Multidrug resistance protein 1 localization in lipid raft domains and prostasomes in prostate cancer cell lines

Background: One of the problems in prostate cancer (CaP) treatment is the appearance of the multidrug resistance phenotype, in which ATP-binding cassette transporters such as multidrug resistance protein 1 (MRP1) play a role. Different localizations of the transporter have been reported, some of them related to the chemoresistant phenotype. Aim: This study aimed to compare the localization of MRP1 in three prostate cell lines (normal, androgen-sensitive, and androgen-independent) in order to understand its possible role in CaP chemoresistance. Methods: MRP1 and caveolae protein markers were detected using confocal microscopy, performing colocalization techniques. Lipid raft isolation made it possible to detect these proteins by Western blot analysis. Caveolae and prostasomes were identified by electron microscopy. Results: We show that MRP1 is found in lipid raft fractions of tumor cells and that the number of caveolae increases with malignancy acquisition. MRP1 is found not only in the plasma membrane associated with lipid rafts but also in cytoplasmic accumulations colocalizing with the prostasome markers Caveolin-1 and CD59, suggesting that in CaP cells, MRP1 is localized in prostasomes. Conclusion: We hypothesize that the presence of MRP1 in prostasomes could serve as a reservoir of MRP1; thus, taking advantage of the release of their content, MRP1 could be translocated to the plasma membrane contributing to the chemoresistant phenotype. The presence of MRP1 in prostasomes could serve as a predictor of malignancy in CaP

Multidrug resistance protein 1 localization in lipid raft domains and prostasomes in prostate cancer cell lines

Background: One of the problems in prostate cancer (CaP) treatment is the appearance of the multidrug resistance phenotype, in which ATP-binding cassette transporters such as multidrug resistance protein 1 (MRP1) play a role. Different localizations of the transporter have been reported, some of them related to the chemoresistant phenotype. Aim: This study aimed to compare the localization of MRP1 in three prostate cell lines (normal, androgen-sensitive, and androgen-independent) in order to understand its possible role in CaP chemoresistance. Methods: MRP1 and caveolae protein markers were detected using confocal microscopy, performing colocalization techniques. Lipid raft isolation made it possible to detect these proteins by Western blot analysis. Caveolae and prostasomes were identified by electron microscopy. Results: We show that MRP1 is found in lipid raft fractions of tumor cells and that the number of caveolae increases with malignancy acquisition. MRP1 is found not only in the plasma membrane associated with lipid rafts but also in cytoplasmic accumulations colocalizing with the prostasome markers Caveolin-1 and CD59, suggesting that in CaP cells, MRP1 is localized in prostasomes. Conclusion: We hypothesize that the presence of MRP1 in prostasomes could serve as a reservoir of MRP1; thus, taking advantage of the release of their content, MRP1 could be translocated to the plasma membrane contributing to the chemoresistant phenotype. The presence of MRP1 in prostasomes could serve as a predictor of malignancy in CaP

Genetic variations of EBV-LMP1 from nasopharyngeal carcinoma biopsies: potential loss of T cell epitopes

To find Epstein-Barr virus (EBV) strains with genetic variations of EBV latent membrane protein 1 (EBV-LMP1) from nasopharyngeal carcinoma (NPC), the full-length DNA of LMP1 genes from 21 NPC biopsies obtained in Hunan province in southern China was amplified and sequenced. Our sequences were compared to those previously reported by the Clustal V method. Results showed that all 21 sequences displayed two amino acid changes most frequently in LMP1 of CD4+ T cell epitopes at codons 144 (F®I, 21/21) and 212 (G®S, 19/21) or (G®N, 2/21). We also show that type A EBV strain is prevalent in the cases of NPC from Hunan province with a 30-bp 18/21 deletion, and we highlight that this deletion resulted in loss of one of the CD4+ T cell-restricted epitopes. The other 3 sequences without this deletion all had a change at codon 344 (G®D). Furthermore, in the major epitope sequence of CD8+ T cells restricted by HLA-A2, all 21 sequences showed changes at codons 126 (L®F) and 129 (M®I). Our study discovered that one of the 21 sequence variations harbored a new change at codon 131 (W®C), and 5/21 specimens showed another novel change at codon 115 (G®A) in the major epitope sequence of CD8+ T cells restricted by HLA-A2. Our study suggests that these sequence variations of NPC-derived LMP1 may lead to a potential escape from host cell immune recognition, protecting latent EBV infection and causing an increase in tumorigenicity.

