The last 10 amino acid residues beyond the hydrophobic motif are critical for the catalytic competence and function of protein kinase Cá*

The segment C-terminal to the hydrophobic motif at the V5 domain of protein kinase C (PKC) is the least conserved both in length and in amino acid identity among all PKC isozymes. By generating serial truncation mutants followed by biochemical and functional analyses, we show here that the very C terminus of PKCα is critical in conferring the full catalytic competence to the kinase and for transducing signals in cells. Deletion of one C-terminal amino acid residue caused the loss of ~60% of the catalytic activity of the mutant PKCα, whereas deletion of 10 C-terminal amino acid residues abrogated the catalytic activity of PKCα in immune complex kinase assays. The PKCα C-terminal truncation mutants were found to lose their ability to activate mitogen-activated protein kinase, to rescue apoptosis induced by the inhibition of endogenous PKC in COS cells, and to augment melatonin-stimulated neurite outgrowth. Furthermore, molecular dynamics simulations revealed that the deletion of 1 or 10 C-terminal residues results in the deformation of the V5 domain and the ATP-binding pocket, respectively. Finally, PKCα immunoprecipitated using an antibody against its C terminus had only marginal catalytic activity compared with that of the PKCα immunoprecipitated by an antibody against its N terminus. Therefore, the very C-terminal tail of PKCα is a novel determinant of the catalytic activity of PKC and a promising target for selective modulation of PKCα function. Molecules that bind preferentially to the very C terminus of distinct PKC isozymes and suppress their catalytic activity may constitute a new class of selective inhibitors of PKC.

The last five amino acid residues at the C-terminus of PRK1 /KKN is essential for full lipid responsiveness

PRK1/PKN is a member of the protein kinase C (PKC) superfamily of serine/threonine protein kinases. Despite its important role as a RhoA effector, limited information is available regarding how this kinase is regulated. We show here that the last seven amino acid residues at the C-terminus is dispensable for the catalytic activity of PRK1 but is critical for the in vivo stability of this kinase. Surprisingly, the intact hydrophobic motif in PRK1 is dispensable for 3-phosphoinositide-dependent kinase-1 (PDK-1) binding and phosphorylation of the activation loop, as the PRK1-Δ940 mutant lacking the last two residues of the hydrophobic motif and the last 5 residues at the C-terminus interacts with PDK-1 in vivo and has a similar specific activity as the wild-type protein. We also found that the last four amino acid residues at the C-terminus of PRK1 is critical for the full lipid responsiveness as the PRK1-Δ942 deletion mutant is no longer activated by arachidonic acid. Our data suggest that the very C-terminus in PRK1 is critically involved in the control of the catalytic activity and activation by lipids. Since this very C-terminal segment is the least conserved among members of the PKC superfamily, it would be a promising target for isozyme-specific pharmaceutical interventions.

The very C-terminus of PRK1/PKN is essential for its activation by RhoA and downstream signaling

PRK1 is a lipid- and Rho GTPase-activated serine/threonine protein kinase implicated in the regulation of receptor trafficking, cytoskeletal dynamics and tumorigenesis. Although Rho binding has been mapped to the HR1 region in the regulatory domain of PRK1, the mechanism involved in the control of PRK1 activation following Rho binding is poorly understood. We now provide the first evidence that the very C-terminus beyond the hydrophobic motif in PRK1 is essential for the activation of this kinase by RhoA. Deletion of the HR1 region did not completely abolish the binding of PRK1-ΔHR1 to GTPγS-RhoA nor the activation of this mutant by GTPγS-RhoA in vitro. In contrast, removing of the last six amino acid residues from the C-terminus of PRK1 or truncating of a single C-terminal residue from PRK1-ΔHR1 completely abrogated the activation of these mutants by RhoA both in vitro and in vivo. The critical dependence of the very C-terminus of PRK1 on the signaling downstream of RhoA was further demonstrated by the failure of the PRK1 mutant lacking its six C-terminal residues to augment lisophosphatidic acid-elicited neurite retraction in neuronal cells. Thus, we show that the HR1 region is necessary but not sufficient in eliciting a full activation of PRK1 upon binding of RhoA. Instead, such activation is controlled by the very C-terminus of PRK1. Our results also suggest that the very C-terminus of PRK1, which is the least conserved among members of the protein kinase C superfamily, is a potential drug target for pharmacological intervention of RhoA-mediated signaling pathways

