Biomedical subjects

Thomas Rudel

Publications and source records attributed to Thomas Rudel.

16 recordsLinked to original sources

PubMed2006

The kinesin KIF1C and microtubule plus ends regulate podosome dynamics in macrophages.

Microtubules are important for the turnover of podosomes, dynamic, actin-rich adhesions implicated in migration and invasion of monocytic cells. The molecular basis for this functional dependency, however, remained unclear. Here, we show that contact by microtubule plus ends critically influences the cellular fate of podosomes in primary human macrophages. In particular, we identify the kinesin KIF1C, a member of the Kinesin-3 family, as a plus-end-enriched motor that targets regions of podosome turnover. Expression of mutation constructs or small interfering RNA-/short hairpin RNA-based depletion of KIF1C resulted in decreased podosome dynamics and ultimately in podosome deficiency. Importantly, protein interaction studies showed that KIF1C binds to nonmuscle myosin IIA via its PTPD-binding domain, thus providing an interface between the actin and tubulin cytoskeletons, which may facilitate the subcellular targeting of podosomes by microtubules. This is the first report to implicate a kinesin in podosome regulation and also the first to describe a function for KIF1C in human cells.

Cell Differentiation↗

PubMed2006

Quantitative proteome analysis of CD95 (Fas/Apo-1)-induced apoptosis by stable isotope labeling with amino acids in cell culture, 2-DE and MALDI-MS.

Proteome analysis of Jurkat T cells induced to undergo apoptosis by CD95 (Fas/Apo-1) treatment was performed to identify modified proteins. We used stable isotope labeling with amino acids in cell culture (SILAC) using leucine to identify proteins of apoptotic and control Jurkat T cells by 2-DE and MALDI-MS. Out of 224 spots analyzed, we quantified 213 spots with 3.5 leucine-containing peptide pairs on average; 28 proteins with a relative abundance of higher than 1.5 were found. Five new modified proteins including calcyclin binding protein, cytosolic acyl coenzyme A thioester hydrolase, heterogeneous ribonucleoprotein M, replication factor C 37-kDa subunit, and tropomyosin 4 chain were identified as being modified in response to apoptosis. In comparison to differential proteome analysis via silver-stained 2-D gels and PMF of total Jurkat T cell lysates, 15 additional apoptosis-modified proteins were identified though 8 proteins were not found. The described approach using SILAC instead of silver staining for relative quantification was simpler to perform regarding the number of required 2-D gels, that cumbersome gel comparisons were avoided, and more apoptosis-modified proteins were identified, but with a higher demand on data interpretation of the mass spectra obtained.

Apoptosis↗

PubMed2006

A new interaction between Abi-1 and betaPIX involved in PDGF-activated actin cytoskeleton reorganisation.

Members of the Rho family of GTPases are key regulators of the actin cytoskeleton. In particular, activated Rac1 stimulates membrane dorsal ruffle formation in response to platelet-derived growth factor (PDGF). Abl-interactor (Abi)-1 and betaPIX, a guanine nucleotide exchange factor for Rac1, localise at these Rac1-induced actin structures and play important roles in the induction of membrane dorsal ruffling in response to PDGF in fibroblasts. Here, we demonstrate a novel interaction between Abi-1 and betaPIX using the yeast two-hybrid system, in vitro pull-down assays, and in vivo co-immunoprecipitation experiments. In vitro, the C-terminal fragment of betaPIX interacted with Abi-1, while in vivo the N-terminal fragment of betaPIX interacted with Abi-1. The biological function of this interaction was investigated in mouse fibroblasts in response to PDGF stimulation. Abi-1 and betaPIX co-localised in the cytoplasm and to membrane dorsal ruffles after PDGF treatment. We show that the co-expression of Abi-1 and truncated forms of betaPIX in mouse fibroblasts blocked PDGF-induced membrane dorsal ruffles. Together, these results show that the interaction between Abi-1 and betaPIX is involved in the formation of growth factor-induced membrane dorsal ruffles.

Actins↗

PubMed2006

NF-kappaB and inhibitor of apoptosis proteins are required for apoptosis resistance of epithelial cells persistently infected with Chlamydophila pneumoniae.

Infection with Chlamydophila pneumoniae (Cpn) renders host cells resistant to apoptosis induced by a variety of stimuli. While modulation of apoptosis has been extensively studied in cells acutely infected with Cpn, very little is known on how persistent chlamydial infection influences host cell survival. Here we show that epithelial cells persistently infected with Cpn resist apoptosis induced with TNFalpha or staurosporine. Cpn induced the activation of nuclear factor kappa B (NF-kappaB) and inhibition of NF-kappaB with a chemical inhibitor or by silencing expression of the p65 subunit sensitized infected cells for apoptosis induction by staurosporine or TNFalpha. Persistent infection resulted in the upregulation of the NF-kappaB regulated inhibitor of apoptosis protein 2 (cIAP-2) but not inhibitor of apoptosis protein 1 (cIAP-1). Interestingly, silencing of either cIAP-1 or cIAP-2 sensitized infected cells, suggesting that IAPs play an important role in the apoptosis resistance of persistently infected cells.