Post-endocytic sorting of calcitonin receptor-like receptor and receptor activity-modifying protein 1

Calcitonin receptor-like receptor (CLR) and the receptor activity-modifying protein 1 (RAMP1) comprise a receptor for calcitonin gene-related peptide (CGRP). Although CGRP induces endocytosis of CLR/RAMP1, little is known about post-endocytic sorting of these proteins. We observed that the duration of stimulation with CGRP markedly affected post-endocytic sorting of CLR/RAMP1. In HEK and SK-N-MC cells, transient stimulation (10(-7) M CGRP, 1 h), induced CLR/RAMP1 recycling with similar kinetics (2-6 h), demonstrated by labeling receptors in living cells with antibodies to extracellular epitopes. Recycling of CLR/RAMP1 correlated with resensitization of CGRP-induced increases in [Ca(2+)](i). Cycloheximide did not affect resensitization, but bafilomycin A(1), an inhibitor of vacuolar H(+)-ATPases, abolished resensitization. Recycling CLR and RAMP1 were detected in endosomes containing Rab4a and Rab11a, and expression of GTPase-defective Rab4aS22N and Rab11aS25N inhibited resensitization. After sustained stimulation (10(-7) M CGRP, >2 h), CLR/RAMP1 trafficked to lysosomes. RAMP1 was degraded approximately 4-fold more rapidly than CLR (RAMP1, 45% degradation, 5 h; CLR, 54% degradation, 16 h), determined by Western blotting. Inhibitors of lysosomal, but not proteasomal, proteases prevented degradation. Sustained stimulation did not induce detectable mono- or polyubiquitination of CLR or RAMP1, determined by immunoprecipitation and Western blotting. Moreover, a RAMP1 mutant lacking the only intracellular lysine (RAMP1K142R) internalized and was degraded normally. Thus, after transient stimulation with CGRP, CLR and RAMP1 traffic from endosomes to the plasma membrane, which mediates resensitization. After sustained stimulation, CLR and RAMP1 traffic from endosomes to lysosomes by ubiquitin-independent mechanisms, where they are degraded at different rates.

Endocytosis of prion protein is required for ERK1/2 signaling induced by stress-inducible protein 1

The secreted cochaperone STI1 triggers activation of protein kinase A (PKA) and ERK1/2 signaling by interacting with the cellular prion (PrPC) at the cell surface, resulting in neuroprotection and increased neuritogenesis. Here, we investigated whether STI1 triggers PrPC trafficking and tested whether this process controls PrPC-dependent signaling. We found that STI1, but not a STI1 mutant unable to bind PrPC, induced PrPC endocytosis. STI1-induced signaling did not occur in cells devoid of endogenous PrPC; however, heterologous expression of PrPC reconstituted both PKA and ERK1/2 activation. In contrast, a PrPC mutant lacking endocytic activity was unable to promote ERK1/2 activation induced by STI1, whereas it reconstituted PKA activity in the same condition, suggesting a key role of endocytosis in the former process. The activation of ERK1/2 by STI1 was transient and appeared to depend on the interaction of the two proteins at the cell surface or shortly after internalization. Moreover, inhibition of dynamin activity by expression of a dominant-negative mutant caused the accumulation and colocalization of these proteins at the plasma membrane, suggesting that both proteins use a dynamin-dependent internalization pathway. These results show that PrPC endocytosis is a necessary step to modulate STI1-dependent ERK1/2 signaling involved in neuritogenesis.

