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PRA1 domain family, member 2 (PRAF2) is a new 19 kDa protein with four putative transmembrane (TM) domains. PRAF2 (formerly designated JM4) belongs to a new protein family, which plays a role in the regulation of intracellular protein transport. Recently, PRAF2 was found to interact with the chemokine receptor CCR5 [Schweneker, M., Bachmann, A.S., Moelling, K., 2005. JM4 is a four-transmembrane protein binding to the CCR5 receptor. FEBS Lett. 579, 1751–1758]. In order to further study the function and regulation of PRAF2, we determined its genomic structure and its protein expression pattern in normal and cancerous human tissues. PRAF2 encodes a 178-residue protein, whose sequence is related to PRAF1 (PRA1/prenylin) and PRAF3 (JWA/GTRAP3–18). The human PRAF2 gene contains three exons separated by two introns and is located on human chromosome Xp11.23. The recombinant PRAF2 protein was readily expressed in Schneider 2 (S2) insect cells, and the native protein was detected in human tissues with strong expression in the brain, small intestine, lung, spleen, and pancreas. The protein was undetectable in tissue of the testes. Strong PRAF2 protein expression was also found in human tumor tissues of the breast, colon, lung, and ovary, with a weaker staining pattern in normal tissues of the same patient. Our studies show for the first time that the CCR5-interacting PRAF2 protein is expressed in several human tissues with a possible function in ER/Golgi transport and vesicular traffic.

Background: Apolipoprotein A5 (ApoA5) originally gained attention as a regulator of serum triglyceride concentrations through transgenic mouse studies. Our group recently developed the first assay to quantify serum ApoA5 protein concentrations and demonstrated that they are increased by administration of a potent peroxisome proliferator-activated receptor-α agonist. Methods: To better characterize the circulating ApoA5, the protein was purified from human serum, and a definitive N-terminal protein sequence was obtained. In light of previous observations that ApoA5 was present in VLDL and not LDL, plasma infranatant and intermediate-density lipoprotein (IDL) were analyzed for ApoA5. Because the mature protein contains a single unpaired cysteine, ApoA5 in human serum was immunoprecipitated, and its migration pattern was examined via Western blotting under reducing and nonreducing conditions to determine whether the protein circulates as a disulfide-linked homodimer or heterodimer. Results: Definitive N-terminal protein sequences obtained from ApoA5 purified from human serum indicated that cleavage of the signal peptide occurs in vivo at the predicted site. We found ApoA5 in VLDL, HDL, and chylomicrons but not in LDL, IDL, or plasma infranatant. Under both reducing and nonreducing conditions, ApoA5 migrated mainly as a single band with a relative molecular mass (Mr) of ∼39 000, indicating that the protein exists in serum as a monomer and not as a disulfide-linked homodimer or heterodimer. Conclusions: Our data help characterize ApoA5 by defining its lipoprotein particle distribution, by determining its N-terminal protein sequence, and by demonstrating that the mature protein circulates mainly as a monomer and not as a disulfide-linked homodimer or heterodimer.

The gene encoding ribosomal protein S19 (RPS19) is mutated in approximately 25% of patients with Diamond-Blackfan anemia (DBA), which is a rare congenital erythroblastopenia. DBA patients have a variety of clinical characteristics, and the role of the RPS19 gene in the pathogenesis of the disease is presently unknown. To investigate a possible role for RPS19 in erythropoiesis, we looked for proteins associated with mouse RPS19 using a yeast two-hybrid system and identified a novel protein, which we named S19 binding protein (S19BP). The deduced amino acid sequence of S19BP derived from cDNA defines a calculated mass of 15,849 and an isoelectric point of 11.3. No known functional motifs were found in S19BP except a short polylysine tract embedded in a putative nucleolar localization signal. Immunolocalization experiments revealed that S19BP was highly concentrated in nucleoli after 6 h of transfection in Cos-7 cells. S19BP was expressed ubiquitously at a basal level but a significantly high level of expression was observed in some tissues.

The low-density lipoprotein (LDL) receptor is a transmembrane glycoprotein that mediates the binding and endocytosis of lipoproteins containing apolipoprotein B and E, especially the cholesterol-rich LDL. Mutations in the LDL receptor gene can produce dysfunctional LDL receptors and cause familial hypercholesterolemia. The expression of the LDL receptor gene is under an intriguing regulation by sterol and nonsterol mediators either at the transcriptional level or at the posttranscriptional level, both of which are linked to cell signaling pathways. Upregulation of liver LDL receptor expression is effective in treating hypercholesterolemia. In this review, we focus on the latest progress on the mechanisms and regulation of the LDL receptor gene expression.

Apolipoprotein E (apoE) associates with lipoproteins and mediates their interaction with members of the LDL receptor family. ApoE exists as three common isoforms that have important distinct functional and biological properties. Two apoE isoforms, apoE3 and apoE4, are recognized by the LDL receptor, whereas apoE2 binds poorly to this receptor and is associated with type III hyperlipidemia. In addition, the apoE4 isoform is associated with the common late-onset familial and sporadic forms of Alzheimer's disease. Although the interaction of apoE with the LDL receptor is well characterized, the specificity of other members of this receptor family for apoE is poorly understood. In the current investigation, we have characterized the binding of apoE to the VLDL receptor and the LDL receptor-related protein (LRP). Our results indicate that like the LDL receptor, LRP prefers lipid-bound forms of apoE, but in contrast to the LDL receptor, both LRP and the VLDL receptor recognize all apoE isoforms. Interestingly, the VLDL receptor does not require the association of apoE with lipid for optimal recognition and avidly binds lipid-free apoE. It is likely that this receptor-dependent specificity for various apoE isoforms and for lipid-free versus lipid-bound forms of apoE is physiologically significant and is connected to distinct functions for these receptors.

▪ Abstract The low-density lipoprotein receptor (LDLR) is responsible for uptake of cholesterol-carrying lipoprotein particles into cells. The receptor binds lipoprotein particles at the cell surface and releases them in the low-pH environment of the endosome. The focus of the current review is on biochemical and structural studies of the LDLR and its ligands, emphasizing how structural features of the receptor dictate the binding of low-density lipoprotein (LDL) and beta-migrating forms of very low-density lipoprotein (β-VLDL) particles, how the receptor releases bound ligands at low pH, and how the cytoplasmic tail of the LDLR interfaces with the endocytic machinery.