Apoptosis↗

PubMed2006

Low-phosphate-dependent invasion resembles a general way for Neisseria gonorrhoeae to enter host cells.

Obligate human-pathogenic Neisseria gonorrhoeae expresses numerous variant surface proteins mediating adherence to and invasion of target cells. The invariant major outer membrane porin PorB of serotype A (P.IA) gonococci triggers invasion into Chang cells only if the medium is devoid of phosphate. Since gonococci expressing PorB(IA) are frequently isolated from patients with severe disseminating infections, the interaction initiated by the porin may be of major relevance for the development of this serious disease. Here, we investigated the low-phosphate-dependent invasion and compared it to the well-known pathways of entry initiated by Opa proteins. P.IA-triggered invasion requires clathrin-coated pit formation and the action of actin and Rho GTPases. However, in contrast to Opa-initiated invasion via heparan sulfate proteoglycans, microtubules, acidic sphingomyelinase, phosphatidylinositol 3-kinase, and myosin light chain kinase are not involved in this entry pathway. Nor are Src kinases required, as they are in invasion, e.g., via the CEACAM3 receptor. Invasion by PorB(IA) occurs in a wide spectrum of cell types, such as primary human epithelial and endothelial cells and in cancer cells of human and animal origin. Low-phosphate-dependent invasion is thus a pathway of gonococcal entry distinct from Opa-mediated invasion.

Animals↗

PubMed2006

IAP-IAP complexes required for apoptosis resistance of C. trachomatis-infected cells.

Host cells infected with obligate intracellular bacteria Chlamydia trachomatis are profoundly resistant to diverse apoptotic stimuli. The molecular mechanisms underlying the block in apoptotic signaling of infected cells is not well understood. Here we investigated the molecular mechanism by which apoptosis induced via the tumor necrosis factor (TNF) receptor is prevented in infected epithelial cells. Infection with C. trachomatis leads to the up-regulation of cellular inhibitor of apoptosis (cIAP)-2, and interfering with cIAP-2 up-regulation sensitized infected cells for TNF-induced apoptosis. Interestingly, besides cIAP-2, cIAP-1 and X-linked IAP, although not differentially regulated by infection, are required to maintain apoptosis resistance in infected cells. We detected that IAPs are constitutively organized in heteromeric complexes and small interfering RNA-mediated silencing of one of these IAPs affects the stability of another IAP. In particular, the stability of cIAP-2 is modulated by the presence of X-linked IAP and their interaction is stabilized in infected cells. Our observations suggest that IAPs are functional and stable as heteromers, a thus far undiscovered mechanism of IAP regulation and its role in modulation of apoptosis.

Apoptosis↗

PubMed2005

Ras-Raf signaling needs prohibitin.

Prohibitin has been connected to diverse cellular functions including cell cycle control, senescence, apoptosis and stabilization of mitochondrial proteins. By employing a loss of function approach using siRNAs we have demonstrated an unexpected role of PHB in the activation of Raf/MEK/ERK pathway by active Ras and in modulating epithelial cell adhesion and migration. PHB directly interacts with C-Raf and is required for the displacement of 14-3-3 from C-Raf by active Ras to facilitate plasma membrane localization and activation. Further, the adherent complex proteins cadherin and beta-catenin were localized to plasma membrane suggesting stabilized adherent junctions in PHB silenced cells. Our findings demonstrated a function of PHB in the control of a central signaling pathway involved in cell growth and malignant transformation.

Animals↗

PubMed2005

Prohibitin is required for Ras-induced Raf-MEK-ERK activation and epithelial cell migration.

Ras proteins control the signalling pathways that are responsible for normal growth and malignant transformation. Raf protein kinases are direct Ras effector proteins that initiate the mitogen-activated protein kinase (MAPK) cascade, which mediates diverse biological functions such as cell growth, survival and differentiation. Here we show that prohibitin, a ubiquitously expressed and evolutionarily conserved protein is indispensable for the activation of the Raf-MEK-ERK pathway by Ras. The membrane targeting and activation of C-Raf by Ras needs prohibitin in vivo. In addition, direct interaction with prohibitin is required for C-Raf activation. C-Raf kinase fails to interact with the active Ras induced by epidermal growth factor in the absence of prohibitin. Moreover, in prohibitin-deficient cells the adhesion complex proteins cadherin and beta-catenin relocalize to the plasma membrane and thereby stabilize adherens junctions. Our data show an unexpected role of prohibitin in the activation of the Ras-Raf signalling pathway and in modulating epithelial cell adhesion and migration.

