Pink1 kinase and its membrane potential (Deltaψ)-dependent cleavage product both localize to outer mitochondrial membrane by unique targeting mode

The Parkinson disease-associated kinase Pink1 is targeted to mitochondria where it is thought to regulate mitochondrial quality control by promoting the selective autophagic removal of dysfunctional mitochondria. Nevertheless, the targeting mode of Pink1 and its submitochondrial localization are still not conclusively resolved. The aim of this study was to dissect the mitochondrial import pathway of Pink1 by use of a highly sensitive in vitro assay. Mutational analysis of the Pink1 sequence revealed that its N terminus acts as a genuine matrix localization sequence that mediates the initial membrane potential (Δψ)-dependent targeting of the Pink1 precursor to the inner mitochondrial membrane, but it is dispensable for Pink1 import or processing. A hydrophobic segment downstream of the signal sequence impeded complete translocation of Pink1 across the mitochondrial inner membrane. Additionally, the C-terminal end of the protein promoted the retention of Pink1 at the outer membrane. Thus, multiple targeting signals featured by the Pink1 sequence result in the final localization of both the full-length protein and its major Δψ-dependent cleavage product to the cytosolic face of the outer mitochondrial membrane. Full-length Pink1 and deletion constructs resembling the natural Pink1 processing product were found to assemble into membrane potential-sensitive high molecular weight protein complexes at the mitochondrial surface and displayed similar cytoprotective effects when expressed in vivo, indicating that both species are functionally relevant.     

Endocytosis of surface bound dopamine β-hydroxylase and plasma membrane following catecholamine secretion by bovine adrenal chromaffin cells

Cultured chromaffin cells were stimulated with either Ba²⁺ or nicotine to secrete catecholamines. This resulted in the appearance of the chromaffin granule membrane protein, dopamine β-hydroxylase (DBH), on the cell surface. The DBH exposed on the cell surface was labeled using fluorescently tagged anti-DBH Fab fragments and the cell surface was simultaneously labeled with fluorescently tagged concanavalin A. Immediately after labeling, both fluorescent markers were localized on or near the cell surface; anti-DBH fluorescence was distributed as patches, but Con A fluorescence was uniformly distributed. Approximately 30 min after labeling, anti-DBH fluorescence appeared to be almost completely internalized without apparent redistribution on the surface whereas much of the Con A fluorescence remained on the cell surface.The rate of DBH endocytosis was quantified using ¹²⁵I labeled anti-IgG to measure surface bound anti-DBH. Following stimulation of catecholamine secretion, DBH and DBH/anti-DBH complexes both disappeared from the cell surface at similar rates. The half-life on the cell surface was approximately 7 min. These results demonstrate that DBH was rapidly and selectively retrieved from the cell surface, probably from the site of exocytosis.     

Phospholipid hydrolysis caused by Clostridium perfringens α-toxin facilitates the targeting of perfringolysin O to membrane bilayers

Clostridium perfringens causes gas gangrene and gastrointestinal disease in humans. These pathologies are mediated by potent extracellular protein toxins, particularly α-toxin and perfringolysin O (PFO). While α-toxin hydrolyzes phosphatidylcholine and sphingomyelin, PFO forms large transmembrane pores on cholesterol-containing membranes. It has been suggested that the ability of PFO to perforate the membrane of target cells is dictated by how much free cholesterol molecules are present. Given that C. perfringens α-toxin cleaves the phosphocholine headgroup of phosphatidylcholine, we reasoned that α-toxin may increase the number of free cholesterol molecules in the membrane. Our present studies reveal that α-toxin action on membrane bilayers facilitates the PFO?cholesterol interaction as evidenced by a reduction in the amount of cholesterol required in the membrane for PFO binding and pore formation. These studies suggest a mechanism for the concerted action of α-toxin and PFO during C. perfringens pathogenesis.     