Members of the Sir2 family of NAD-dependent protein deacetylases regulate diverse cellular processes including aging, gene silencing, and cellular differentiation. Here, we report that the distant mammalian Sir2 homolog SIRT6 is a broadly expressed, predominantly nuclear protein. Northern analysis of embryonic samples and multiple adult tissues revealed mouse SIRT6 (mSIRT6) mRNA peaks at day E11, persisting into adulthood in all eight tissues examined. At the protein level, mSIRT6 was readily detectable in the same eight tissue types, with the highest levels in muscle, brain, and heart. Subcellular localization studies using both C- and N-terminal green fluorescent protein fusion proteins showed mSIRT6-green fluorescent protein to be a predominantly nuclear protein. Indirect immunofluorescence using antibodies to two different mSIRT6 epitopes confirmed that endogenous mSIRT6 is also largely nuclear. Consistent with previous findings, we did not observe any NAD+-dependent protein deacetylase activity in preparations of mSIRT6. However, purified recombinant mSIRT6 did catalyze the robust transfer of radiolabel from [32P]NAD to mSIRT6. Two highly conserved residues within the catalytic core of the protein were required for this reaction. This reaction is most likely mono-ADP-ribosylation because only the modified form of the protein was recognized by an antibody specific to mono-ADP-ribose. Surprisingly, we observed that the catalytic mechanism of this reaction is intra-molecular, with individual molecules of mSIRT6 directing their own modification. These results provide the first characterization of a Sir2 protein from phylogenetic class IV.

Oxidized low-density lipoprotein (LDL) has been shown to be a powerful regulator of gene expression in monocyte-derived macrophage. To determine the effects of oxidized LDL on macrophage gene expression, macrophages incubated with native or oxidized LDL were analyzed by differential display technique. The differentially expressed cDNA (387 bp) fragment by oxidized LDL showed 100% homology to the part of 3’-untranslated region of the gene of human GM2 activator protein. Quantitative RT-PCR data using specific TaqMan probe showed the increased GM2 activator protein mRNA expression in macrophages incubated with oxidized LDL by about twice (215±4 31.6 %) of that in macrophages incubated with native LDL. Our results shows that ox-LDL upregulates GM2 activator protein gene expression, indicating that GM2 activator protein may be associated with the pathogenesis of atherosclerosis.

The purpose of this study was to identify in vitro and then prioritize a tractable set of protein biomarker candidates of atherosclerosis that may eventually be developed to measure the extent, progression, regression, and stability of atherosclerotic lesions. A study was conducted using an in vitro"foam cell" model based on the stimulation of differentiated THP1 cells with oxidized low-density lipoprotein (oxidized LDL) as compared with low-density lipoprotein (LDL). Analysis of the proteins contained in the cell supernatant using proteome scanning technology identified 59 proteins as being increased, 57 with no statistically measurable difference, and 17 decreasing in abundance following treatment with oxidized LDL, as compared with LDL. From the up-regulated list, proteins were prioritized based on their analytical confidence as well as their relevance to atherosclerosis pathways. Within the group of increased abundance, seven families of proteins were of particular interest: fatty acid-binding proteins, chitinase-like enzymes, cyclophilins, cathepsins, proteoglycans, urokinase-type plasminogen activator receptor, and a macrophage scavenger receptor.

Human plasma phospholipid transfer protein (PLTP) plays an important role in lipoprotein metabolism. In this study, we investigated the effects of lipoproteins on the secretion of PLTP in cultured BeWo choriocarcinoma cells. Low-density lipoproteins (LDLs) decreased PLTP secretion in a dose- and time-dependent manner, whereas very low density lipoproteins and high-density lipoproteins (HDLs) had little effect. LDL suppression of PLTP secretion was not altered by the inhibition of both LDL receptor and LDL receptor–related protein with receptor-associated protein. Mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor, U0126, could abolish the LDL-mediated inhibition of PLTP secretion. Furthermore, LDL, but not HDL, could stimulate the expression of MAPK phosphatase-1 (MKP-1) in BeWo cells that resulted in the inactivation of p44/p42 extracellular signal-regulated kinase (ERK) 1 and 2, the family members of MAPKs. These results support the conclusion that LDL-mediated suppression of PLTP secretion in BeWo cells is through a LDL receptor-independent MAPK signaling pathway.

Niemann-Pick type C (NPC) is a lysosomal storage disorder, characterized by intracellular accumulation of low-density lipoprotein (LDL)-derived cholesterol and neurodegeneration leading to premature death. The most common form of the disease, NPC1, results from mutations in the NPC1 gene. Thus, the NPC1 protein is the focus of intense investigation to elucidate the function of this protein and its role in the disease pathogenesis. Recent studies have revealed the NPC1 subcellular location, topology and potential functions of the NPC1 protein. In lieu of direct experimental evidence, certain hypotheses about the function of NPC1 can be inferred by analyzing disease-causing mutations, NPC1 protein sequence homology to other related proteins, and the potential tertiary structure similarity between NPC1 and its prokaryotic ancestors, such as the E. coli RND permease AcrB. This review will discuss recent work on the characterization and function of the NPC1 protein and highlight structural features that may be important in assisting in the elucidation of NPC1 function and role in subcellular lipid transport and homeostasis.

Histone modification is emerging as a major regulatory mechanism for modulating gene expression by altering the accessibility of transcription factors to DNA. This study unravels the relationship between histone H3 modifications and LDL receptor induction, focusing also on routes by which phosphorylation is mediated in human hepatoma HepG2 cells. We show that while histone H3 is constitutively acetylated at LDL receptor chromatin, 12-O-tetradecanoylphorbol-13-acetate (TPA) causes rapid hyperphosphorylation of histone H3 on serine 10 (histone H3-Ser10), despite global reduction in its phosphorylation levels. Ser10 hyperphosphorylation precedes LDL receptor induction and is independent of the p42/44MAPK, p38MAPK, pp90RSK, or MSK-1 cascade. Interestingly, inhibition of protein kinase C (PKC) blocks Ser10 hyperphosphorylation and also compromises LDL receptor induction by TPA. Consistent with its role, recombinant purified PKC phosphorylate purified histone H3-Ser10. Collectively, our findings highlight a novel role for PKC in regulating histone H3-Ser10 phosphorylation and suggest that histone modification provides numerous regulatory opportunities to set the overall range of control attainable for LDL receptor gene induction.