Untersuchungen zur Induktion von Filopodien durch das Amyloid Precursor Like Protein 1 (APLP1), ein Mitglied der APP-Genfamilie

Die Alzheimer Krankheit ist eine fortschreitendende Demenzerkrankung von der in Deutschland ca. 1,6 Millionen Menschen betroffen sind. Im Gehirn der Patienten finden sich sogenannte amyloide Plaques, deren Hauptbestandteil das Aβ-Protein ist. Dieses Peptid ist ein Spaltprodukt des APP-Proteins (engl. amyloid precursor protein). APP ist das namensgebende Mitglied der APP-Proteinfamilie zu der neben APP die beiden APP-Homologen APLP1 und APLP2 (engl. amyloid precursor like protein) gehören. Obwohl inzwischen über die pathologische Rolle dieser Proteinfamilie bei der Alzheimer Krankheit vieles bekannt ist, bleiben die physiologischen Funktionen dieser Proteine bisher größtenteils ungeklärt. Die vorliegende Arbeit beschreibt erstmals einen APLP1-spezifischen Effekt auf die Ausbildung von Filopodien. Sowohl das humane als auch das murine APLP1 induzierten nach transienter Überexpression die Bildung zahlreicher filopodialer Fortsätze auf der Membran von PC12-Zellen. Vergleichbare Resultate konnten mit beiden APLP1-Proteinen auch auf der Membran von embryonalen (E18.5), cortikalen Neuronen der Ratte gezeigt werden. Dass APLP1 einen derartigen Effekt auf Neuronen und PC12-Zellen zeigt, begründet die Annahme, dass APLP1 in vivo eine Funktion bei der Entwicklung und Differenzierung von Neuronen übernimmt. Anhand von Versuchen mit deletierten APLP1-Proteinen und APLP1/APLP2-Chimärproteinen konnte gezeigt werden, dass die von Exon 5 und Exon 6 codierten Bereiche des APLP1 für die Induktion der Filopodien essentiell sind. Unter Einbeziehung von in ihrer räumlichen Struktur bereits bekannten Domänen und aufgrund von Homologievergleichen der primären Aminosäuresequenz dieser Region mit entsprechenden Bereichen der APP- bzw. APLP2-Proteine wurde die wahrscheinliche Lage der Filopodien-induzierenden Domäne innerhalb des von Exon 6 codierten Bereiches diskutiert. Es konnte ferner gezeigt werden, dass die untersuchte Induktion von Filopodien durch die sogenannte α-Sekretierung moduliert werden kann. Unter den gewählten Versuchsbedingungen war nur membranständiges APLP1, nicht aber sekretiertes APLP1 in der Lage, Filopodien zu induzieren. Abschliessend wurden Ergebnisse gezeigt, die erste Einblicke in Signalkaskaden erlauben, die von APLP1 angesteuert werden und so die Enstehung der Filopodien auslösen. Bezüglich des primären Prozesses der Signalkaskade, der Bindung von APLP1 an einen bisher unbekannten Rezeptor, wurde die Möglichkeit diskutiert, ob APP oder APLP2 oder sogar APLP1 selbst als Rezeptor fungieren könnten. Die beobachteten Prozesse nach Überexpression von APLP1 entsprechen vermutlich einer physiologischen Funktion bei der Differenzierung von Neuronen, die mit der Interaktion einer extrazellulär gelegenen Domäne mit einem Rezeptor beginnt, die Aktivierung einer Signalkaskade zur Akrinreorganisation zu Folge hat und die Entstehung filopodialer Strukturen auslöst.

A tool for the qualitative comparison of membrane-embedded and detergent-solubilized membrane protein structures in projection

The calculation of projection structures (PSs) from Protein Data Bank (PDB)-coordinate files of membrane proteins is not well-established. Reports on such attempts exist but are rare. In addition, the different procedures are barely described and thus difficult if not impossible to reproduce. Here we present a simple, fast and well-documented method for the calculation and visualization of PSs from PDB-coordinate files of membrane proteins: the projection structure visualization (PSV)-method. The PSV-method was successfully validated using the PS of aquaporin-1 (AQP1) from 2D crystals and cryo-transmission electron microscopy, and the PDB-coordinate file of AQP1 determined from 3D crystals and X-ray crystallography. Besides AQP1, which is a relatively rigid protein, we also studied a flexible membrane transport protein, i.e. the L-arginine/agmatine antiporter AdiC. Comparison of PSs calculated from the existing PDB-coordinate files of substrate-free and L-arginine-bound AdiC indicated that conformational changes are detected in projection. Importantly, structural differences were found between the PSV-method calculated PSs of the detergent-solubilized AdiC proteins and the PS from cryo-TEM of membrane-embedded AdiC. These differences are particularly exciting since they may reflect a different conformation of AdiC induced by the lateral pressure in the lipid bilayer.