Type I PIPK-α regulates directed cell migration by modulating Rac1 plasma membrane targeting and activation

Phosphatidylinositol-4,5-bisphosphate (PI4,5P₂) is a critical regulator of cell migration, but the roles of the type I phosphatidylinositol-4-phosphate 5-kinases (PIPKIs), which synthesize PI4,5P₂, have yet to be fully defined in this process. In this study, we report that one kinase, PIPKI-α, is a novel upstream regulator of Rac1 that links activated integrins to the regulation of cell migration. We show that PIPKI-α controls integrin-induced translocation of Rac1 to the plasma membrane and thereby regulates Rac1 activation. Strikingly, this function is not shared with other PIPKI isoforms, is independent of catalytic activity, and requires physical interaction of PIPKI-α with the Rac1 polybasic domain. Consistent with its role in Rac1 activation, depletion of PIPKI-α causes pronounced defects in membrane ruffling, actin organization, and focal adhesion formation, and ultimately affects the directional persistence of migration. Thus, our study defines the role of PIPKI-α in cell migration and describes a new mechanism for the spatial regulation of Rac1 activity that is critical for cell migration.     

The mitochondrial targeting chaperone 14-3-3ε regulates a RIG-I translocon that mediates membrane association and innate antiviral immunity

RIG-I is a cytosolic pathogen recognition receptor that initiates immune responses against RNA viruses. Upon viral RNA recognition, antiviral signaling requires RIG-I redistribution from the cytosol to membranes where it binds the adaptor protein, MAVS. Here we identify the mitochondrial targeting chaperone protein, 14-3-3ε, as a RIG-I-binding partner and essential component of a translocation complex or "translocon" containing RIG-I, 14-3-3ε, and the TRIM25 ubiquitin ligase. The RIG-I translocon directs RIG-I redistribution from the cytosol to membranes where it mediates MAVS-dependent innate immune signaling during acute RNA virus infection. 14-3-3ε is essential for the stable interaction of RIG-I with TRIM25, which facilitates RIG-I ubiquitination and initiation of innate immunity against hepatitis C virus and other pathogenic RNA viruses. Our results define 14-3-3ε as a key component of a RIG-I translocon required for innate antiviral immunity.     

Determination of the unstable drug otilonium bromide in human plasma by LC–ESI-MS and its application to a pharmacokinetic study

Otilonium bromide (OB) degrades rapidly in plasma and readily undergoes hydrolysis by the plasma esterase. In this paper, an LC–ESI-MS method has been developed for the determination of OB in human plasma. The rapid degradation of OB in plasma was well prevented by immediate addition of potassium fluoride (KF, an inhibitor of plasma esterase) to the freshly collected plasma before prompt treatment with acetonitrile. The method was validated over the concentration range of 0.1–20 ng/ml. The data of intra-run and inter-run precision and accuracy were within ±15%. The mean extraction recoveries for OB and the internal standard were higher than 93.0% and the matrix effects were negligible. The method has been successfully used in a pharmacokinetic study.     

The signal distinguishing between targeting of outer membrane β-barrel protein to plastids and mitochondria in plants

The proteome of the outer membrane of mitochondria and chloroplasts consists of membrane proteins anchored by α-helical or β-sheet elements. While proteins with α-helical transmembrane domains are present in all cellular membranes, proteins with β-barrel structure are specific for these two membranes. The organellar β-barrel proteins are encoded in the nuclear genome and thus, have to be targeted to the outer organellar membrane where they are recognized by surface exposed translocation complexes. In the last years, the signals that ensure proper targeting of these proteins have been investigated as essential base for an understanding of the regulation of cellular protein distribution. However, the organellar β-barrel proteins are unique as most of them do not contain a typical targeting information in form of an N-terminal cleavable targeting signal. Recently, it was discovered that targeting and surface recognition of mitochondrial β-barrel proteins in yeast, humans and plants depends on the hydrophobicity of the last β-hairpin of the β-barrel. However, we demonstrate that the hydrophobicity is not sufficient for the discrimination of targeting to chloroplasts or mitochondria. By domain swapping between mitochondrial and chloroplast targeted β-barrel proteins atVDAC1 and psOEP24 we demonstrate that the presence of a hydrophilic amino acid at the C-terminus of the penultimate β-strand is required for mitochondrial targeting. A mutation of the chloroplast β-barrel protein psOEP24 which mimics such profile is efficiently targeted to mitochondria. Thus, we present the properties of the signal for mitochondrial targeting of β-barrel proteins in plants.     

