Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 43
Filter
1.
FEBS Lett ; 2024 Aug 07.
Article in English | MEDLINE | ID: mdl-39160442

ABSTRACT

The human Atg8 family member GABARAP is involved in numerous autophagy-related and -unrelated processes. We recently observed that specifically the deficiency of GABARAP enhances epidermal growth factor receptor (EGFR) degradation upon ligand stimulation. Here, we report on two putative LC3-interacting regions (LIRs) within EGFR, the first of which (LIR1) is selected as a GABARAP binding site in silico. Indeed, in vitro interaction studies reveal preferential binding of LIR1 to GABARAP and GABARAPL1. Our X-ray data demonstrate interaction of core LIR1 residues FLPV with both hydrophobic pockets of GABARAP suggesting canonical binding. Although LIR1 occupies the LIR docking site, GABARAP Y49 and L50 appear dispensable in this case. Our data support the hypothesis that GABARAP affects the fate of EGFR at least in part through direct binding.

2.
Article in English | MEDLINE | ID: mdl-36768039

ABSTRACT

Objectives: Work-related mental distress is one of the most dominant reasons for sick leave and early retirement. Specialized therapy programs for work-related mental health problems are rare, especially in a group setting. This study evaluates the severity of depression, anxiety, somatization and burnout symptoms before and after a work-related group therapy program. Methods: Patients of a psychosomatic outpatient clinic with work-related mental disorders completed 12 sessions of a manual-based group training with reference to the workplace. Data were collected using the Patient Health Questionnaire-9 (PHQ-9), Patient Health Questionnaire-15 (PHQ-15), General Anxiety Disorder Scale-7 (GAD-7) and the Maslach Burnout Inventory (MBI) before (T1) and directly after the intervention (T2). Results: Overall, 48 participants completed the intervention. The participants' symptoms of depression (T1: M = 11.06, SD = 6.19, T2: M = 8.92, SD = 8.17; p < 0.001, d = 0.53) and anxiety (T1: M = 9.94, SD = 5.18, T2: M = 7.13, SD = 5.69; p = 0.001, d = 0.49) as well as their emotional exhaustion (T1: M = 4.63, SD = 0.95, T2: M = 4.05, SD = 1.35; p < 0.001, d = 0.55) decreased significantly, and the difference was clinically relevant at T2. For cynicism (T1: M = 3.93, SD = 0.99, T2: M = 3.70, SD = 1.32; p = 0.14, d = 0.22) and personal fulfillment at work (T1: M = 4.30, SD = 0.83, T2: M = 4.41, SD = 0.94; p = 0.24, d = 0.17), the difference between T1 and T2 was not significant. Women benefited more than men (PHQ-9: p < 0.001, d = 0.96; GAD-7: p < 0.001, d = 0.91; PHQ-15: p < 0.001, d = 0.76) from the training. Conclusions: Participants' mental health symptoms were substantially reduced during the course of the work-related group therapy. As mental health problems account for the largest group of work disability days, the potential of group therapy should be better exploited in health care services.


Subject(s)
Burnout, Professional , Psychotherapy, Group , Male , Humans , Female , Anxiety/therapy , Anxiety Disorders/epidemiology , Anxiety Disorders/therapy , Mental Health , Workplace/psychology , Depression/therapy , Depression/psychology
3.
J Vet Pharmacol Ther ; 45(5): 467-472, 2022 Sep.
Article in English | MEDLINE | ID: mdl-35652132