14-3-3 Proteins↗

PubMed2005

Shotgun proteome analysis of protein cleavage in apoptotic cells.

A new shotgun proteomics approach was employed to identify degraded proteins. Jurkat T-cells were induced to undergo apoptosis by Fas (CD95/Apo-1) stimulation. The proteins were separated by large (30 cm) sodium dodecyl sulphate-polyacrylamide gel electrophoresis and identified by liquid chromatography-tandem mass spectrometry after digestion of 100 gel slices with trypsin. The molecular masses of the individual gel slices were calculated through the known theoretical masses of the identified proteins. Proteins were defined as degradation candidates if either the empirical determined molecular mass was at most 80% of the theoretical value, or if proteins were identified in clearly different gel slices. In this manner, the degradation of 11 already identified apoptosis-modified proteins was confirmed and nine until now unknown degradation candidate proteins identified. Degradation during apoptosis must be verified by additional techniques such as in vitro caspase assays as shown for nucleolin and Rho GDI 2. The results presented confirm the suitability of a shotgun approach for the identification of putative protease targets.

Apoptosis↗

PubMed2005

Cloning and biochemical characterization of APIT, a new l-amino acid oxidase from Aplysia punctata.

The purple ink of the sea hare Aplysia punctata contains a 60 kDa protein with tumoricidal activity. This A. punctata ink toxin (APIT) kills tumor cells within 6--8h in an apoptosis independent manner by the production of high amounts of hydrogen peroxide which induce a necrotic form of oxidative stress. Here, we describe the biochemical features of APIT associated with its anti-tumor activity. APIT is a weakly glycosylated FAD-binding L-amino acid oxidase that catalyzes the oxidative deamination of L-lysine and L-arginine and thereby produces hydrogen peroxide (H(2)O(2)), ammonia (NH(4)(+)) and the corresponding alpha-keto acids. The tumoricidal effect is completely abrogated in the absence of the amino acids L-lysine and L-arginine. The enzyme is stable at temperatures from 0 to 50 degrees C. Similar to other FAD-binding enzymes, it is resistant against tryptic digest. Even digest with proteinase K fails to degrade the enzyme. Cloning of the APIT gene and subsequent sequencing revealed a FAD-binding domain followed by a so-called GG-motif, which is typical for L-amino acid oxidases. Strongest homology exists to escapin, aplysianin A precursor, the cyplasins L and S and achacin.

Amino Acid Sequence↗

PubMed2005

Inhibition of Marburg virus protein expression and viral release by RNA interference.

High mortality rates and lack of an available vaccine against Marburg haemorrhagic fever (MHF) highlight the need for a defensive therapy against MHF and greater knowledge of the causative agent, the Marburg virus (MARV). Here, RNA interference (RNAi) is employed to destroy MARV transcripts, disrupting replication and allowing analysis of various roles of MARV proteins. Small interfering RNAs (siRNAs) homologous to three MARV transcripts (NP, VP35 and VP30) were co-transfected into cells with plasmids encoding the corresponding nucleocapsid proteins. The resulting decrease in MARV nucleocapsid-protein levels was shown to be specific, as siRNA that was not homologous to the MARV genome did not decrease the levels of viral nucleocapsid proteins. Additionally, transcript levels of double-stranded RNA (dsRNA)-sensor proteins, the dsRNA-activated protein kinase and 2',5'-oligoadenylate synthetase 1 remained unchanged, suggesting that the decrease in viral proteins was not a result of activation of the antiviral properties of the interferon system. Subsequently, siRNAs were shown to reduce intracellular viral proteins in MARV-infected cells and viral material released into the medium. Targeted reduction of VP30 downregulated the intracellular levels of all other viral proteins, suggesting that VP30 plays an essential role for transcription/replication. The efficient reduction of MARV replication also suggests that RNAi may provide an agent against MHF.

Animals↗

PubMed2004

A global approach combining proteome analysis and phenotypic screening with RNA interference yields novel apoptosis regulators.

Global approaches like proteome or transcriptome analyses have been performed extensively to identify candidate genes or proteins involved in biological and pathological processes. Here we describe the identification of proteins implicated in the regulation of apoptosis using proteome analysis and the functional validation of targets by RNA interference. A high-throughput platform for the validation of synthetic small interfering RNAs (siRNAs) by quantitative real-time PCR was established. Genes of the identified factors were silenced by automated siRNA transfection, and their role in apoptotic signaling was investigated. Using this strategy, nine new modulators of apoptosis were identified. A subsequent detailed study demonstrated that hepatoma-derived growth factor (HDGF) is required for TNFalpha-induced release of pro-apoptotic factors from mitochondria. The strategy described here may be used for hypothesis-free, global gene function analysis.