Low-density lipoprotein receptor-related protein 1B (LRP1B) is frequently deleted in tumors of various types, but its status and expression in esophageal squamous cell carcinomas (ESCs) have never been reported. In the course of a program to screen ESC cell lines for copy-number aberrations using array-based comparative genomic hybridization, we identified a homozygous deletion of LRP1B. Genomic PCR experiments revealed homozygous deletions of LRP1B in additional ESC cell lines (total, 6 of 43; 14.0%) and in primary esophageal tumors (30 of 70; 42.9%). Moreover, expression of LRP1B mRNA was frequently silenced in ESC lines without homozygous deletions (14 of 37; 37.8%). Using bisulfite-PCR analysis and sequencing, we found that LRP1B-nonexpressing cells without homozygous deletions were highly methylated at a CpG island of LRP1B, a sequence possessing promoter activity. Treatment with 5-aza-2′-deoxycytidine restored expression of LRP1B in those ESC lines. Histone acetylation status correlated directly with expression of LRP1B and inversely with the methylation status of the CpG island. Methylation of LRP1B was also detected in primary esophageal tumors. Restoration of LRP1B expression in ESC cells reduced colony formation. These results suggest that loss of LRP1B function in esophageal carcinogenesis most often occurs either by homozygous deletion or by transcriptional silencing through hypermethylation of its CpG island.

A soluble form of human low-density lipoprotein receptor (LDL-R) fused in frame with rabbit transferrin (LDL-Rs(hu)/Tf(rab)) is assessed in vivo as a therapeutic tool for lowering plasma LDL cholesterol. The cDNA encoding LDL-Rs(hu)/Tf(rab) is expressed in mice, using a hydrodynamics-based gene transfer procedure. The transgene is transcribed in the liver of transduced animals and the corresponding protein is secreted into the bloodstream. Circulating LDL-Rs(hu)/Tf(rab) binds LDL specifically, thus indicating that it is correctly processed through the cellular compartments in vivo. More importantly, the expression of LDL-Rs(hu)/Tf(rab) allows the removal of injected human (125)I-labeled LDL ((123)I-LDL) from the bloodstream of transduced CD1 mice, which show faster LDL plasma clearance, anticipating by approximately 90 min the same clearance value observed in control animals. A similar effect is observed in transduced LDL-R(-/-) mice, in which the clearance of injected human LDL depends solely on the presence of circulating LDL-Rs(hu) /Tf(rab). In these animals the extent of plasma LDL clearance is directly related to the concentration of LDL-Rs(hu)/Tf(rab) in the blood. Finally, LDL-Rs(hu)/Tf(rab) does not alter the pattern of LDL organ distribution: in transduced animals, as well as in control animals, liver and bladder are the predominantly labeled organs after (123)I-LDL injection. However, LDL-Rs(hu)/Tf(rab) has a quantitative effect on LDL tissue deposition: in treated animals LDL-Rs(hu)/Tf(rab) determines an increase in radioactivity in the liver at early times after (123)I-LDL injection and a progressive labeling of the bladder, starting 20 min after injection.

The transcription factor sterol regulatory element binding protein (SREBP)-2 plays a pivotal role in lipid metabolism. Previously, we have shown that the mature form of SREBP-2 is a substrate of Erk-mitogen-activated protein kinases (MAPK). The aim of the present study was to identify Erk-specific phosphorylation sites. Using a protein chemistry approach, we could identify Ser-432 and Ser-455 as major phosphorylation sites. Further characterization by electrophoretic mobility shift assay and promoter reporter gene analyses revealed that phosphorylation does not influence protein/DNA interaction, but enhances trans-activity. In intact cells, SREBP-2 is phosphorylated by insulin, which seems to be related to their bio-responses on low density lipoprotein receptor activity. These results suggest that activation of Erk-MAPK pathways by hormones such as insulin might be related to a novel regulatory principle of SREBP-2.

We highlight the importance of the low-density lipoprotein (LDL) receptor family and its pharmaceutical implications in the field of drug delivery. The members of the LDL receptor family are a group of cell surface receptors that transport a number of macromolecules into cells through a process called receptor-mediated endocytosis. This process involves the receptor recognizing a ligand from the extracellular membrane (ECM), internalizing it through clathrin-coated pits and degrading it upon fusion with lysosomes. There are nine members of the receptor family, which include the LDL receptor, low-density lipoprotein-related protein (LRP), megalin, very low-density lipoprotein (VLDL) receptor, apoER2 and sorLA/LRP11, LRP1b, MEGF7, LRP5/6; the former six having been identified in humans. Each member is expressed in a number of different tissues and has a wide range of different ligands, not specific to the recognition of the LDL particle. Thus, rather than the original hypothesis that the receptor is only a mediator of cholesterol uptake, it may also be involved in a number of other physiological functions, including the progression of certain disease states and, potentially, cellular drug uptake. A number of studies have suggested that the LDL receptors are involved in endocytosis of drugs and drug formulations including aminoglycosides, anionic liposomes and cyclosporine A (CsA). This article reviews the importance of lipoproteins as a drug delivery system and how LDL receptors are relevant to the design and targeting of specific drugs.

One common feature of the more than 1,000 complement-type repeats (or low density lipoprotein (LDL)-A modules) found in LDL receptor and the other members of the LDL receptor superfamily is a cluster of five highly conserved acidic residues in the C-terminal region, DXXXDXXDXXDE. However, the role of the third conserved aspartate of these LDL-A modules in protein folding and ligand recognition has not been elucidated. In this report, using a model LDL-A module and several experimental approaches, we demonstrate that this acidic residue, like the other four conserved acidic residues, is involved in calcium-dependent protein folding. These results suggest an alternative calcium coordination conformation for the LDL-A modules. The proposed model provides a plausible explanation for the conservation of this acidic residue among the LDL-A modules. Furthermore, the model can explain why mutations of this residue in human LDL receptor cause familial hypercholesterolemia.

Beginning from a mouse EST (GenBank accession No. BE644537) which was significantly up-regulated in cryptorchidism and represented a novel gene, we cloned a new gene (GenBank accession No. AY138810) which is related to apoptosis in human spermatogenic cells by means of GeneScan program and PCR technology. The gene whose full cDNA length is 1875 bp containing 8 exons and 7 introns is located in human chromosome 11q13.3. Its protein containing 316 amino acid residues is a new member of HSP40 protein family because the sequence contains the highly conserved J domain which is present in all DnaJ-like proteins and is considered to have a critical role in DnaJ-DnaK protein-protein interactions. TSARG6 protein displays a 45% identity in a 348-amino acid overlap with DJB5_HUMAN protein. The result of RT-PCR and Northern blot analysis showed that TSARG6 is specifically expressed in adult testis and the transcript is 1.8 kb. Based upon all these observations, it is considered that we cloned a new gene which probably inhibited human testis spermatogenesis apoptosis.