ABSTRACT

Grapiprant is a prostaglandin E2 receptor antagonist that has been found to be an effective anti-inflammatory in dogs and that is devoid of some of the adverse effects associated with traditional NSAIDs that elicit their effects through inhibition of PGE2 production. Previously published reports have described the pharmacokinetics of this drug in horses when administered at 2 mg/kg; however, pharmacodynamic effects in this species have yet to be described. The objective of the current study was to describe the pharmacokinetics and pharmacodynamics of grapiprant at a higher dose. Eight horses received a single oral administration of 15 mg/kg. Plasma concentrations were determined for 96 h using liquid chromatography-tandem mass spectrometry. Non-compartmental analysis was used to determine pharmacokinetic parameters. Pharmacodynamic effects were assessed ex vivo by stimulating blood samples with PGE2 and determining TNF-ɑ concentrations. Maximum concentration, time to maximum concentration and area under the curve were 327.5 (188.4-663.0) ng/ml, 1 (0.75-2.0) hour and 831.8 (512.6-1421.6) h*ng/ml, respectively. The terminal half-life was 11.1 (8.27-21.2) hr. Significant stimulation of TNF alpha was noted for 2-4 h post-drug administration. Results of this study suggest a short duration of EP4 receptor engagement when administered at a dose of 15 mg/kg.


Subject(s)
Horses , Sulfonylurea Compounds , Tumor Necrosis Factor-alpha , Administration, Oral , Animals , Area Under Curve , Half-Life , Horses/blood , Imidazoles , Prostaglandins E , Pyridines , Sulfonylurea Compounds/pharmacokinetics
4.
Int J Mol Sci ; 22(1)2020 Dec 23.
Article in English | MEDLINE | ID: mdl-33374830

ABSTRACT

GABARAP (γ-aminobutyric acid type A receptor-associated protein) and its paralogues GABARAPL1 and GABARAPL2 comprise a subfamily of autophagy-related Atg8 proteins. They are studied extensively regarding their roles during autophagy. Originally, however, especially GABARAPL2 was discovered to be involved in intra-Golgi transport and homotypic fusion of post-mitotic Golgi fragments. Recently, a broader function of mammalian Atg8s on membrane trafficking through interaction with various soluble N-ethylmaleimide-sensitive factor-attachment protein receptors SNAREs was suggested. By immunostaining and microscopic analysis of the Golgi network, we demonstrate the importance of the presence of individual GABARAP-type proteins on Golgi morphology. Furthermore, triple knockout (TKO) cells lacking the whole GABARAP subfamily showed impaired Golgi-dependent vesicular trafficking as assessed by imaging of fluorescently labelled ceramide. With the Golgi apparatus being central within the secretory pathway, we sought to investigate the role of the GABARAP-type proteins for cell surface protein trafficking. By analysing the surfaceome compositionofTKOs, we identified a subset of cell surface proteins with altered plasma membrane localisation. Taken together, we provide novel insights into an underrated aspect of autophagy-independent functions of the GABARAP subfamily and recommend considering the potential impact of GABARAP subfamily proteins on a plethora of processes during experimental analysis of GABARAP-deficient cells not only in the autophagic context.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Apoptosis Regulatory Proteins/metabolism , Autophagy-Related Protein 8 Family/metabolism , Golgi Apparatus/metabolism , Microtubule-Associated Proteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Apoptosis Regulatory Proteins/genetics , Autophagy-Related Protein 8 Family/genetics , Cell Membrane/metabolism , Ceramides/metabolism , Golgi Apparatus/ultrastructure , HEK293 Cells , Humans , Microtubule-Associated Proteins/genetics , Protein Transport
5.
Cells ; 9(6)2020 06 16.
Article in English | MEDLINE | ID: mdl-32560054

ABSTRACT

The autophagy-related ATG8 protein GABARAP has not only been shown to be involved in the cellular self-degradation process called autophagy but also fulfils functions in intracellular trafficking processes such as receptor transport to the plasma membrane. Notably, available mass spectrometry data suggest that GABARAP is also secreted into extracellular vesicles (EVs). Here, we confirm this finding by the immunoblotting of EVs isolated from cell culture supernatants and human blood serum using specific anti-GABARAP antibodies. To investigate the mechanism by which GABARAP is secreted, we applied proximity labelling, a method for studying the direct environment of a protein of interest in a confined cellular compartment. By expressing an engineered peroxidase (APEX2)-tagged variant of GABARAP-which, like endogenous GABARAP, was present in EVs prepared from HEK293 cells-we demonstrate the applicability of APEX2-based proximity labelling to EVs. The biotinylated protein pool which contains the APEX2-GABARAP co-secretome contained not only known GABARAP interaction partners but also proteins that were found in APEX2-GABARAP's proximity inside of autophagosomes in an independent study. All in all, we not only introduce a versatile tool for co-secretome analysis in general but also uncover the first details about autophagy-based pathways as possible biogenesis mechanisms of GABARAP-containing EVs.