The C-terminus of PRK2/PKNγ is required for optimal activation by RhoA in a GTP-dependent manner

PRK2/PKNγ is a Rho effector and a member of the protein kinase C superfamily of serine/threonine kinases. Here, we explore the structure–function relationship between various motifs in the C-terminal half of PRK2 and its kinase activity and regulation. We report that two threonine residues at conserved phosphoacceptor position in the activation loop and the turn motif are essential for the catalytic activity of PRK2, but the phosphomimetic Asp-978 at hydrophobic motif is dispensable for kinase catalytic  competence. Moreover, the PRK2-Δ958 mutant with the turn motif truncated still interacts with 3-phosphoinositide-dependent kinase-1 (PDK-1). Thus, both the intact hydrophobic motif and the turn motif in PRK2 are dispensable for the binding of PDK-1. We also found that while the last seven amino acid residues at the C-terminus of PRK2 are not required for the activation of the kinase by RhoA in vitro, however, the extreme C-terminal segment is critical for the full activation of PRK2 by RhoA in cells in a GTP-dependent manner. Our data suggest that the extreme C-terminus of PRK2 may represent a potential drug target for effector-specific pharmacological intervention of Rho-medicated biological processes.

Arg1098 is critical for the chloride dependence of human angiotensin I-converting enzyme C-domain catalytic activity

Angiotensin (Ang) I-converting enzyme (ACE) is a Zn²+ metalloprotease with two homologous catalytic domains. Both the N- and C-terminal domains are peptidyl dipeptidases. Hydrolysis by ACE of its decapeptide substrate Ang I is increased by Cl−, but the molecular mechanism of this regulation is unclear. A search for single substitutions to Gln among all conserved basic residues (Lys/Arg) in human ACE C-domain identified R1098Q as the sole mutant that lacked Cl− dependence. Cl−dependence is also lost when the equivalent Arg in the N-domain, Arg⁵⁰⁰, is substituted with Gln. The Arg¹⁰⁹⁸ to Lys substitution reduced Cl− binding affinity by ∼100-fold. In the absence of Cl−, substrate binding affinity (1/K m) of and catalytic efficiency (k cat/K m) for Ang I hydrolysis are increased 6.9- and 32-fold, respectively, by the Arg¹⁰⁹⁸ to Gln substitution, and are similar (<2-fold difference) to the respective wild-type C-domain catalytic constants in the presence of optimal [Cl−]. The Arg¹⁰⁹⁸ to Gln substitution also eliminates Cl− dependence for hydrolysis of tetrapeptide substrates, but activity toward these substrates is similar to that of the wild-type C-domain in the absence of Cl−. These findings indicate that: 1) Arg¹⁰⁹⁸ is a critical residue of the C-domain Cl−-binding site and 2) a basic side chain is necessary for Cl− dependence. For tetrapeptide substrates, the inability of R1098Q to recreate the high affinity state generated by the Cl−-C-domain interaction suggests that substrate interactions with the enzyme-bound Cl− are much more important for the hydrolysis of short substrates than for Ang I. Since Cl− concentrations are saturating under physiological conditions and Arg¹⁰⁹⁸ is not critical for Ang I hydrolysis, we speculate that the evolutionary pressure for the maintenance of the Cl−-binding site is its ability to allow cleavage of short cognate peptide substrates at high catalytic efficiencies.

A critical role for the short intracellular C terminus in receptor activity-modifying protein function

Receptor activity-modifying proteins (RAMPs) interact with and modify the behavior of the calcitonin receptor (CTR) and calcitonin receptor-like receptor (CLR). We have examined the contribution of the short intracellular C terminus, using constructs that delete the last eight amino acids of each RAMP. C-Terminal deletion of individual RAMPs had little effect on the signaling profile induced when complexed with CLR in COS-7 or human embryonic kidney (HEK)293 cells. Likewise, confocal microscopy revealed each of the mutant RAMPs translocated hemagglutinin-tagged CLR to the cell surface. In contrast, a pronounced effect of RAMP C-terminal truncation was seen for RAMP/CTRa complexes, studied in COS-7 cells, with significant attenuation of amylin receptor phenotype induction that was stronger for RAMP1 and -2 than RAMP3. The loss of amylin binding upon C-terminal deletion could be partially recovered with overexpression of Gαs, suggesting an impact of the RAMP C terminus on coupling of G proteins to the receptor complex. In HEK293 cells the c-Myc-RAMP1 C-terminal deletion mutant showed high receptor-independent cell surface expression; however, this construct showed low cell surface expression when expressed alone in COS-7 cells, indicating interaction of RAMPs with other cellular components via the C terminus. This mutant also had reduced cell surface expression when coexpressed with CTR. Thus, this study reveals important functionality of the RAMP C-terminal domain and identifies key differences in the role of the RAMP C terminus for CTR versus CLR-based receptors.