In this study, the ageing-specific expression genes of the murine cerebrum were investigated by employing differential-display reverse transcription polymerase chain reaction (DDRT-PCR) in three senescence-accelerated mouse (SAM) strains: SAMP8/Ta, SAMP10/Ta and SAMR1TA. In the comparison of gene expression profile in different strain mice, 16 differential fragments have been detected, 3, 6 and 7 of them belong to SAMP10/Ta, SAMP8/Ta and SAMR1TA, respectively. Sequencing data indicated that those fragments are homologous with heat shock protein cognate protein 70, ATP-dependent mitochondrial RNA helicase, Dleu2 mRNA, Mouse DNA sequence from clone RP23-334C3 on chromosome X, ubiquinol-cytochrome c reductase complex (7.2 kDa), 60S ribosomal protein L21, FIS, phenylalkylamine Ca2+ antagonist (emopamil) binding protein, fucosyltransferase 9, glial cell line derived neurotrophic factor family receptor alpha 1, endonuclease/reverse transcriptase, PER1 interacting protein of the suprachiamatic nucleus homolog, centrosomal protein CG-NAP, ferritin heavy chain gene, NID-2 gene, and prkdc gene for DNA-dependent protein kinase catalytic subunit.

Recently, a new class of lipid-lowering agents has been described that upregulate LDL receptor (LDLr) activity. These agents are proposed to activate sterol-regulated gene expression through binding to the sterol regulatory element binding protein (SREBP) cleavage-activating protein (SCAP). Here, we show that the steroidal LDLr upregulator, GW707, induces accumulation of lysosomal free cholesterol and inhibits LDL-stimulated cholesterol esterification, similar to that observed in U18666A-treated cells and in Niemann-Pick type C1 (NPC1) mutants. Moreover, we demonstrate that induction of the NPC-like phenotype by GW707 is independent of SCAP function. We find that treatment with GW707 does not increase SREBP-dependent gene expression above that observed in lipoprotein-starved cells. Rather, we show that the apparent increase in SREBP-dependent activity in GW707-treated cells is attributable to a failure to appropriately suppress sterol-regulated gene expression, as has been shown previously for U18666A-treated cells and NPC mutant fibroblasts. We further demonstrate that cells treated with either GW707 or U18666A fail to appropriately generate 27-hydroxycholesterol in response to LDL cholesterol. Taken together, these findings support a mechanism in which GW707 exerts its hypolipidemic effects through disruption of late endosomal/lysosomal sterol trafficking and subsequent stimulation of LDLr activity.

A commercial antibody (clone 22) directed against the apoptosis-linked gene 2 (alg2, pdcd6) encoded protein has been used by several groups. Up-regulated expression of the antigen was observed in primary tumours and in metastatic tissue and also during rat brain ischemia. Furthermore, antigen down-regulation was found in human atherosclerotic plaques. Recently, we found that the clone 22 antibody does not recognise ALG-2. In the present study the antigen of the clone 22 antibody was identified as the heat shock protein 90 (HSP90) co-chaperone protein p23, identical to the cytosolic prostaglandin E2 synthase, by immunoprecipitation followed by tryptic in-gel digests and mass spectrometry of the purified peptides. Moreover, the heterogeneous ribonuclear protein A2/B1 was found to be a part of the p23 co-immunoprecipitated protein complex.

DNA screening for LDL receptor mutations was performed in 170 unrelated hyperlipidemic Chinese patients and two clinically diagnosed familial hypercholesterolemia patients. Two deletions (Del e3-5 and Del e6-8), eight point mutations (W-18X, D69N, R94H, E207K, C308Y, I402T, A410T, and A696G), and two polymorphisms (A370T and I602V) were identified. Of these mutations, C308Y and Del e6-8 were found in homozygosity, and D69N and C308Y were seen in unrelated patients. The effects of mutations on LDL receptor function were characterized in COS-7 cells. The LDL receptor level and activity were close to those of wild type in A696G transfected cells. A novel intermediate protein and reduction of LDL receptor activity were seen in D69N transfected cells. For R94H, E207K, C308Y, I402T, and A410T mutations, only approximately 20-64% of normal receptor activities were seen. Conversely, Del e3-5 and Del e6-8 lead to defective proteins with approximately 0-13% activity. Most of the mutant receptors were localized intracellularly, with a staining pattern resembling that of the endoplasmic reticulum and Golgi apparatus (D69N, R94H, E207K, C308Y, and I402T) or endosome/lysosome (A410T and Del e6-8). Molecular analysis of the LDL receptor gene will clearly identify the cause of the patient's hyperlipidemia and allow appropriate early treatment as well as antenatal and family studies.

Altered regulation of 5-HT1A receptors is implicated in mood disorders such as anxiety and major depression. To provide insight into its transcriptional regulation, we previously identified a novel DNA element [14 bp 5′-repressor element (FRE)] of the 5-HT1A receptor gene that mediates repression in neuronal and non-neuronal cells (Ou et al., 2000). We have now cloned a novel DNA binding protein [five′ repressor element under dual repression binding protein-1 (Freud-1)] that binds to FRE to mediate repression of the 5-HT1A receptor or heterologous promoters. Freud-1 is evolutionarily conserved and contains two DM-14 basic repeats, a predicted helix-loop-helix DNA binding domain, and a protein kinase C conserved region 2 (C2)/calcium-dependent lipid binding (CalB) calcium/phospholipid binding domain. An intact CalB domain was required for Freud-1-mediated repression. In serotonergic raphe cells, overexpression of Freud-1 repressed the 5-HT1A promoter and decreased 5-HT1A receptor protein levels, whereas transfection of antisense to Freud-1 derepressed the 5-HT1A gene and increased 5-HT1A receptor protein expression. Calcium-dependent signaling blocked Freud-1-FRE binding and derepressed the 5-HT1A promoter. Treatment with inhibitors of calmodulin or CAM-dependent protein kinase reversed calcium-mediated inhibition of Freud-1. Freud-1 RNA and protein were present in raphe nuclei, hippocampus, cortex, and hypothalamus, and Freud-1 protein was colocalized with 5-HT1A receptors, suggesting its importance in regulating 5-HT1A receptorsin vivo. Thus, Freud-1 represents a novel calcium-regulated repressor that negatively regulates basal 5-HT1A receptor expression in neurons and may play a role in the altered regulation of 5-HT1A receptors associated with anxiety or major depression.