Subject(s)
Autophagy-Related Proteins/metabolism , Autophagy/physiology , Extracellular Vesicles/metabolism , Microtubule-Associated Proteins/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Apoptosis Regulatory Proteins/metabolism , Autophagosomes/metabolism , Cell Membrane/metabolism , HEK293 Cells , Humans , Protein Transport/physiology
6.
Cells ; 9(5)2020 05 22.
Article in English | MEDLINE | ID: mdl-32456010

ABSTRACT

The γ-aminobutyric acid type A receptor-associated protein (GABARAP) and its close paralogs GABARAPL1 and GABARAPL2 constitute a subfamily of the autophagy-related 8 (Atg8) protein family. Being associated with a variety of dynamic membranous structures of autophagic and non-autophagic origin, Atg8 proteins functionalize membranes by either serving as docking sites for other proteins or by acting as membrane tethers or adhesion factors. In this study, we describe that deficiency for GABARAP alone, but not for its close paralogs, is sufficient for accelerated EGF receptor (EGFR) degradation in response to EGF, which is accompanied by the downregulation of EGFR-mediated MAPK signaling, altered target gene expression, EGF uptake, and EGF vesicle composition over time. We further show that GABARAP and EGFR converge in the same distinct compartments at endogenous GABARAP expression levels in response to EGF stimulation. Furthermore, GABARAP associates with EGFR in living cells and binds to synthetic peptides that are derived from the EGFR cytoplasmic tail in vitro. Thus, our data strongly indicate a unique and novel role for GABARAP during EGFR trafficking.


Subject(s)
Apoptosis Regulatory Proteins/deficiency , Epidermal Growth Factor/metabolism , Microtubule-Associated Proteins/deficiency , Proteolysis , Sequence Homology, Amino Acid , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Sequence , Apoptosis Regulatory Proteins/metabolism , Autophagy-Related Protein 8 Family/metabolism , Cell Line, Tumor , Endocytosis/drug effects , Endosomes/drug effects , Endosomes/metabolism , ErbB Receptors/chemistry , ErbB Receptors/metabolism , Fluorescent Dyes/metabolism , Gene Expression Regulation/drug effects , HEK293 Cells , Humans , Lysosomes/drug effects , Lysosomes/metabolism , Microtubule-Associated Proteins/metabolism , Models, Biological , Phosphorylation/drug effects , Proteasome Inhibitors/pharmacology , Proteolysis/drug effects , Signal Transduction/drug effects , Transport Vesicles/drug effects , Transport Vesicles/metabolism
7.
Sci Rep ; 9(1): 19221, 2019 Dec 11.
Article in English | MEDLINE | ID: mdl-31822784

ABSTRACT

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

8.
Molecules ; 24(9)2019 May 13.
Article in English | MEDLINE | ID: mdl-31086037

ABSTRACT

Subcellular structures containing autophagy-related proteins of the Atg8 protein family have been investigated with conventional wide-field fluorescence and single molecule localisation microscopy. Fusion proteins of GABARAP and LC3B, respectively, with EYFP were overexpressed in HEK293 cells. While size distributions of structures labelled by the two proteins were found to be similar, shape distributions appeared quite disparate, with EYFP-GABARAP favouring circular structures and elliptical structures being dominant for EYFP-LC3B. The latter also featured a nearly doubled fraction of U-shape structures. The experimental results point towards highly differential localisation of the two proteins, which appear to label structures representing distinct stages or even specific channels of vesicular trafficking pathways. Our data also demonstrate that the application of super-resolution techniques expands the possibilities of fluorescence-based methods in autophagy studies and in some cases can rectify conclusions obtained from conventional fluorescence microscopy with diffraction-limited resolution.