c-Cbl facilitates endocytosis and lysosomal degradation of cystic fibrosis transmembrane conductance regulator in human airway epithelial cells

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl− channel expressed in the apical membrane of fluid-transporting epithelia. The apical membrane density of CFTR channels is determined, in part, by endocytosis and the postendocytic sorting of CFTR for lysosomal degradation or recycling to the plasma membrane. Although previous studies suggested that ubiquitination plays a role in the postendocytic sorting of CFTR, the specific ubiquitin ligases are unknown. c-Cbl is a multifunctional molecule with ubiquitin ligase activity and a protein adaptor function. c-Cbl co-immunoprecipitated with CFTR in primary differentiated human bronchial epithelial cells and in cultured human airway cells. Small interfering RNA-mediated silencing of c-Cbl increased CFTR expression in the plasma membrane by inhibiting CFTR endocytosis and increased CFTR-mediated Cl− currents. Silencing c-Cbl did not change the expression of the ubiquitinated fraction of plasma membrane CFTR. Moreover, the c-Cbl mutant with impaired ubiquitin ligase activity (FLAG-70Z-Cbl) did not affect the plasma membrane expression or the endocytosis of CFTR. In contrast, the c-Cbl mutant with the truncated C-terminal region (FLAG-Cbl-480), responsible for protein adaptor function, had a dominant interfering effect on the endocytosis and plasma membrane expression of CFTR. Moreover, CFTR and c-Cbl co-localized and co-immunoprecipitated in early endosomes, and silencing c-Cbl reduced the amount of ubiquitinated CFTR in early endosomes. In summary, our data demonstrate that in human airway epithelial cells, c-Cbl regulates CFTR by two mechanisms: first by acting as an adaptor protein and facilitating CFTR endocytosis by a ubiquitin-independent mechanism, and second by ubiquitinating CFTR in early endosomes and thereby facilitating the lysosomal degradation of CFTR.

Diverse eukaryotes have retained mitochondrial homologues of the bacterial division protein FtsZ

Mitochondrial fission requires the division of both the inner and outer mitochondrial membranes. Dynamin-related proteins operate in division of the outer membrane of probably all mitochondria, and also that of chloroplasts – organelles that have a bacterial origin like mitochondria. How the inner mitochondrial membrane divides is less well established. Homologues of the major bacterial division protein, FtsZ, are known to reside inside mitochondria of the chromophyte alga Mallomonas, a red alga, and the slime mould Dictyostelium discoideum, where these proteins are likely to act in division of the organelle. Mitochondrial FtsZ is, however, absent from the genomes of higher eukaryotes (animals, fungi, and plants), even though FtsZs are known to be essential for the division of probably all chloroplasts. To begin to understand why higher eukaryotes have lost mitochondrial FtsZ, we have sampled various diverse protists to determine which groups have retained the gene. Database searches and degenerate PCR uncovered genes for likely mitochondrial FtsZs from the glaucocystophyte Cyanophora paradoxa, the oomycete Phytophthora infestans, two haptophyte algae, and two diatoms – one being Thalassiosira pseudonana, the draft genome of which is now available. From Thalassiosira we also identified two chloroplast FtsZs, one of which appears to be undergoing a C-terminal shortening that may be common to many organellar FtsZs. Our data indicate that many protists still employ the FtsZ-based ancestral mitochondrial division mechanism, and that mitochondrial FtsZ has been lost numerous times in the evolution of eukaryotes.