High-resolution imaging of 2D outer membrane protein F (OmpF) crystals by atomic force microscopy

In this chapter the methodological bases are provided to achieve subnanometer resolution on two-dimensional (2D) membrane protein crystals by atomic force microscopy (AFM). This is outlined in detail with the example of AFM studies of the outer membrane protein F (OmpF) from the bacterium Escherichia coli (E. coli). We describe in detail the high-resolution imaging of 2D OmpF crystals in aqueous solution and under near-physiological conditions. The topographs of OmpF, and stylus effects and artifacts encountered when imaging by AFM are discussed.

Tocopherol-associated protein-1 accelerates apoptosis induced by alpha-tocopheryl succinate in mesothelioma cells

Alpha-tocopheryl succinate (alpha-TOS), a redox-silent analogue of vitamin E, induces apoptosis in multiple cell lines in a selective manner, by activating the intrinsic pathway. Since it is a highly hydrophobic compound, it may require a carrier protein for its trafficking to intracellular targets like mitochondria. We studied the role of the ubiquitous tocopherol-associated protein-1 (TAP1 or sec14-like 2) in apoptosis induction by alpha-TOS in malignant mesothelioma (MM) cells. Over-expression of TAP1 in MM cells sensitised them to apoptosis by low doses of alpha-TOS which were sub-apoptotic for the parental cells. Apoptosis induced in TAP1-over-expressing cells was mitochondria- and caspase-dependent, as suggested by dissipation of mitochondrial trans-membrane potential and inhibition by zVAD-fmk, respectively. Binding assays showed affinity of alpha-TOS for TAP1. Finally, TAP1 over-expressing cells accumulated alpha-TOS at higher levels compared to their normal counterparts. We suggest that TAP1 may act as an intracellular shuttle for alpha-TOS, promoting apoptosis initiated by this vitamin E analogue, as shown here for MM cells.

Outer membrane protein UspA1 and lipooligosaccharide are involved in invasion of human epithelial cells by Moraxella catarrhalis

Invasion of non-professional phagocytes is a strategy employed by several mucosal pathogens, but has not been investigated in detail for Moraxella catarrhalis, a major cause of human respiratory tract infections. We investigated the role of outer membrane protein (OMP) UspA1 and lipooligosaccharide (LOS) in M. catarrhalis invasion into epithelial cells. An isogenic mutant of strain O35E, which lacked expression of the UspA1 adhesin, demonstrated not only severely impaired adherence (86%) to but also reduced invasion (77%) into Chang conjunctival cells in comparison with the wild-type strain. The isogenic, LOS-deficient mutant strain O35E.lpxA was attenuated in adherence (93%) and its capacity to invade was severely reduced (95%), but not abolished. Inhibition assays using sucrose and cytochalasin D, respectively, demonstrated that clathrin and actin polymerization contribute to internalization of M. catarrhalis by Chang cells. Furthermore, inhibition of UspA1-mediated binding to cell-associated fibronectin and alpha5beta1 integrin decreased invasion of M. catarrhalis strain O35E (72% and 41%, respectively). These data indicate that OMP UspA1 and LOS profoundly affect the capacity of M. catarrhalis to invade epithelial cells.

Glycoprotein biosynthesis in a eukaryote lacking the membrane protein Rft1

Mature dolichol-linked oligosaccharides (mDLOs) needed for eukaryotic protein N-glycosylation are synthesized by a multistep pathway in which the biosynthetic lipid intermediate Man5GlcNAc2-PP-dolichol (M5-DLO) flips from the cytoplasmic to the luminal face of the endoplasmic reticulum. The endoplasmic reticulum membrane protein Rft1 is intimately involved in mDLO biosynthesis. Yeast genetic analyses implicated Rft1 as the M5-DLO flippase, but because biochemical tests challenged this assignment, the function of Rft1 remains obscure. To understand the role of Rft1, we sought to analyze mDLO biosynthesis in vivo in the complete absence of the protein. Rft1 is essential for yeast viability, and no Rft1-null organisms are currently available. Here, we exploited Trypanosoma brucei (Tb), an early diverging eukaryote whose Rft1 homologue functions in yeast. We report that TbRft1-null procyclic trypanosomes grow nearly normally. They have normal steady-state levels of mDLO and significant N-glycosylation, indicating robust M5-DLO flippase activity. Remarkably, the mutant cells have 30-100-fold greater steady-state levels of M5-DLO than wild-type cells. All N-glycans in the TbRft1-null cells originate from mDLO indicating that the M5-DLO excess is not available for glycosylation. These results suggest that rather than facilitating M5-DLO flipping, Rft1 facilitates conversion of M5-DLO to mDLO by another mechanism, possibly by acting as an M5-DLO chaperone.