Subject(s)
Adaptor Proteins, Signal Transducing/analysis , Microscopy/methods , Microtubule-Associated Proteins/analysis , Apoptosis Regulatory Proteins , HEK293 Cells , Humans
9.
J Biol Chem ; 294(18): 7388-7402, 2019 05 03.
Article in English | MEDLINE | ID: mdl-30862675

ABSTRACT

The hepatitis C virus (HCV) nonstructural protein 5A (NS5A) plays a key role in viral replication and virion assembly, and the regulation of the assembly process critically depends on phosphorylation of both serine and threonine residues in NS5A. We previously identified SRC proto-oncogene, nonreceptor tyrosine kinase (c-Src), as an essential host component of the HCV replication complex consisting of NS5A, the RNA-dependent RNA polymerase NS5B, and c-Src. Pulldown assays revealed an interaction between NS5A and the Src homology 2 (SH2) domain of c-Src; however, the precise binding mode remains undefined. In this study, using a variety of biochemical and biophysical techniques, along with molecular dynamics simulations, we demonstrate that the interaction between NS5A and the c-Src SH2 domain strictly depends on an intact phosphotyrosine-binding competent SH2 domain and on tyrosine phosphorylation within NS5A. Detailed analysis of c-Src SH2 domain binding to a panel of phosphorylation-deficient NS5A variants revealed that phosphorylation of Tyr-93 located within domain 1 of NS5A, but not of any other tyrosine residue, is crucial for complex formation. In line with these findings, effective replication of subgenomic HCV replicons as well as production of infectious virus particles in mammalian cell culture models were clearly dependent on the presence of tyrosine at position 93 of NS5A. These findings indicate that phosphorylated Tyr-93 in NS5A plays an important role during viral replication by facilitating NS5A's interaction with the SH2 domain of c-Src.


Subject(s)
Hepacivirus/physiology , Tyrosine/metabolism , Viral Nonstructural Proteins/metabolism , Virus Replication , src-Family Kinases/metabolism , Cell Line, Tumor , Humans , Phosphorylation , Protein Binding , Proto-Oncogene Mas , Viral Nonstructural Proteins/chemistry , src Homology Domains
10.
Sci Rep ; 9(1): 526, 2019 01 24.
Article in English | MEDLINE | ID: mdl-30679523

ABSTRACT

The determination of unique functions of GABARAP (gamma-aminobutyric acid type A receptor-associated protein), a member of the highly conserved protein family of mammalian autophagy-related 8 protein (mATG8), within diverse cellular processes remains challenging. Because available anti-GABARAP antibodies perform inadequate, especially within various microscopy-based applications, we aimed to develop an antibody that targets GABARAP but not its close orthologs. Following the latest recommendations for antibody validation including fluorescence protein tagging, genetic and orthogonal strategies, we characterized the resulting anti-GABARAP (8H5) antibody during confocal immunofluorescence imaging in-depth. We compared the antibody staining pattern with that obtained for fluorescence protein tagged GABARAP, GABARAPL1 or GABARAPL2 each ectopically expressed in GABARAP knockout cells. Furthermore, we imaged cells expressing all mATG8 family members at endogenous levels and checked GABARAP knockout cells for unspecific staining under fed or macroautophagy-inducing conditions. Finally, we simultaneously stained cells for endogenous GABARAP and the common autophagosomal marker LC3B. Summarized, the presented antibody shows high specificity for GABARAP without cross-reactivity to other mATG8 family members in immunofluorescence imaging making it a valuable tool for the identification of unique GABARAP functions.