Creatine transporters: a reappraisal

Creatine (Cr) plays a key role in cellular energy metabolism and is found at high concentrations in metabolically active cells such as skeletal muscle and neurons. These, and a variety of other cells, take up Cr from the extra cellular fluid by a high affinity Na⁺/Cl^--dependent creatine transporter (CrT). Mutations in the crt gene, found in several patients, lead to severe retardation of speech and mental development, accompanied by the absence of Cr in the brain.
In order to characterize CrT protein(s) on a biochemical level, antibodies were raised against synthetic peptides derived from the N- and C-terminal cDNA sequences of the putative CrT-1 protein. In total homogenates of various tissues, both antibodies, directed against these different epitopes, recognize the same two major polypetides on Western blots with apparent Mr of 70 and 55 kDa. The C-terminal CrT antibody (α-CrTCOOH) immunologically reacts with proteins located at the inner membrane of mitochondria as determined by immuno-electron microscopy, as well as by subfractionation of mitochondria. Cr-uptake experiments with isolated mitochondria showed these organelles were able to transport Cr via a sulfhydryl-reagent-sensitive transporter that could be blocked by anti-CrT antibodies when the outer mitochondrial membrane was permeabilized. We concluded that mitochondria are able to specifically take-up Cr from the cytosol, via a low-affinity CrT, and that the above polypeptides would likely represent mitochondrial CrT(s). However, by mass spectrometry techniques, the immunologically reactive proteins, detected by our anti-CrT antibodies, were identified as E2 components of the agr-keto acid dehydrogenase multi enzyme complexes, namely pyruvate dehydrogenase (PDH), branched chain keto acid dehydrogenase (BC-KADH) and α-ketoglutarate dehydrogenase (α-KGDH). The E2 components of PDH are membrane associated, whilst it would be expected that a mitochondrial CrT would be a transmembrane protein. Results of phase partitioning by Triton X-114, as well as washing of mitochondrial membranes at basic pH, support that these immunologically cross-reactive proteins are, as expected for E2 components, membrane associated rather than transmembrane. On the other hand, the fact that mitochondrial Cr uptake into intact mitoplast could be blocked by our α-CrTCOOH antibodies, indicate that our antisera contain antibodies reactive to proteins involved in mitochondrial transport of Cr. The presence of specific antibodies against CrT is also supported by results from plasma membrane vesicles isolated from human and rat skeletal muscle, where both 55 and 70 kDa polypeptides disappeared and a single polypeptide with an apparent electrophoretic mobility of ~ 60 kDa was enriched This latter is most likely representing the genuine plasma membrane CrT.
Due to the fact that all anti-CrT antibodies that were independently prepared by several laboratories seem to cross-react with non-CrT polypeptides, specifically with E2 components of mitochondrial dehydrogenases, further research is required to characterise on a biochemical/biophysical level the CrT polypeptides, e.g. to determine whether the ~ 60 kDa polypeptide is indeed a bona-fide CrT and to identify the mitochondrial transporter that is able to facilitate Cr-uptake into these organelles. Therefore, the anti-CrT antibodies available so far should only be used with these precautions in mind. This holds especially true for quantitation of CrT polypeptides by Western blots, e.g. when trying to answer whether CrT's are up- or down-regulated by certain experimental interventions or under pathological conditions.
In conclusion, we still hold to the scheme that besides the high-affinity and high-efficiency plasmalemma CrT there exists an additional low affinity high Km Cr uptake mechanism in mitochondria. However, the exact biochemical nature of this mitochondrial creatine transport, still remains elusive. Finally, similar to the creatine kinase (CK) isoenzymes, which are specifically located at different cellular compartments, also the substrates of CK are compartmentalized in cytosolic and mitochondrial pools. This is in line with ¹⁴C-Cr-isotope tracer studies and a number of [³¹P]-NMR magnetization transfer studies, as well as with recent [¹H]-NMR spectroscopy data.

Identification of two proteins, S14 and UIP1, that interact with UCH37

By the use of the yeast two-hybrid screen we have identified two proteins that interacted with UCH37: S14, which is a subunit of PA700 and a novel protein, UIP1 (UCH37 interacting protein 1). The interaction of UCH37 with S14 or UIP1 was confirmed by in vitro binding assay and in vivo co-immunoprecipitation analysis. The C-terminal extension of UCH37 is essential for interaction with S14 or UIP1 as shown by the yeast two-hybrid assay and the in vitro binding assay. Furthermore, UIP1 blocked the interaction between UCH37 and S14 in vitro.