Cloning and characterization of an Actinobacillus pleuropneumoniae outer membrane protein belonging to the family of PAL lipoproteins

A 14-kDa outer membrane protein (OMP) was purified from Actinobacillus pleuro-pneumoniae serotype 2. The protein strongly reacts with sera from pigs experimentally or naturally infected with any of the 12 serotypes of A. pleuropneumoniae. The gene encoding this protein was isolated from a gene library of A. pleuropneumoniae serotype 2 reference strain by immunoscreening. Expression of the cloned gene in Escherichia coli revealed that the protein is also located in the outer membrane fraction of the recombinant host. DNA sequence analysis of the gene reveals high similarity of the protein's amino acid sequence to that of the E. coli peptidoglycan-associated lipoprotein PAL, to the Haemophilus influenzae OMP P6 and to related proteins of several other Gram-negative bacteria. We have therefore named the 14-kDa protein PalA, and its corresponding gene, palA. The 20 amino-terminal amino acid residues of PalA constitute a signal sequence characteristic of membrane lipoproteins of prokaryotes with a recognition site for the signal sequence peptidase II and a sorting signal for the final localization of the mature protein in the outer membrane. The DNA sequence upstream of palA contains an open reading frame which is highly similar to the E. coli tolB gene, indicating a gene cluster in A. pleuropneumoniae which is very similar to the E. coli tol locus. The palA gene is conserved and expressed in all A. pleuropneumoniae serotypes and in A. lignieresii. A very similar palA gene is present in A. suis and A. equuli.

Rapid method to express and purify human membrane protein using the Xenopus oocyte system for functional and low-resolution structural analysis.

Progress toward elucidating the 3D structures of eukaryotic membrane proteins has been hampered by the lack of appropriate expression systems. Recent work using the Xenopus oocyte as a novel expression system for structural analysis demonstrates the capability of providing not only the significant amount of protein yields required for structural work but also the expression of eukaryotic membrane proteins in a more native and functional conformation. There is a long history using the oocyte expression system as an efficient tool for membrane transporter and channel expression in direct functional analysis, but improvements in robotic injection systems and protein yield optimization allow the rapid scalability of expressed proteins to be purified and characterized in physiologically relevant structural states. Traditional overexpression systems (yeast, bacteria, and insect cells) by comparison require chaotropic conditions over several steps for extraction, solubilization, and purification. By contrast, overexpressing within the oocyte system for subsequent negative-staining transmission electron microscopy studies provides a single system that can functionally assess and purify eukaryotic membrane proteins in fewer steps maintaining the physiological properties of the membrane protein.

Structural details of an interaction between cardiolipin and an integral membrane protein

Anionic lipids play a variety of key roles in biomembrane function, including providing the immediate environment for the integral membrane proteins that catalyze photosynthetic and respiratory energy transduction. Little is known about the molecular basis of these lipid–protein interactions. In this study, x-ray crystallography has been used to examine the structural details of an interaction between cardiolipin and the photoreaction center, a key light-driven electron transfer protein complex found in the cytoplasmic membrane of photosynthetic bacteria. X-ray diffraction data collected over the resolution range 30.0–2.1 Å show that binding of the lipid to the protein involves a combination of ionic interactions between the protein and the lipid headgroup and van der Waals interactions between the lipid tails and the electroneutral intramembrane surface of the protein. In the headgroup region, ionic interactions involve polar groups of a number of residues, the protein backbone, and bound water molecules. The lipid tails sit along largely hydrophobic grooves in the irregular surface of the protein. In addition to providing new information on the immediate lipid environment of a key integral membrane protein, this study provides the first, to our knowledge, high-resolution x-ray crystal structure for cardiolipin. The possible significance of this interaction between an integral membrane protein and cardiolipin is considered.