Subject(s)
Antibodies, Monoclonal/analysis , Apoptosis Regulatory Proteins/analysis , Fluorescent Antibody Technique/methods , Microtubule-Associated Proteins/analysis , Amino Acid Sequence , Animals , Cell Line , Humans , Optical Imaging/methods , Rats
11.
Sci Rep ; 7(1): 5979, 2017 07 20.
Article in English | MEDLINE | ID: mdl-28729737

ABSTRACT

HIV-1 Nef is an important pathogenic factor for HIV/AIDS pathogenesis. Studies have shown that the association of Nef with the inner leaflet of the plasma membrane and with endocytic and perinuclear vesicles is essential for most activities of Nef. Using purified recombinant proteins in pull-down assays and by co-immunoprecipitation assays we demonstrate that Nef binds directly and specifically to all GABARAP family members, but not to LC3 family members. Based on nuclear magnetic resonance (NMR) experiments we showed that Nef binds to GABARAP via two surface exposed hydrophobic pockets. S53 and F62 of GABARAP were identified as key residues for the interaction with Nef. During live-cell fluorescence microscopy an accumulation of Nef and all GABARAP family members in vesicular structures throughout the cytoplasm and at the plasma membrane was observed. This plasma membrane accumulation was significantly reduced after knocking down GABARAP, GABARAPL1 and GABARAPL2 with respective siRNAs. We identified GABARAPs as the first known direct interaction partners of Nef that are essential for its plasma membrane localization.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Autophagy-Related Protein 8 Family/metabolism , Cell Membrane/metabolism , HIV-1/metabolism , Microtubule-Associated Proteins/metabolism , nef Gene Products, Human Immunodeficiency Virus/metabolism , Adaptor Proteins, Signal Transducing/chemistry , Amino Acid Sequence , Apoptosis Regulatory Proteins , Autophagy-Related Protein 8 Family/chemistry , Binding Sites , Cell Extracts , Conserved Sequence , HEK293 Cells , Humans , Hydrophobic and Hydrophilic Interactions , Ligands , Microtubule-Associated Proteins/chemistry , Protein Binding , Protein Transport , RNA, Small Interfering/metabolism
12.
Viruses ; 7(7): 4119-30, 2015 Jul 21.
Article in English | MEDLINE | ID: mdl-26197333

ABSTRACT

Dengue virus (DENV) is an important human pathogen causing millions of disease cases and thousands of deaths worldwide. Non-structural protein 4A (NS4A) is a vital component of the viral replication complex (RC) and plays a major role in the formation of host cell membrane-derived structures that provide a scaffold for replication. The N-terminal cytoplasmic region of NS4A(1-48) is known to preferentially interact with highly curved membranes. Here, we provide experimental evidence for the stable binding of NS4A(1-48) to small liposomes using a liposome floatation assay and identify the lipid binding sequence by NMR spectroscopy. Mutations L6E;M10E were previously shown to inhibit DENV replication and to interfere with the binding of NS4A(1-48) to small liposomes. Our results provide new details on the interaction of the N-terminal region of NS4A with membranes and will prompt studies of the functional relevance of the curvature sensitive membrane anchor at the N-terminus of NS4A.


Subject(s)
Dengue Virus/metabolism , Dengue/virology , Liposomes/metabolism , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism , Cell Membrane/metabolism , Cell Membrane/virology , Dengue/metabolism , Dengue Virus/chemistry , Dengue Virus/genetics , Humans , Protein Structure, Tertiary , Viral Nonstructural Proteins/genetics
13.
Biochim Biophys Acta ; 1848(5): 1119-26, 2015 May.
Article in English | MEDLINE | ID: mdl-25644870

ABSTRACT

Dengue virus (DENV) infection is a growing public health threat with more than one-third of the world's population at risk. Non-structural protein 4A (NS4A), one of the least characterized viral proteins, is a highly hydrophobic transmembrane protein thought to induce the membrane alterations that harbor the viral replication complex. The NS4A N-terminal (amino acids 1-48), has been proposed to contain an amphipathic α-helix (AH). Mutations (L6E; M10E) designed to reduce the amphipathic character of the predicted AH, abolished viral replication and reduced NS4A oligomerization. Nuclear magnetic resonance (NMR) spectroscopy was used to characterize the N-terminal cytoplasmic region (amino acids 1-48) of both wild type and mutant NS4A in the presence of SDS micelles. Binding of the two N-terminal NS4A peptides to liposomes was studied as a function of membrane curvature and lipid composition. The NS4A N-terminal was found to contain two AHs separated by a non-helical linker. The above mentioned mutations did not significantly affect the helical secondary structure of this domain. However, they reduced the affinity of the N-terminal NS4A domain for lipid membranes. Binding of wild type NS4A(1-48) to liposomes is highly dependent on membrane curvature.