Molecular characterization of Osh6p, an oxysterol binding protein homolog in the yeast Saccharomyces cerevisiae

Oxysterol binding protein (OSBP) and its homologs have been shown to regulate lipid metabolism and vesicular transport. However, the exact molecular function of individual OSBP homologs remains uncharacterized. Here we demonstrate that the yeast OSBP homolog, Osh6p, bound phosphatidic acid and phosphoinositides via its N-terminal half containing the conserved OSBP-related domain (ORD). Using a green fluorescent protein fusion chimera, Osh6p was found to localize to the cytosol and patch-like or punctate structures in the vicinity of the plasma membrane. Further examination by domain mapping demonstrated that the N-terminal half was associated with FM4-64 positive membrane compartments; however, the C-terminal half containing a putative coiled-coil was localized to the nucleoplasm. Functional analysis showed that the deletion of OSH6 led to a significant increase in total cellular ergosterols, whereas OSH6 overexpression caused both a significant decrease in ergosterol levels and resistance to nystatin. Oleate incorporation into sterol esters was affected in OSH6 overexpressing cells. However, Lucifer yellow internalization, and FM4-64 uptake and transport were unaffected in both OSH6 deletion and overexpressing cells. Furthermore, osh6Δ exhibited no defect in carboxypeptidase Y transport and maturation. Lastly, we demonstrated that both the conserved ORD and the putative coiled-coil motif were indispensable for the in vivo function of Osh6p. These data suggest that Osh6p plays a role primarily in regulating cellular sterol metabolism, possibly stero transport.

ORP3 splice variants and their expression in human tissues and hematopoietic cells

ORP3 is a member of the newly described family of oxysterol-binding protein (OSBP)-related proteins (ORPs). We previously demonstrated that this gene is highly expressed in CD34+ hematopoietic progenitor cells, and deduced that the "full-length" ORP3 gene comprises 23 exons and encodes a predicted protein of 887 amino acids with a C-terminal OSBP domain and an N-terminal pleckstrin homology domain. To further characterize the gene, we cloned ORP3 cDNA from PCR products and identified multiple splice variants. A total of eight isoforms were demonstrated with alternative splicing of exons 9, 12, and 15. Isoforms with an extension to exon 15 truncate the OSBP domain of the predicted protein sequence. In human tissues there was specific isoform distribution, with most tissues expressing varied levels of isoforms with the complete OSBP domain; while only whole brain, kidney, spleen, thymus, and thyroid expressed high levels of the isoforms associated with the truncated OSBP domain. Interestingly, the expression in cerebellum, heart, and liver of most isoforms was negligible. These data suggest that differential mRNA splicing may have resulted in functionally distinct forms of the ORP3 gene.

Targeted disruption of the basic Krüppel-like factor gene (Klf3) reveals a role in adipogenesis

Krüppel-like factors (KLFs) recognize CACCC and GC-rich sequences in gene regulatory elements. Here, we describe the disruption of the murine basic Krüppel-like factor gene (Bklf or Klf3). Klf3 knockout mice have less white adipose tissue, and their fat pads contain smaller and fewer cells. Adipocyte differentiation is altered in murine embryonic fibroblasts from Klf3 knockouts. Klf3 expression was studied in the 3T3-L1 cellular system. Adipocyte differentiation is accompanied by a decline in Klf3 expression, and forced overexpression of Klf3 blocks 3T3-L1 differentiation. Klf3 represses transcription by recruiting C-terminal binding protein (CtBP) corepressors. CtBPs bind NADH and may function as metabolic sensors. A Klf3 mutant that does not bind CtBP cannot block adipogenesis. Other KLFs, Klf2, Klf5, and Klf15, also regulate adipogenesis, and functional CACCC elements occur in key adipogenic genes, including in the C/ebpα promoter. We find that C/ebpα is derepressed in Klf3 and Ctbp knockout fibroblasts and adipocytes from Klf3 knockout mice. Chromatin immunoprecipitations confirm that Klf3 binds the C/ebpα promoter in vivo. These results implicate Klf3 and CtBP in controlling adipogenesis.

Use of calcium, folate and vitamin D3-fortified milk for 6 months improves nutritional status but not bone mass or turnover, in a group of Australia aged care