Delineating the complex mechanistic interplay between NF-κβ driven mTOR depedent autophagy and monocyte to macrophage differentiation: A functional perspective

Autophagy is an extremely essential cellular process aimed to clear redundant and damaged materials, namely organelles, protein aggregates, invading pathogens, etc. through the formation of autophagosomes which are ultimately targeted to lysosomal degradation. In this study, we demonstrated that mTOR dependent classical autophagy is ubiquitously triggered in differentiating monocytes. Moreover, autophagy plays a decisive role in sustaining the process of monocyte to macrophage differentiation. We have delved deeper into understanding the underlying mechanistic complexities that trigger autophagy during differentiation. Intrigued by the significant difference between the protein profiles of monocytes and macrophages, we investigated to learn that autophagy directs monocyte differentiation via protein degradation. Further, we delineated the complex cross-talk between autophagy and cell-cycle arrest in differentiating monocytes. This study also inspects the contribution of adhesion on various steps of autophagy and its ultimate impact on monocyte differentiation. Our study reveals new mechanistic insights into the process of autophagy associated with monocyte differentiation and would undoubtedly help to understand the intricacies of the process better for the effective design of therapeutics as autophagy and autophagy-related processes have enormous importance in human patho-physiology.     

Effective inhibition of melanoma by BI-69A11 is mediated by dual targeting of the AKT and NF-κB pathways

In melanoma, the activation of pro-survival signaling pathways, such as the AKT and NF-κB pathways, is critical for tumor growth. We have recently reported that the AKT inhibitor BI-69A11 causes efficient inhibition of melanoma growth. Here, we show that in addition to its AKT inhibitory activity, BI-69A11 also targets the NF-κB pathway. In melanoma cell lines, BI-69A11 inhibited TNF-α-stimulated IKKα/β and IκB phosphorylation as well as NF-κB reporter gene expression. Furthermore, the effective inhibition of melanoma growth by BI-69A11 was attenuated upon NF-κB activation. Mechanistically, reduced NF-κB signaling by BI-69-A11 is mediated by the inhibition of sphingosine kinase 1, identified in a screen of 315 kinases. Significantly, we demonstrate that BI-69A11 is well tolerated and orally active against UACC 903 and SW1 melanoma xenografts. Our results demonstrate that BI-69A11 inhibits both the AKT and the NF-κB pathways and that the dual targeting of these pathways may be efficacious as a therapeutic strategy in melanoma.     

Tripartite motif-containing protein 38 negatively regulates TLR3/4- and RIG-I-mediated IFN-β production and antiviral response by targeting NAP1

Recognition of RNA virus through TLR and RIG-I-like receptor results in rapid expression of type I IFNs, which play an essential role in host antiviral responses. However, the mechanisms to terminate the production of type I IFNs are not well defined. In the current study, we identified a member of the tripartite motif (TRIM) family, TRIM38, as a negative regulator in TLR3/4- and RIG-I-mediated IFN-β signaling. Knockdown of TRIM38 expression by small interfering RNA resulted in augmented activation of IFN regulatory factor 3 and enhanced expression of IFN-β, whereas overexpression of TRIM38 had opposite effects. Coimmunoprecipitation and colocalization experiments demonstrated that TRIM38 interacted with NF-κB-activating kinase-associated protein 1 (NAP1), which is required for TLR-induced IFN regulatory factor 3 activation and IFN-β production. As an E3 ligase, TRIM38 promoted K48-linked polyubiquitination and proteasomal degradation of NAP1. Thus, knockdown of TRIM38 expression resulted in higher protein level of NAP1 in primary macrophages. Consistent with the inhibitory roles in TLR3/4- and RIG-I-mediated IFN-β signaling, knockdown of TRIM38 significantly inhibited the replication of vesicular stomatitis virus. Overexpression of TRIM38 resulted in enhanced replication of vesicular stomatitis virus. Therefore, our results demonstrate that TRIM38 is a negative regulator for TLR and RIG-I-mediated IFN-β production by targeting NAP1 for ubiquitination and subsequent proteasome-mediated degradation.     