Subject(s)
Dengue Virus/metabolism , Membrane Lipids/metabolism , Viral Nonstructural Proteins/metabolism , Circular Dichroism , Dengue Virus/growth & development , Liposomes , Membrane Lipids/chemistry , Micelles , Mutation , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Multimerization , Protein Structure, Secondary , Protein Structure, Tertiary , Structure-Activity Relationship , Surface Plasmon Resonance , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Virus Replication
14.
J Pept Sci ; 20(5): 334-40, 2014 May.
Article in English | MEDLINE | ID: mdl-24616074

ABSTRACT

We studied the interaction of the SH3 domain of Bin1 with a 15-mer peptide of HCV NS5A and show its potency to competitively displace a 15-mer human c-Myc fragment, which is a physiological ligand of Bin1, using NMR spectroscopy. Fluorescence spectroscopy and ITC were employed to determine the affinity of Bin1 SH3 to NS5A(347-361), yielding a submicromolar affinity to NS5A. Our study compares the binding dynamics and affinities of the relevant regions for binding of c-Myc and NS5A to Bin1 SH3. The result gives further insights into the potential role of NS5A in Bin1-mediated apoptosis.


Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Tumor Suppressor Proteins/chemistry , Tumor Suppressor Proteins/metabolism , Viral Nonstructural Proteins/metabolism , src Homology Domains , Apoptosis , Binding, Competitive , Calorimetry , Magnetic Resonance Spectroscopy , Models, Molecular , Proto-Oncogene Proteins c-myc/chemistry , Spectrometry, Fluorescence , Viral Nonstructural Proteins/chemistry
15.
PLoS One ; 9(1): e86482, 2014.
Article in English | MEDLINE | ID: mdl-24466115

ABSTRACT

BACKGROUND: Dengue virus (DENV) is a mosquito-transmitted positive single strand RNA virus belonging to the Flaviviridae family. DENV causes dengue fever, currently the world's fastest-spreading tropical disease. Severe forms of the disease like dengue hemorrhagic fever and dengue shock syndrome are life-threatening. There is no specific treatment and no anti-DENV vaccines. Our recent data suggests that the amino terminal cytoplasmic region of the dengue virus non-structural protein 4A (NS4A) comprising amino acid residues 1 to 48 forms an amphipathic helix in the presence of membranes. Its amphipathic character was shown to be essential for viral replication. NMR-based structure-function analysis of the NS4A amino terminal region depends on its milligram-scale production and labeling with NMR active isotopes. METHODOLOGY/PRINCIPAL FINDINGS: This report describes the optimization of a uniform procedure for the expression and purification of the wild type NS4A(1-48) peptide and a peptide derived from a replication-deficient mutant NS4A(1-48; L6E, M10E) with disrupted amphipathic nature. A codon-optimized, synthetic gene for NS4A(1-48) was expressed as a fusion with a GST-GB1 dual tag in E. coli. Tobacco etch virus (TEV) protease mediated cleavage generated NS4A(1-48) peptides without any artificial overhang. Using the described protocol up to 4 milligrams of the wild type or up to 5 milligrams of the mutant peptide were obtained from a one-liter culture. Isotopic labeling of the peptides was achieved and initial NMR spectra were recorded. CONCLUSIONS/SIGNIFICANCE: Small molecules targeting amphipathic helices in the related Hepatitis C virus were shown to inhibit viral replication, representing a new class of antiviral drugs. These findings highlight the need for an efficient procedure that provides large quantities of the amphipathic helix containing NS4A peptides. The double tag strategy presented in this manuscript answers these needs yielding amounts that are sufficient for comprehensive biophysical and structural studies, which might reveal new drug targets.


