS7 knockout mice show no gross histological abnormalities and are fertile. Disruption of the tis7 gene by homologous recombination delayed muscle regeneration and altered the isometric contractile properties of skeletal muscles after muscle crush damage in TIS7(-/-) mice. Cultured primary myogenic satellite cells (MSCs) from TIS7(-/-) mice displayed marked reductions in differentiation potential and fusion index in a strictly cell-autonomous fashion. Loss of TIS7 caused the down-regulation of muscle-specific genes, such as those for MyoD, myogenin, and laminin-alpha2. Fusion potential in TIS7(-/-) MSCs could be rescued by TIS7 expression or laminin supplementation. Therefore, TIS7 is not essential for mouse development but plays a novel regulatory role during adult muscle regeneration.

Animals↗