Lactobacillus gasseri LF221 and K7 — from isolation to application

The article presents research findings on two human strains with probiotic activity. On the basis of API 50 CHL fermentation pattern, PCR by species-specific primers and sequencing of the V2–V3 region of 16S rRNA both strains designated as LF221 and K7 were identified as members of the Lactobacillus gasseri species. Two LF221 bacteriocins, acidocin LF221 A and B were purified and sequenced. They were classified as members of the two-component class II bacteriocins. Among basic probiotic properties, the survival under conditions in gastro-intestinal tract, ability to adhere to cultured intestinal enterocytes and pig’s mucosa and stimulation of the immune response were demonstrated. In in vivo study of 24 weaned piglets, the survival rate of K7 Rif^r and LF221 Rif^r was quantified by selective enumeration on MRS agar with rifampicin. The survival of both strains was good (2.9 × 10⁵ cfu of K7 Rif^r /g faeces; 4.8 × 10⁵ cfu of LF221 Rif^r /g) and the LF221 Rif^r /K7 Rif^r viable cells were found either in the mucosa of duodenum, jejunum or in the ileum. The possible effect of K7 to inhibit adhesion of E. coli O8:K88 to enterocytes was studied on Caco-2 cultured cells, on tissue obtained from small intestines of pigs and in vivo on gnotobiotic piglets. Lactobacilli were found to be effective in reducing E. coli adhesion to enterocytes in Caco-2 model, but not on mucosa of pig’s jejunum under ex vivo conditions. Competitive exclusion, production of organic acids and stimulation of immune response, were involved in inhibition of E. coli by K7 strain in gnotobiotic piglets. Any inflammatory change in intestines of piglets treated with K7 was observed, which confirmed its safe use. Among the technological parameters the survival and activity of the strains during cheese-making are presented. Presented at the Second Probiotic Conference, Košice, 15–19 September 2004, Slovakia.     

SVEP1 influences monocyte to macrophage differentiation via integrin α4β1/α9β1 and Rho/Rac signalling

BackgroundThe large extracellular matrix protein SVEP1 mediates cell adhesion via integrin α9β1. Recent studies have identified an association between a missense variant in SVEP1 and increased risk of coronary artery disease (CAD) in humans and in mice Svep1 deficiency alters the development of atherosclerotic plaques. However how SVEP1 functionally contributes to CAD pathogenesis is not fully understood. Monocyte recruitment and differentiation to macrophages is a key step in the development of atherosclerosis. Here, we investigated the requirement for SVEP1 in this process.MethodsSVEP1 expression was measured during monocyte–macrophage differentiation in primary monocytes and THP-1 human monocytic cells. SVEP1 knockout THP-1 cell lines and the dual integrin α4β1/α9β1 inhibitor, BOP, were utilised to investigate the effect of these proteins in THP-1 cell adhesion, migration and cell spreading assays. Subsequent activation of downstream integrin signalling intermediaries was quantified by western blotting.ResultsSVEP1 gene expression increases in monocyte to macrophage differentiation in human primary monocytes and THP-1 cells. Using two SVEP1 knockout THP-1 cells we observed reduction in monocyte adhesion, migration, and cell spreading compared to control cells. Similar results were found with integrin α4β1/α9β1 inhibition. We demonstrate reduced activity of Rho and Rac1 in SVEP1 knockout THP-1 cells.ConclusionsSVEP1 regulates monocyte recruitment and differentiation phenotypes through an integrin α4β1/α9β1 dependent mechanism.General significanceThese results describe a novel role for SVEP1 in monocyte behaviour relevant to CAD pathophysiology.     