Both estrogen and selective estrogen receptor modulators (SERMs) such as tamoxifen and raloxifene have been demonstrated to lower plasma low density lipoprotein (LDL)-cholesterol concentrations by stimulation of LDL receptor gene expression. To determine the molecular mechanisms of estradiol- and tamoxifen-induced LDL receptor expression, we performed transient transfection experiments with luciferase reporter gene-constructs under transcriptional control of the human LDL receptor promoter. We demonstrate, that estradiol and tamoxifen stimulate LDL receptor gene expression in human HepG2 hepatoma cells only when estrogen receptor (ER)-alpha but not when ER-beta is cotransfected. Deletion mutants and point mutations of the LDL receptor promoter reveal that estradiol- and tamoxifen-stimulated expression of this gene depends on an intact repeat 3 in the LDL receptor promoter, a cis-element previously shown to interact with Sp1. Gel mobility analyses demonstrated estradiol- and tamoxifen-stimulated binding of nuclear proteins to repeat 3 (bp -56 to bp -36) of the LDL receptor promoter. These data provide an alternative mechanism of LDL receptor gene expression by non-classical estradiol- and tamoxifen-stimulated induction through an ER-alpha/Sp1 complex.

The low density lipoprotein (LDL) receptor family comprises several proteins with similar structures including the LDL receptor and apoE receptor 2 (apoER2). The human brain expresses two major splice variants of apoER2 mRNA, one of which includes an additional exon that encodes 59 residues in the cytoplasmic domain. This exon is absent from the LDL receptor and contains three proline-rich (PXXP) motifs that may allow apoER2 to function as a signal transducer. To investigate the role of this insert, we took advantage of the well characterized low density lipoprotein receptor pathway. Chimeras comprising the ectodomain and transmembrane domain of the LDL receptor fused to the cytoplasmic domain of apoER2 lacking the PXXP-containing residues are able to mediate clathrin-dependent endocytosis of LDL as effectively as cells expressing the LDL receptor but not if the PXXP insert is present in the protein. Although expressed on the cell surface, the PXXP-containing chimeric receptor is excluded from clathrin vesicles as judged by its failure to co-localize with adaptor protein-2 possibly due to interaction with intracellular adaptors or scaffolding proteins. Chimeras with the transmembrane domain of apoER2, predicted to be longer than that of the LDL receptor by several residues, fail to mediate endocytosis of LDL or to co-localize with adaptor protein-2 regardless of the presence or absence of the PXXP insert. Thus features of apoER2 that distinguish it as a signaling receptor, rather than as an endocytosis receptor like the LDL receptor, reside in or near the transmembrane domain and in the proline-rich motifs.

Low-density lipoprotein receptor-related proteins (LRPs) are evolutionarily ancient cell-surface receptors with diverse biological functions. All are expressed in the central nervous system and, for most receptors, animal models have shown that they are indispensable for successful neurodevelopment. The mechanisms by which they regulate the formation of the nervous system are varied and include the transduction of extracellular signals and the modulation of intracellular signal propagation, as well as cargo transport, the function most commonly attributed to this gene family. Here, we will summarize recent advances in our understanding of the molecular basis on which these receptors function during development.

For many years, the low-density lipoprotein (LDL) receptor and the LDL receptor-related protein (LRP) have been considered to be prototypes of cargo receptors which deliver, via endocytosis, macromolecules into cells. However, the recent identification of additional members of this gene family and examination of their biology has revealed that at least some of these proteins are also signaling receptors. Very low density lipoprotein receptor and ApoER2 transmit the extracellular reelin signal into migrating neurons, and thus are key components of the reelin pathway which governs neuronal layering of the forebrain during embryonic brain development. LRP5 and LRP6 are integral components of the Wnt signaling pathway which is central to many processes of metazoan development, cell proliferation, and tumor formation. Adaptor proteins interacting with the cytosolic domains of these receptors might orchestrate their ability to deliver their cargo or a signal.

In the present work, the complete amino acid sequence of the Entamoeba histolyticaribosomal protein L10 (EhL10) is reported. cDNA of 630 bp revealed an open reading frame that encodes a protein of 210 amino acids. Analysis of EhL10 ribosomal protein revealed 75% similarity and 57% identity with QM protein from Homo sapiensand 78 and 60%, respectively, with Arabidopsis thaliana. Western blot analysis of ribosomal proteins from E. histolytica showed that EhL10 protein is part of the ribosomal complex. Immunofluorescence analysis of EhL10 distribution in a transfected E. histolytica strain showed that EhL10 protein was mainly localized in the nucleus of trophozoites. Overexpression of EhL10 ribosomal protein in trophozoites transfected with the pExEhNeo/EhL10 vector exhibited a 60% reduction in cellular growth. DNA mobility-shift assays demonstrated that EhL10 ribosomal protein was able to destabilize the activating protein 1 (AP-1) complex binding specifically to the c-Jun-like protein. It is proposed in this study that the complex formation of EhL10 with c-Jun-like protein interferes with transcriptional activation of genes controlled by Jun (i.e. gene involved in cell growth). It is also being reported identification of a member of the AP-1 complex, the c-Jun-like protein, in nuclear extracts of E. histolytica using human-specific antibodies against this protein. The observations suggest that EhL10 may have an extraribosomal function in E. histolytica involved in suppression of cell proliferation in E. histolytica similar to the QM protein. © 2003 Elsevier Science B.V. All rights reserved.

The bovine papillomavirus type 1 (BPV-1) genome has been shown to contain a small open-reading frame designated E5B (nucleotides 4013–4167) which is predicted to encode a hydrophobic, 52 amino acid protein. In order to detect and characterize the E5B protein, an 18 nucleotide sequence encoding a 6 amino acid epitope was added to the 3′ end of the E5B open-reading frame which was then expressed in COS-1 cells using a SV40 vector. Immunoprecipitation, immunofluorescence, and cell fractionation studies identified the E5B protein as a 4-kDa protein and localized it primarily to membranes of the endoplasmic reticulum and nucleus. Unlike the E5A protein of BPV-1, E5B did not form dimers (despite containing a cysteine residue) or form complexes with growth factor receptors such as the PDGF receptor or erb B-2 receptor. Interestingly, the E5B protein formed physical complexes with the hydrophobic E5A oncoprotein, apparently via transmembrane interactions. Additionally, expression of E5B inhibited the transforming capability of BPV-1 E5A. These observations suggest that the expression of this viral protein may play a significant role in BPV/host cell interactions.