Apoptosis↗

PubMed2004

Proteomic and gene profiling approaches to study host responses to bacterial infection.

Infectious disease is the result of an intimate relationship between the pathogen and host, which involves cross-talk. After an initial flood of mainly descriptive reports on the influence of acute bacterial infection on cells, transcriptome and proteome studies are now becoming more refined in their approach, and are shedding light on the role of pathogen-specific mechanisms/structures in pathogenesis. In addition, studies of gene expression in vivo have shed new light on how the host influences the niche occupied by bacteria. Elegant refinements to proteomics using beads coated with bacterial invasins, or purifying subcellular fractions are producing a picture of invasion specific processes. Such approaches combined with modern functional genomics technologies such as RNAi represent the next phase in understanding host-bacteria interactions.

Animals↗

PubMed2002

VDAC and the bacterial porin PorB of Neisseria gonorrhoeae share mitochondrial import pathways.

The human pathogen Neisseria gonorrhoeae induces host cell apoptosis during infection by delivering the outer membrane protein PorB to the host cell's mitochondria. PorB is a pore-forming beta-barrel protein sharing several features with the mitochondrial voltage-dependent anion channel (VDAC), which is involved in the regulation of apoptosis. Here we show that PorB of pathogenic Neisseria species produced by host cells is efficiently targeted to mitochondria. Imported PorB resides in the mitochondrial outer membrane and forms multimers with similar sizes as in the outer bacterial membrane. The mitochondria completely lose their membrane potential, a characteristic previously observed in cells infected with gonococci or treated with purified PorB. Closely related bacterial porins of non-pathogenic Neisseria mucosa or Escherichia coli remain in the cytosol. Import of PorB into mitochondria in vivo is independent of a linear signal sequence. Insertion of PorB into the mitochondrial outer membrane in vitro depends on the activity of Tom5, Tom20 and Tom40, but is independent of Tom70. Our data show that human VDAC and bacterial PorB are imported into mitochondria by a similar mechanism.

Bacterial Outer Membrane Proteins↗

PubMed2002

Prediction of translocation and cleavage of heterogeneous ribonuclear proteins and Rho guanine nucleotide dissociation inhibitor 2 during apoptosis by subcellular proteome analysis.

Jurkat T cells induced to undergo apoptosis by the CD95(Fas/Apo-1) pathway were investigated by proteome analysis. The most prominent differing protein spots of apoptotic and nonapoptotic cells were identified as various heterogeneous ribonuclear proteins (hnRNPs) and Rho guanin nucleotide dissociation inhibitor (GDI) 2. In apoptotic cells, four spots slightly differing in molecular mass and/or isoelectric point were identified as Rho GDI 2 with the mass and pI as expected after caspase-3 cleavage near the N-terminus. Subcellular proteome analysis revealed that Rho GDI 2 was highly enriched in the cytosolic fraction, present in minor amounts in the nuclear fraction and absent from the mitochondrial fraction. In apoptotic cells however, the spots representing processed and modified Rho GDI 2 were found in the cytosol, in the nucleus and also the mitochondria at different spot positions. In addition, twelve different hnRNPs were identified to be altered after induction of cell death of which hnRNPs A/B, D, F, H, I and L were hitherto unknown to be modified during apoptosis. Most of the hnRNP spots were found in the nucleus of nonapoptotic cells, whereas these proteins, either modified or unmodified, relocated to the cytosol and/or the mitochondria in apoptotic cells. Our results demonstrate that modification of proteins during apoptosis is often accompanied by their relocalisation between cellular compartments.

Active Transport, Cell Nucleus↗

PubMedDate not supplied

Proteome analysis of apoptotic cells.

Apoptosis, a genetically determined form of cell death, is a central and complex process involved in the development of multicellular organisms in the maintenance of cell homeostasis. During apoptosis, a large number of proteins involved in transducing signals are posttranslationally modified. Classical proteomics, the combination of protein separation by two-dimensional gel electrophoresis (2DGE) and protein identification by mass spectrometry (MS), enabled the discovery of more than 100 proteins altered during apoptosis. Functional data about protein degradation, modification, translocation, and synthesis were obtained. In addition to classical proteomics, some specifically designed proteome studies were carried out to analyze specific apoptotic components such as the mitochondrial releasing factors, death-inducing signaling complex (DISC), inhibitor of apoptosis (IAP) interacting proteins, and caspases. The identification of main regulators significantly influenced the elucidation of the concept underlying apoptosis signaling. Thus, the application of detailed protein analytical methods in the young field of apoptosis research was particularly fruitful.

Animals↗