Unified QSAR approach to antimicrobials. 4. Multi-target QSAR modeling and comparative multi-distance study of the giant components of antiviral drug–drug complex networks

One limitation of almost all antiviral Quantitative Structure–Activity Relationships (QSAR) models is that they predict the biological activity of drugs against only one species of virus. Consequently, the development of multi-tasking QSAR models (mt-QSAR) to predict drugs activity against different species of virus is of the major vitally important. These mt-QSARs offer also a good opportunity to construct drug–drug Complex Networks (CNs) that can be used to explore large and complex drug-viral species databases. It is known that in very large CNs we can use the Giant Component (GC) as a representative sub-set of nodes (drugs) and but the drug–drug similarity function selected may strongly determines the final network obtained. In the three previous works of the present series we reported mt-QSAR models to predict the antimicrobial activity against different fungi [Gonzalez-Diaz, H.; Prado-Prado, F. J.; Santana, L.; Uriarte, E. Bioorg. Med. Chem. 2006, 14, 5973], bacteria [Prado-Prado, F. J.; Gonzalez-Diaz, H.; Santana, L.; Uriarte E. Bioorg. Med. Chem. 2007, 15, 897] or parasite species [Prado-Prado, F.J.; González-Díaz, H.; Martinez de la Vega, O.; Ubeira, F.M.; Chou K.C. Bioorg. Med. Chem. 2008, 16, 5871]. However, including these works, we do not found any report of mt-QSAR models for antivirals drug, or a comparative study of the different GC extracted from drug–drug CNs based on different similarity functions. In this work, we used Linear Discriminant Analysis (LDA) to fit a mt-QSAR model that classify 600 drugs as active or non-active against the 41 different tested species of virus. The model correctly classifies 143 of 169 active compounds (specificity = 84.62%) and 119 of 139 non-active compounds (sensitivity = 85.61%) and presents overall training accuracy of 85.1% (262 of 308 cases). Validation of the model was carried out by means of external predicting series, classifying the model 466 of 514, 90.7% of compounds. In order to illustrate the performance of the model in practice, we develop a virtual screening recognizing the model as active 92.7%, 102 of 110 antivirus compounds. These compounds were never use in training or predicting series. Next, we obtained and compared the topology of the CNs and their respective GCs based on Euclidean, Manhattan, Chebychey, Pearson and other similarity measures. The GC of the Manhattan network showed the more interesting features for drug–drug similarity search. We also give the procedure for the construction of Back-Projection Maps for the contribution of each drug sub-structure to the antiviral activity against different species.     

Live-cell imaging of membrane proteins by a coiled-coil labeling method—Principles and applications

In situ investigations in living cell membranes are important to elucidate the dynamic behaviors of membrane proteins in complex biomembrane environments. Protein-specific labeling is a key technique for the detection of a target protein by fluorescence imaging. The use of post-translational labeling methods using a genetically encodable tag and synthetic probes targeting the tag offer a smaller label size, labeling with synthetic fluorophores, and precise control of the labeling ratio in multicolor labeling compared with conventional genetic fusions with fluorescent proteins. This review focuses on tag–probe labeling studies for live-cell analysis of membrane proteins based on heterodimeric peptide pairs that form coiled-coil structures. The robust and simple peptide–peptide interaction enables not only labeling of membrane proteins by noncovalent interactions, but also covalent crosslinking and acyl transfer reactions guided by coiled-coil assembly. A number of studies have demonstrated that membrane protein behaviors in live cells, such as internalization of receptors and the oligomeric states of various membrane proteins (G-protein-coupled receptors, epidermal growth factor receptors, influenza A M2 channel, and glycopholin A), can be precisely analyzed using coiled-coil labeling, indicating the potential of this labeling method in membrane protein research.     