Protein 4.1 is an important structural protein that is expressed in erythroid and in a variety of nonerythroid tissues. In mammalian erythrocytes, it plays a key role in regulating the physical properties of mechanical stability and deformability in membranes by stabilizing the spectrin-actin interaction. The protein 4.1 family mainly comprises 4.1R, 4.1G (general type), 4.1B (brain type), and 4.1N (neuron type). We identified a novel human 4.1 ( 4.1O) gene that is 2312 bp in length and encodes a protein of 553 amino acid residues. The protein shared homology with mouse protein 4.1B (identity 38%, similarity 55%) with a FERM domain. The expression pattern of the human 4.1O gene in 16 tissues showed that there was a transcript only in ovary, whereas in the remaining 15 tissues, specific bands of the transcript could not be detected. In eight human fetal tissues, the specific bands of the transcript could be detected in skeletal muscle, with lower levels detected in thymus and brain. The 4.1O gene consists of 14 exons and 13 introns and was mapped to Chromosome 9q21-9q22 by bioinformatics analysis.

During a large-scale screen of a human fetal brain cDNA library, a novel human gene GNB2L1 encoding a novel RACK (receptor of activated protein kinase C) protein was isolated and sequenced. The cDNA is 1142 bp long and has a predicted open reading frame encoding 316 aa. The predicted protein shows higher similarity to rat RACK1 and many RACK proteins of different organisms including Drosophila, C. elegans, mouse, rat, human, C. fasciculata, zebrafish, A. thaliana, S. cerevisiae and so on, suggesting it is conserved during evolution. The gene was mapped to human chromosome 5q35.3, the telomer position of chromosome 5q, in which the disease gene for early-onset primary congenital lymphedema was mapped. Also, 5q35.3 is a frequently reported location for cytogenetic and molecular abnormalities in renal cell carcinomas. The gene has 8 exons and 7 introns. It is expressed ubiquitously in many human tissues detected by northern blot analysis and RT-PCR.

14-3-3 proteins were discovered by Moore and Perez in the soluble extract of bovine brain. These proteins are highly abundant in the brain. In this review 14-3-3 cDNA cloning, nucleotide sequence of 14-3-3 cDNA, the structure of 14-3-3 gene and 14-3-3 gene expression, in situ hybridization of 14-3-3 mRNA in the brain, the function and regulation of 14-3-3 protein, the binding of 14-3-3 protein to other proteins, the effects of 14-3-3 protein on the binding of a protein to other proteins, and the effect on protein kinase, etc., are concisely described. From the recent rapid development of proteom technology, markedly more target proteins of 14-3-3 protein should be discovered.

The low density lipoprotein (LDL) receptor-related protein 1B (LRP1B) is a newly identified member of the LDL receptor family and is closely related to LRP. It was discovered as a putative tumor suppressor and is frequently inactivated in lung cancer cells. In the present study, we used an LRP1B minireceptor (mLRP1B4), which mimics the function and trafficking of LRP1B, to explore the roles of LRP1B on the plasminogen activation system. We found that mLRP1B4 and urokinase plasminogen activator receptor (uPAR) form immunoprecipitable complexes on the cell surface in the presence of complexes of uPA and its inhibitor, plasminogen activator inhibitor type-1 (PAI-1). However, compared with cells expressing the analogous LRP minireceptor (mLRP4), cells expressing mLRP1B4 display a substantially slower rate of uPA.PAI-1 complex internalization. Expression of mLRP1B4, or an mLRP4 mutant deficient in endocytosis, leads to an accumulation of uPAR at the cell surface and increased cell-associated uPA and PAI-1 when compared with cells expressing mLRP4. In addition, we found that expression of mLRP1B or the mLRP4 endocytosis mutant impairs the regeneration of unoccupied uPAR on the cell surface and that this correlates with a diminished rate of cell migration. Taken together, these results demonstrate that LRP1B can function as a negative regulator of uPAR regeneration and cell migration.

We have identified a novel mammalian gene, termed nicolin 1 gene (NICN1), that is present in human, dog and mouse, whereas it is absent from the available genome sequences of nonmammalian organisms. The NICN1 gene consists of six exons and spans about 6 kb of genomic DNA. It encodes a 213 amino acid protein that does not belong to any known protein family. Experiments using green fluorescent protein (GFP)-tagged nicolin 1 fusion proteins indicate that nicolin 1 is a nuclear protein. Northern analysis and semiquantitative RT-PCR demonstrated that the 2.5 kb NICN1 mRNA is expressed in a tissue-specific manner. The highest NICN1 expression levels are found in brain, testis, liver, and kidney. On the other hand the NICN1 expression is weak in spleen, leukocytes, small intestine and colon. The NICN1 gene is also expressed during development.

The low-density lipoprotein (LDL) receptor family consists of several related scavenger receptors that not only function as important cargo transporters, but also inform the cell of changes in its environment by mediating signaling responses. The LDL receptor was the first family member to be characterized and its function seems to be restricted to lipoprotein metabolism. By contrast, lipoprotein metabolism does not appear to be the exclusive function of the other characterized LDL receptor family members. It is now apparent that cargo transport by members of the LDL receptor family is closely associated with regulation of cellular physiology and cellular signaling events. Here, we focus on the diverse biological activities of certain members of this family.

Apolipoprotein E (apoE) is a ligand for members of the low density lipoprotein (LDL) receptor family, receptors highly expressed in neurons. A study of one of the mechanisms by which apoE might affect neuronal cell metabolism is reported herein. ApoE can induce Akt/protein kinase B phosphorylation in Neuro-2a via two different pathways. Both pathways are mediated by phosphatidylinositol 3-kinase and cAMP-dependent protein kinase. The first pathway is stimulated by apoE3 and E4, but not by E2, after a 1-h incubation. The process requires the binding of apoE to the heparan sulfate proteoglycan/LDL receptor-related protein complex. The second pathway is activated after a 2-h incubation of the cells, in another isoform-dependent manner (E2 = E3 dbl greater-than sign E4) and is mediated by calcium. Our results suggest that apoE might affect cell metabolism and survival in neurons in an isoform-specific manner by inducing novel signaling pathways.