Subject(s)
Dengue Virus/physiology , Mutation/genetics , Peptide Fragments/metabolism , Recombinant Fusion Proteins/metabolism , Viral Nonstructural Proteins/metabolism , Virus Replication , Amino Acid Sequence , Antiviral Agents/pharmacology , Base Sequence , Blotting, Western , Dengue/prevention & control , Dengue/virology , Endopeptidases/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Glutathione Transferase/genetics , Humans , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Mutant Proteins/genetics , Mutant Proteins/isolation & purification , Mutant Proteins/metabolism , Peptide Fragments/genetics , Plasmids/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Sequence Homology, Nucleic Acid , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Ubiquitin/genetics , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics
16.
J Biol Chem ; 288(52): 37204-15, 2013 Dec 27.
Article in English | MEDLINE | ID: mdl-24240096

ABSTRACT

Apoptosis and autophagy are fundamental homeostatic processes in eukaryotic organisms fulfilling essential roles in development and adaptation. Recently, the anti-apoptotic factor Bcl-2 has been reported to also inhibit autophagy, thus establishing a potential link between these pathways, but the mechanistic details are only beginning to emerge. Here we show that Bcl-2 directly binds to the phagophore-associated protein GABARAP. NMR experiments revealed that the interaction critically depends on a three-residue segment (EWD) of Bcl-2 adjacent to the BH4 region, which is anchored to one of the two hydrophobic pockets on the GABARAP molecule. This is at variance with the majority of GABARAP interaction partners identified previously, which occupy both hydrophobic pockets simultaneously. Bcl-2 affinity could also be detected for GEC1, but not for other mammalian Atg8 homologs. Finally, we provide evidence that overexpression of Bcl-2 inhibits lipidation of GABARAP, a key step in autophagosome formation, possibly via competition with the lipid conjugation machinery. These results support the regulatory role of Bcl-2 in autophagy and define GABARAP as a novel interaction partner involved in this intricate connection.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Autophagy/physiology , Cytoskeletal Proteins/metabolism , Lipoylation/physiology , Membrane Proteins/metabolism , Microtubule-Associated Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/genetics , Amino Acid Motifs , Animals , Apoptosis/physiology , Apoptosis Regulatory Proteins , Cell Line, Transformed , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/genetics , Humans , Hydrophobic and Hydrophilic Interactions , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mice , Microtubule-Associated Proteins/chemistry , Microtubule-Associated Proteins/genetics , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Structure, Quaternary , Protein Structure, Tertiary , Proto-Oncogene Proteins c-bcl-2/chemistry , Proto-Oncogene Proteins c-bcl-2/genetics , Rats
17.
Biochemistry ; 52(36): 6160-8, 2013 Sep 10.
Article in English | MEDLINE | ID: mdl-23947833

ABSTRACT

Src homology 3 (SH3) domains are widely known for their ability to interact with other proteins using the canonical PxxP binding motif. Besides those well-characterized interaction modes, there is an increasing number of SH3 domain-containing complexes that lack this motif. Here we characterize the interaction of SH3 domains, in particular the Bin1-SH3 domain, with the intrinsically disordered part of nonstructural protein 5A of the hepatitis C virus using noncanonical binding sites in addition to its PxxP motif. These binding regions partially overlap with regions that have previously been identified as having an increased propensity to form α-helices. Remarkably, upon interaction with the Bin1-SH3 domain, the α-helical propensity decreases and a fuzzy complex is formed.