Elemental microanalysis of fibroblasts by a scanning proton microprobe and application to Menkes’ disease

A scanning proton microprobe has been used for the elemental microanalysis of individual fibroblast cells. Both normal fibroblasts and fibroblasts cultured from patients with Menkes' disease, an X-linked genetic disorder known to be associated with defective copper metabolism, were examined by the probe. The cells were cultured on a thin ultra-clean nylon foil and retained on that surface for analysis. The focused high-energy proton beam was used to irradiate selected individual cells and elemental information was derived from X-ray and backscattered proton data. The sensitivity of the scanning proton microprobe to trace concentrations of heavy elements has allowed this elemental information to be used to identify individual cells as being either normal or a Menkes' mutant. The cell identification was based on the application of discriminate analysis to a data set formed from the ratios of copper to each of the macroelements present in the cell. This method of cell identification offers the promise of rapid diagnosis of Menkes' disease.     

On the relationship between drug’s size,cell membrane mechanical properties and high levels of multi drug resistance: a comparison to published data

Multi drug resistance (MDR) or cross resistance to drugs was initially explained on the basis that MDR cells express drug transporters that expel membrane-embedded drugs. However, it is now clear that transporters are a single piece from a complex puzzle. An issue that has been solved recently is, given that these transporters have to handle drugs, why should a membrane-embedded drug and a transporter meet? To solve this problem, a theory has been suggested considering the interaction between the cell membrane mechanical properties and the size of drugs. In simple terms, this theory proposes that an excess in the packing of lipid in the inner leaflet of the membrane of MDR cells is responsible for blocking drugs mechanically as a function of their sizes at the membrane level, thus impairing their flux into the cytosol. In turn it is expected that this would allow any membrane embedded drug to diffuse toward transporters. The study concluded that the size of drugs is necessarily important regarding the mechanical interaction between the drug and the membrane, and likely to be central to MDR. Remarkably, an experimental study based on MDR and published years ago concluded that the molecular weight (MW) of drugs was central to MDR (Biedler and Riehm in Cancer Res 30:1174–1184, 1970). Given that size and MW are linked together, I have compared the former theory to the latter experimental data and demonstrate that, indeed, basic membrane mechanics is involved in high levels of cross resistance to drugs in Pgp expressing cells.     

M-CSF (monocyte colony stimulating factor) and M-CSF receptor expression by breast tumour cells: M-CSF mediated recruitment of tumour infiltrating monocytes?

Infiltrating immune cells in 30 primary human epithelial breast tumours were studied using specific anti-CD3 (T cells), anti-CD68 (macrophages), anti-CD57 (NK cells), and an anti-pan-B cell antibody (L26). The majority of tumour infiltrating inflammatory cells are T cells (40-50%) and monocytes/macrophages (15-35%). The macrophage specific chemo-attractant and growth factor CSF-1 is detected by immunohistochemical techniques (IHC) at the level of invasive breast cancer cells in 46/50 tumours but not at the level of in-situ (pre-invasive) cancer. A mosaic staining pattern was usually observed, with a very high expression in areas of obvious stromal invasion (90% cells positive) and absent or trace staining in intraductal carcinoma. Macrophages and plasma cells are equally intensely positive. In-situ hybridisation experiments confirm the production of CSF-1 (mRNA) by tumour cells and show the same pattern of expression. Expression of the CSF-1 receptor protein (fms) was also observed by IHC in 41/48 invasive tumours, albeit at weaker intensities than in tumour infiltrating monocytes/macrophages. A concomitant expression of both CSF-1 and fms in in-situ carcinoma was never seen (n = 14). It is therefore proposed that the associated expression of CSF-1 and its receptor may be linked to the invasive potential of breast cancer, the monocytic infiltrate being an indication of the quantitative importance of CSF-1 production by the tumour.