The plasma profile of major lipoprotein classes and its subdivision into particular fractions plays a crucial role in the pathogenesis of atherosclerosis and is a major predictor of coronary artery disease. Our aim was to identify genomic determinants of triglyceride and cholesterol distribution into lipoprotein fractions and lipoprotein particle sizes in the recombinant inbred rat set PXO, in which alleles of two rat models of the metabolic syndrome (SHR and PD inbred strains) segregate together with those from Brown Norway rat strain. Adult male rats of 15 PXO strains (n = 8–13/strain) and two progenitor strains SHR-Lx (n = 13) and BXH2/Cub (n = 18) were subjected to one-week of high-sucrose diet feeding. We performed association analyses of triglyceride (TG) and cholesterol (C) concentrations in 20 lipoprotein fractions and the size of major classes of lipoprotein particles utilizing 704 polymorphic microsatellite markers, the genome-wide significance was validated by 2,000 permutations per trait. Subsequent in silico focusing of the identified quantitative trait loci was completed using a map of over 20,000 single nucleotide polymorphisms. In most of the phenotypes we identified substantial gradient among the strains (e.g. VLDL-TG from 5.6 to 66.7 mg/dl). We have identified 14 loci (encompassing 1 to 65 genes) on rat chromosomes 3, 4, 7, 8, 11 and 12 showing suggestive or significant association to one or more of the studied traits. PXO strains carrying the SHR allele displayed significantly higher values of the linked traits except for LDL-TG and adiposity index. Cholesterol concentrations in large, medium and very small LDL particles were significantly associated to a haplotype block spanning part of a single gene, low density lipoprotein receptor-related protein 1B (Lrp1b). Using genome-wide association we have identified new genetic determinants of triglyceride and cholesterol distribution into lipoprotein fractions in the recombinant inbred panel of rat model strains.

The HEK293 human cell lineage is widely used in cell biology and biotechnology. Here we use whole-genome resequencing of six 293 cell lines to study the dynamics of this aneuploid genome in response to the manipulations used to generate common 293 cell derivatives, such as transformation and stable clone generation (293T); suspension growth adaptation (293S); and cytotoxic lectin selection (293SG). Remarkably, we observe that copy number alteration detection could identify the genomic region that enabled cell survival under selective conditions (i.c. ricin selection). Furthermore, we present methods to detect human/vector genome breakpoints and a user-friendly visualization tool for the 293 genome data. We also establish that the genome structure composition is in steady state for most of these cell lines when standard cell culturing conditions are used. This resource enables novel and more informed studies with 293 cells, and we will distribute the sequenced cell lines to this effect., The human embryonic kidney 293 (HEK293) cell lineage is widely used in cell biology and biotechnology. Here, the authors apply whole genome resequencing methods to characterise genomic variation in six HEK293 cell lines and suggest that this variation could affect experiments using these cell lines.

Review on EPAS1, with data on DNA/RNA, on the protein encoded and where the gene is implicated. Identity

Background: Kawasaki disease (KD) is an acute and systemic vasculitis. Its complications in coronary artery aneurysms (CAA) make KD one of the leading causes of acquired cardiovascular diseases in childhood. Low density lipoprotein receptor-related protein 1B (LRP1B) is abundantly expressed in the medial layer of coronary arteries and involved in endothelium inflammations. Purpose: We aimed to identify the role of LRP1B in CAA formation during KD progression. Methods: we investigated genetic variations in LRP1B in a Taiwanese cohort of 258 KD patients (83 with CAA and 175 without CAA complications). We used univariate and multivariate regression analyses to identify the associations between LRP1B genetic variations and KD patients. Results: CAA formation in KD was significantly associated with the LRP1B (rs6707826) genetic variant (p = 0.007). By using multivariate regression analysis, significant correlations were observed between KD with CAA complications and the presence of the TT+TG genotypes for the LRP1B rs6707826 single-nucleotide polymorphism (full model: odds ratio = 2.82; 95% CI = 1.33–5.78). Conclusion: Our results suggest that genetic polymorphism of LRP1B gene may be used as a genetic marker for the diagnosis and prognosis of the CAA formation in KD and contribute to genetic profiling studies for personalized medicine.

Review on LRP1B, with data on DNA/RNA, on the protein encoded and where the gene is implicated.

The heterodimeric hypoxia-inducible factor (HIF)-1 is a master transcriptional regulator of oxygen homeostasis and a possible target for gene therapy of ischemic disease. Although the role of oxygen concentration in HIF-1a protein stabilization is well established, it is less clear whether and how oxygen-regulated mechanisms contribute to HIF-1a protein modifications, nuclear translocation, heterodimerization with the b-subunit, recruitment of cofactors, and gene trans-activation. Because the HIF-1a protein is proteolytically degraded under normoxic conditions, we established two HeLa Tet-Off cell lines (HT42 and HT43), which inducibly overexpress high levels of HIF-1a under normoxic conditions, allowing to distinguish hypoxia-dependent from hypoxia-independent activation mechanisms. Using these cells, we found that normoxically induced HIF-1a is localized to the nucleus, binds DNA, and trans-activates reporter and endogenous target genes. The levels of p53 expression remained unaffected. The MAP kinase inhibitor PD98059 attenuated HIF-1a protein modifications and trans-activation ability but not protein stabilization and DNA-binding activity. Because overexpressed HIF-1a is fully localized to the nucleus but displays only partial DNA-binding and trans-activation activity, mitogen-activated protein kinase-dependent phosphorylation might be required for full HIF-1 activation. HIF-1a protein was also overexpressed in vivo, following the transplantation of HT42 cells into nude mice, demonstrating the feasibility of HIF-1a gene transfer.

The 39-44 kDa protein known as the receptor-associated protein binds to members of the low density lipoprotein receptor family and is found within cells that express these receptors. The receptor-associated protein has been shown to prevent premature binding of ligands to the receptors in the endoplasmic reticulum and to promote proper folding and transport of the receptors in the secretory pathway. In the thyroid, megalin (a low-density lipoprotein receptor family member) serves as an endocytic receptor for thyroglobulin. Here we present evidence that the receptor-associated protein can bind to thyroglobulin, which suggests a novel function of the receptor-associated protein, namely binding of certain megalin ligands possibly during the biosynthetic pathway. In solid-phase assays thyroglobulin was shown to bind to the receptor-associated protein with moderately high affinity (mean between K(d) and K(i) = 39.8 nM), in a calcium-dependent and saturable manner. The receptor-associated protein also bound to a native carboxyl-terminal 230-kDa thyroglobulin polypeptide, which markedly reduced binding of intact thyroglobulin to the receptor associated protein, indicating that the receptor-associated protein binding sites of thyroglobulin are located in the carboxyl-terminal portion of the molecule. In addition to thyroglobulin, the receptor-associated protein specifically bound to another megalin ligand, namely lipoprotein lipase. Because lipoprotein lipase markedly reduced receptor-associated protein binding to thyroglobulin, we concluded that the receptor-associated protein uses the same binding site/s to bind to thyroglobulin and lipoprotein lipase. Evidence of thyroglobulin binding to the receptor-associated protein was also obtained in vivo and in cultured thyroid cells. Thus, anti-receptor-associated protein antibodies precipitated intact thyroglobulin from extracts prepared from rat thyroids and cultured thyroid cells (FRTL-5 cells). Chase experiments after inhibition of protein synthesis in FRTL-5 cells showed that thyroglobulin interacts with the receptor-associated protein shortly after the beginning of thyroglobulin biosynthesis.