Subject(s)
Viral Nonstructural Proteins/chemistry , src Homology Domains/physiology , Adaptor Proteins, Signal Transducing/metabolism , Binding Sites , Hepacivirus/metabolism , Models, Molecular , Viral Nonstructural Proteins/metabolism
18.
Lab Chip ; 13(18): 3588-98, 2013 Sep 21.
Article in English | MEDLINE | ID: mdl-23743770

ABSTRACT

A chip-based system mimicking the transport function of the human cardiovascular system has been established at minute but standardized microsystem scale. A peristaltic on-chip micropump generates pulsatile shear stress in a widely adjustable physiological range within a microchannel circuit entirely covered on all fluid contact surfaces with human dermal microvascular endothelial cells. This microvascular transport system can be reproducibly established within four days, independently of the individual endothelial cell donor background. It interconnects two standard tissue culture compartments, each of 5 mm diameter, through microfluidic channels of 500 µm width. Further vessel branching and vessel diameter reduction down to a microvessel scale of approximately 40 µm width was realised by a two-photon laser ablation technique applied to inserts, designed for the convenient establishment of individual organ equivalents in the tissue culture compartments at a later time. The chip layout ensures physiological fluid-to-tissue ratios. Moreover, an in-depth microscopic analysis revealed the fine-tuned adjustment of endothelial cell behaviour to local shear stresses along the microvasculature of the system. Time-lapse and 3D imaging two-photon microscopy were used to visualise details of spatiotemporal adherence of the endothelial cells to the channel system and to each other. The first indicative long-term experiments revealed stable performance over two and four weeks. The potential application of this system for the future establishment of human-on-a-chip systems and basic human endothelial cell research is discussed.


Subject(s)
Cell Culture Techniques/instrumentation , Microfluidic Analytical Techniques/instrumentation , Antigens, CD/metabolism , Cadherins/metabolism , Cells, Cultured , Endothelial Cells/cytology , Endothelial Cells/metabolism , Humans , Hydrodynamics , Microvessels/pathology , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Shear Strength , von Willebrand Factor/metabolism
19.
J Virol ; 87(7): 4080-5, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23325687

ABSTRACT

Dengue virus (DENV) causes dengue fever, a major health concern worldwide. We identified an amphipathic helix (AH) in the N-terminal region of the viral nonstructural protein 4A (NS4A). Disruption of its amphipathic nature using mutagenesis reduced homo-oligomerization and abolished viral replication. These data emphasize the significance of NS4A in the life cycle of the dengue virus and demarcate it as a target for the design of novel antiviral therapy.


Subject(s)
Dengue Virus/genetics , Viral Nonstructural Proteins/chemistry , Virus Replication/genetics , Amino Acid Sequence , Blotting, Western , Gene Components , Microscopy, Confocal , Molecular Sequence Data , Mutagenesis , Polymerization , Sequence Alignment , Viral Nonstructural Proteins/metabolism
20.
Altern Lab Anim ; 40(5): 235-57, 2012 Oct.
Article in English | MEDLINE | ID: mdl-23215661

ABSTRACT

Various factors, including the phylogenetic distance between laboratory animals and humans, the discrepancy between current in vitro systems and the human body, and the restrictions of in silico modelling, have generated the need for new solutions to the ever-increasing worldwide dilemma of substance testing. This review provides a historical sketch on the accentuation of this dilemma, and highlights fundamental limitations to the countermeasures taken so far. It describes the potential of recently-introduced microsystems to emulate human organs in 'organ-on-a-chip' devices. Finally, it focuses on an in-depth analysis of the first devices that aimed to mimic human systemic organ interactions in 'human-on-a-chip' systems. Their potential to replace acute systemic toxicity testing in animals, and their inability to provide alternatives to repeated dose long-term testing, are discussed. Inspired by the latest discoveries in human biology, tissue engineering and micro-systems technology, this review proposes a paradigm shift to overcome the apparent challenges. A roadmap is outlined to create a new homeostatic level of biology in 'human-on-a-chip' systems in order to, in the long run, replace systemic repeated dose safety evaluation and disease modelling in animals.


Subject(s)
Animal Testing Alternatives , Animals, Laboratory , Microfluidic Analytical Techniques/methods , Toxicity Tests/methods , Animals , Humans , Stem Cell Research
SELECTION OF CITATIONS
SEARCH DETAIL