In residential care, inadequate calcium and folate intakes and low serum vitamin D (25(OH)D) concentrations are common. We assessed whether daily provision of calcium, folate, and vitamin D3-fortified milk for 6 months improved nutritional status (serum micronutrients), bone quality (heel ultrasound), bone turnover markers (parathyroid hormone, C-terminal collagen I telopeptide, terminal propeptide of type I procollagen), and/or muscle strength and mobility in a group of Australian aged care residents. One hundred and seven residents completed the study (mean (SD) age: 79.9 (10.1) years; body weight: 68.4 (15.4) kg). The median (inter-quartile range) volume of fortified milk consumed was 160 (149) ml/day. At the end of the study, the median daily vitamin D intake increased to 10.4 (8.7) μg (P < .001), which is 70% of the adequate intake (15 μg); and calcium density (mg/MJ) was higher over the study period compared with baseline (161 ± 5 mg/MJ vs. 142 ± 4 mg/MJ, P < .001). Serum 25(OH)D concentrations increased by 23 ± 2 nmol/L (83 (107)%, P < .001), yet remained in the insufficient range (mean 45 ± 2 nmol/L). Consumption of greater than the median intake of milk (160 ml/day) (n = 54, 50%) increased serum 25(OH)D levels into the adequate range (53 ± 2 nmol/L) and reduced serum parathyroid hormone by 24% (P = .045). There was no effect on bone quality, bone turnover markers, muscle strength, or mobility. Consumption of fortified milk increased dietary vitamin D intake and raised serum 25(OH)D concentrations, but not to the level thought to reduce fracture risk. If calcium-fortified milk also was used in cooking and milk drinks, this approach could allow residents to achieve a dietary calcium intake close to recommended levels. A vitamin D supplement would be recommended to ensure adequate vitamin D status for all residents.

Identification of novel genes differentially expressed in haemopoietic progenitor cells