Insulin and insulin-like growth factor I (IGF-I) can amplify gonadotropin-stimulated steroidogenesis by augmenting the expression of key sterol regulatory genes in ovarian cells, viz. low density lipoprotein (LDL) receptor, steroidogenic acute regulatory protein, and P450 cholesterol side-chain cleavage enzyme (CYP11A). The mechanisms underlying the foregoing bihormonal interactions are not known. Accordingly, in relation to the LDL receptor gene, the present study tests the hypothesis that insulin/IGF-I and LH can act via concerted transcriptional control of promoter expression. To this end, we transiently transfected primary monolayer cultures of porcine granulosa-luteal cells with a reporter vector containing the putative 5'-upstream full-length (pLDLR1076/luc) regulatory region (-1076 to +11 bp) of the homologous LDL receptor gene driving firefly luciferase in the presence or absence of insulin (or IGF-I) and/or LH (each 100 ng/ml). Combined exposure to LH and insulin (or IGF-I) stimulated LDL receptor transcriptional activity maximally at 4 h by 8- to 20-fold, as normalized by coexpression of Renilla luciferase. Further analysis of multiple 5'-nested deletional constructs of the LDL receptor gene promoter showed that deletion of -139 bp upstream of the transcriptional start site virtually abolished basal expression and promoter responsiveness to LH and insulin/IGF-I. In contrast, full basal activity and 60-80% of maximal monohormonal and bihormonal drive were retained by the -255 to +11 bp fragment. As LDL receptor gene expression in other tissues is negatively regulated by the abundance of intracellular free cholesterol, we assessed the impact of concomitant pretreatment of granulosa-luteal cells with an exogenous soluble sterol (25-hydroxycholesterol, 1 and 10 microM). Excess sterol markedly (50-70%) attenuated bihormonally and, in lesser measure, LH-stimulated and basal LDL receptor promoter expression, thus affirming a feedback-sensitive sterol-repressive region in this gene. Non-LH receptor-dependent agonists of protein kinase A (PKA), 8-bromo-cAMP (1 mM), and forskolin (10 microM) with or without insulin/IGF-I costimulation likewise augmented LDL receptor promoter expression with similar strong dependency on the -255 to -139 bp 5'-upstream region. To assess more specific PKA-dependent mediation of LH's contribution to combined hormonal drive, the LDL receptor (-1076 to +11 bp) reporter plasmid was cotransfected with a full-sequence rabbit muscle protein kinase inhibitor (PKI) minigene driven constitutively by a Rous sarcoma virus promoter. Expression of the latter PKA antagonist blocked transcriptional stimulation by LH alone as well as that by LH combined with insulin (or IGF-I) by 70-85% without reducing basal transcriptional activity. Transfection of a mutant inactive (Arg to Gly) Rous sarcoma virus/PKI gene confirmed the specificity of the PKI effect. To investigate the convergent role of the insulin/IGF-I effector pathway mediating bihormonal stimulation of LDL receptor promoter expression, transfected granulosa-luteal cells were pretreated for 30 min with two specific inhibitors of phophatidylinositol 3-kinase, wortmannin (100 nM) and LY 294002 (10 microM), or of mitogen-activated protein kinase kinase, PD 98059 (50 microM), U0126 (10 microM), or the latter's inactive derivative, U0124 (10 microM). Both classes of antagonists impeded the ability of insulin or IGF-I to enhance LH-stimulated LDL receptor promoter expression by 60-80%. In conclusion, the present analyses indicate that LH and insulin (or IGF-I) can up-regulate LDL receptor transcriptional activity supraadditively in porcine granulosa-luteal cells 1) via one or more agonistic cis-acting DNA regions located between -255 and -139 bp 5'- upstream of the transcriptional start site, 2) without abrogating sterol-sensitive repressive of this promoter, and 3) by way of intracellular mechanisms that include the PKA, phophatidylinositol 3-kinase, and mitogen-activated protein kinase signaling pathways.

Heterozygosity for a C8524T transition in the protein C gene converting Ser270(TCG) to Leu(TTG) in the protease domain was identified in a family with venous thrombosis. The mutation was associated with parallel reduction in plasma levels of protein C anticoagulant activity and protein C antigen, which is consistent with a type 1 deficiency. Transient expression of mutant protein C cDNA in human kidney 293 cells and analysis of protein C antigen in culture media and cell lysates showed that the secretion of mutant protein compared with wild-type protein was reduced by at least 97% while the intracellular content of mutant and wild-type protein was similar. Northern blot analysis of total mRNA from transfected cells showed no reduction of the mutant protein C mRNA compared with wild-type protein C mRNA. Collectively, these results indicate that the Ser270Leu mutation in the affected family caused the plasma protein C deficiency and are consistent with a disease mechanism that involves synthesis of mutant protein followed by intracellular degradation before its secretion into the extracellular space. The mutation was not present in the parents of the proband, suggesting a de novo mutation. Non-paternity was excluded after the analysis of three intragenic protein C polymorphisms and six dinucleotide repeat allele sets located in five different chromosomes.

Mutations in RP2 cause the second most frequent form of X-linked retinitis pigmentosa, a severe retinal degeneration that leads to loss of visual acuity and blindness. The RP2 gene encodes a protein with homology to cofactor C, a tubulin-folding chaperone. By searching protein sequence databases, we identified a whole set of similar molecules from diverse organisms. Protein sequence alignments show that RP2 and cofactor C represent members of two distinct orthologous groups. All previously identified missense mutations affect amino acid residues which are conserved in all RP2 orthologues or both orthologous groups. Intracellular localization of the wild-type protein and mutated variants was determined by fluorescence microscopy of cells expressing RP2 with a green fluorescent protein tag. A mutation in the N-terminus of RP2 abolishes localization to the plasma membrane in HeLa cells. C-terminal protein truncation mutations, which account for 2/3 of the pathogenic RP2 variants, lead to scattered fluorescent foci in the cytoplasm of COS-7 and HeLa cells. Analysis of protein extracts from the respective cells with anti-RP2 antibodies identified truncated proteins of expected size in a low-speed centrifugation pellet while the wild-type protein appeared in the supernatant. Moreover, no protein was detected in immortalized cell lines from patients with protein truncation mutations while mRNA was still present. Thus, loss of the protein and/or aberrant intracellular distribution might be the basis for the photoreceptor cell degeneration in most RP2 cases.