The biochemical and molecular processes that maintain the stem cell pool, and govern the proliferation and differentiation of haemopoietic stem/progenitor cells (HSPCs) have been widely investigated but are incompletely understood. The purpose of this study was to identify and characterise novel genes that may play a part in regulating the mechanisms that control the proliferation, differentiation and self-renewal of human HSPCs. Reverse transcription differential display polymerase chain reaction (dd-PCR) was used to identify differences in gene expression between a HSPC population defined by expression of the CD34 phenotype, and the more mature CD34 depleted populations. A total of 6 differentially expressed complementary deoxyribonucleic acid (cDNA) sequences were identified. Four of these transcripts were homologous to well characterised genes, while two (band 1 and band 20) were homologous to unknown and uncharacterised partial gene sequences on the GenBank database and were thus chosen for further investigation. The partial cDNA sequences for band 1 and band 20 were designated ORP-3 and MERP-1 (respectively) due to homologies with other well-characterised gene families. Differential expression of the ORP-3 and MERP-1 genes was confirmed using Taqman™ real-time polymerase chain reaction (PCR) with 3 - 4-fold and 4-10 -fold higher levels in the CD34+ fractions of haemopoietic cells compared to CD34- populations respectively. Additionally, expression of both these genes was down regulated with proliferation and differentiation of CD34+ cells further confirming higher expression in a less differentiated subset of haemopoietic cells. The full coding sequences of ORP-3 and MERP-1 were elucidated using bioinformatics, rapid amplification of cDNA ends (RACE) and PCR amplification. The MERP-1 cDNA is 2600 nucleotides (nt) long, and localizes by bioinformatics to chromosome 7.. It consists of three exons and 2 introns spanning an entire length of 31.4 kilobases (kb). The MERP-1 open reading frame (ORF) codes for a putative 344 amino acid (aa) type II transmembrane protein with an extracellular C-terminal ependymin like-domain and an intracellular N-terminal sequence with significant homology to the cytoplasmic domains of members of the protocadherin family of transmembrane glycoproteins. Ependymins and protocadherins are well-characterised calcium-dependant cell adhesion glycoproteins. Although the function of MERP-1 remains to be elucidated, it is possible that MERP-1 like its homologues plays a role in calcium dependent cell adhesion. Differential expression of the MERP-1 gene in haemopoietic cells suggests a role in haemopoietic stem cell proliferation and differentiation, however, its broad tissue distribution implies that it may also play a role in many cell types. Characterization of the MERP-1 protein is required to elucidate these possible roles. The ORP-3 cDNA is 6631nt long, and localizes by bioinformatics to chromosome 7pl5-p21. It consists of 23 exons and 22 introns spanning an entire length of 183.5kb. The ORP-3 ORF codes for a putative 887aa protein which displays the consensus sequence for a highly conserved oxysterol-binding domain. Other well-characterised proteins expressing these domains have been demonstrated to bind oxysterols (OS) in a dose dependant fashion. OS are hydroxylated derivatives of cholesterol Their biological activities include inhibition of cholesterol biosynthesis and cell proliferation in a variety of cell types, including haemopoietic cells. Differential expression of the ORP-3 gene in haemopoietic cells suggests a possible role in the transduction of OS effects on haemopoietic cells, however, its broad tissue distribution implies that it may also play a role in many cell types. Further investigation of ORP-3 gene expression demonstrates a significant correlation with CD34+ sample purity, and 2-fold higher expression in a population of haemopoietic cells defined by the CD34+38- phenotype compared to more mature CD34+38+ cells. This finding, taken together with the previous observation of down-regulation of ORP-3 expression with proliferation and differentiation of CD34+ cells, indicates that ORP-3 expression may be higher in a less differentiated subset of cells with a higher proliferative capacity. This hypothesis is supported by the observation that expression of the ORP-3 gene is approximately 2-fold lower in differentiated HL60 promyelocytic cells compared to control, undifferentiated cells. ORP-3 expression in HL60 cells during normal culture conditions was also found to vary with expression positively correlated with cell number. This indicates a possible cell cycle effect on ORP-3 gene expression with levels highest when cell density, and therefore the percentage of cells in G(0)/G(1) phase of the cell cycle is highest. This observation also correlates with the observation of higher ORP-3 expression in CD34+38-cells, and in CD34+ and HL60 cells undergoing OS induced and camptothecin induced apoptosis that is preceded by cell cycle arrest at G(0)/G(1). Expression of the ORP-3 gene in CD34+ HSPCs from UCB was significantly decreased to approximately half the levels observed in control cells after 24 hours incubation in transforming growth factor beta-1 (TGFâl). As ≥90% of these cells are stimulated into cell cycle entry by TGFâl, this observation further supports the hypothesis that ORP-3 expression is highest when cells reside in the G(0)/G(1) phase of the cell cycle. Data obtained from investigation of ORP-3 gene expression in synchronised HL60 cells however does not support nor disprove this hypothesis. Culture of CD34+ enriched HSPCs and HL60 cells with 25-OHC significantly increased ORP-3 gene expression to approximately 1.5 times control levels. However, as 25-OHC treatment also increased the percentage of apoptotic cells in these experiments, it is not valid to make any conclusions regarding the regulation of ORP-3 gene expression by OS. Indeed, the observation that camptothecin induced apoptosis also increased ORP-3 gene expression in HL60 cells raises the possibility that up-regulation of ORP-3 gene expression is also associated with apoptosis, Taken together, expression of the ORP-3 gene appears to be regulated by differentiation and apoptosis of haemopoietic progenitors, and may also be positively associated with proliferative and G(0)/G(1) cell cycle status indicating a possible role in all of these processes. Given the important regulatory role of apoptosis in haemopoiesis and differential expression of the ORP-3 gene in haemopoietic progenitors, final investigations were conducted to examine the effects OS on human HSPCs. Granulocyte/macrophage colony forming units (CFU-GM) generated from human bone marrow (ABM) and umbilical cord blood (UCB) were grown in the presence of varying concentrations of three different OS - 7keto-cholesterol (7K-C), 7beta-hydroxycholesterol (7p-OHC) and 25-hydroxycholesterol (25-OHC). Similarly, the effect of OS on HL60 and CD34+ cells was investigated using annexin-V staining and flow cytometry to measure apoptosis. Reduction of nitroblue tetrazolium (NBT) was used to assess differentiative status of HL60 cells. CFU-GM from ABM and HL60 growth was inhibited by all three OS tested, with 25-OHC being the most potent. 25-OHC inhibited ≥50% of bone marrow CFU-GM and ≥95% of HL60 cell growth at a level of 1 ug/ml. Compared to UCB, CFU-GM derived from ABM were more sensitive to the effects of all OS tested. Only 25-OHC and 7(5-OHC significantly inhibited growth of UCB derived CFU-GM. OS treatment increased the number of annexin-V CD34+ cells and NBT positive HL60 cells indicating that OS inhibition of CFU-GM and HL60 cell growth can be attributed to induction of apoptosis and differentiation. From these studies, it can be concluded that dd-PCR is an excellent tool for the discovery of novel genes expressed in human HSPCs. Characterisation of the proteins encoded by the novel genes ORP-3 and MERP-1 may reveal a regulatory role for these genes in haemopoiesis. Finally, investigations into the effects of OS on haemopoietic progenitor cells has revealed that OS are a new class of inhibitors of HSPC proliferation of potential relevance in vivo and in vitro.