Unique C-terminal Domain Research Articles

A functional link between the human cell cycle-regulatory phosphatase Cdc14A and the atypical mitogen-activated kinase Erk3

Cdc14 is a member of the dual-specificity phosphatase family, which is essential for faithful cell cycle progression in eukaryotic cells of different origin. The function of human Cdc14A (hCdc14A), however, has not been fully elucidated as only few physiological substrates have been identified. To gain insight into the biological role of Cdc14A, we performed a yeast two-hybrid screen designed to isolate substrates of this human phosphatase. Using this genetic approach, we here report the identification of Erk3, an atypical mitogen-activated protein kinase (MAPK), as a specific binding partner of hCdc14A. GST pull-down assays show that Erk3 interacts directly with hCdc14A in vitro via its unique C-terminal domain. Furthermore, biochemical analysis reveals that hCdc14A can remove cyclin-dependent kinase (Cdk)-mediated phosphorylation of Erk3 in vitro raising the possibility that Erk3 may be a potential substrate for hCdc14A in vivo. Consistent with a physiologically relevant cross-talk in vivo, we find that Cdc14A forms a stable complex with Erk3 in human cells independent of its intrinsic phosphatase activity but mediated by its regulatory C-terminal domain. We show that hCdc14A impacts the emerging signaling pathway between Erk3 and MK5, a MAPK-activated protein kinase. We document that hCdc14A upregulation leads to redistribution of the Erk3 substrate MK5 from the nucleus to the cytoplasm. In addition, we find that hCdc14A stabilizes complex formation between Erk3 and its binding partner cyclin D3, a D-type cyclin implicated in both cellular proliferation and differentiation. Collectively, our findings suggest an intimate functional relationship between the Cdc14A phosphatase and the Erk3 kinase in signaling pathways that regulate key cell-fate decisions in human cells.

A Flexible Peptide Tether Controls Accessibility of a Unique C-terminal RNA-binding Domain in Leucyl-tRNA Synthetases

A unique C-terminal domain extension is required by most leucyl-tRNA synthetases (LeuRS) for aminoacylation. In one exception, the enzymatic activity of yeast mitochondrial LeuRS is actually impeded by its own C-terminal domain. It was proposed that the yeast mitochondrial LeuRS has compromised its aminoacylation activity to some extent and adapted its C terminus for a second role in RNA splicing, which is also essential. X-ray crystal structures of the LeuRS–tRNA complex show that the 60 residue C-terminal domain is tethered to the main body of the enzyme via a flexible peptide linker and allows interactions with the tRNALeu elbow. We hypothesized that this short peptide linker would facilitate rigid body movement of the C-terminal domain as LeuRS transitions between an aminoacylation and editing complex or, in the case of yeast mitochondrial LeuRS, an RNA splicing complex. The roles of the C-terminal linker peptide for Escherichia coli and yeast mitochondrial LeuRS were investigated via deletion mutagenesis as well as by introducing chimeric swaps. Deletions within the C-terminal linker of E. coli LeuRS determined that its length, rather than its sequence, was critical to aminoacylation and editing activities. Although deletions in the yeast mitochondrial LeuRS peptide linker destabilized the protein in general, more stable chimeric enzymes that contained an E. coli LeuRS C-terminal domain showed that shortening its tether stimulated aminoacylation activity. This suggested that limiting C-terminal domain accessibility to tRNALeu facilitates its role in protein synthesis and may be a unique adaptation of yeast mitochondrial LeuRS that accommodates its second function in RNA splicing.

Filamin A negatively regulates the transcriptional activity of p73α in the cytoplasm

The transcription regulator p73α is structurally different from p53 in that it possesses a unique C-terminal domain, which has been implicated in transcriptional repression. To dissect the mechanism of repression by this domain, we performed a yeast two-hybrid screen of a HeLa cDNA library using residues 487–636 of p73α as bait and isolated a cDNA clone encoding the C-terminal portion (residues 2210–2647) of filamin A, a 280-kDa actin-binding protein. Additional yeast two-hybrid assays indicated that filamin A specifically interacts with the p73α C-terminus, which is lacking in p53 and p73β. The interaction was confirmed by GST pull-down assays in vitro and by immunoprecipitation analysis in vivo. Immunofluorescence microscopy revealed that p73α remained in the cytoplasm in A7 melanoma cells stably expressing filamin A, whereas it was localized in the nucleus of filamin A-deficient M2 cells. Deletion of the C-terminus of p73α (residues 487–636) resulted in nuclear localization in both cell types. Consistent with our interaction data, transient co-expression of filamin A resulted in the down-regulation of p73α, but not of p53, transcriptional activity on various p53-responsive promoters. Taken together, our data suggest that p73α is sequestered in the cytoplasm by filamin A, thereby inhibiting its transcriptional activity.

The C-terminal domain of T4 RNA ligase 1 confers specificity for tRNA repair

T4 RNA ligase 1 (Rnl1) is a tRNA repair enzyme that thwarts a tRNA-damaging host response to virus infection. The 374-aa Rnl1 protein consists of an N-terminal nucleotidyltransferase domain fused to a unique C-terminal domain composed of 10 alpha helices. We exploited an in vitro tRNA splicing system to demonstrate that Rnl1 has an inherent specificity for sealing tRNA with a break in the anticodon loop. The tRNA specificity is imparted by the C domain, any deletion of which caused the broken tRNA to be sealed as poorly as the linear intron in vitro and also abolished Rnl1 tRNA splicing activity in vivo. Deletion analysis demarcated Rnl1-(1-254) as a minimal catalytic domain of Rnl1, capable of all chemical steps of the nonspecific RNA ligation reaction. Alanine scanning of the N domain identified Ser103, Leu104, Lys117, and Ser118 as important for pRNA ligation in vitro and tRNA repair in vivo.

Expression and characterization of mutations in human very long-chain acyl-CoA dehydrogenase using a prokaryotic system

Very long-chain acyl-CoA dehydrogenase (VLCAD) catalyzes the first enzymatic step in the mitochondrial β-oxidation of fatty acids 14–20 carbons in length. More than 100 cases of VLCAD deficiency have been reported with the disease varying from a severe, often fatal neonatal form to a mild adult-onset form. VLCAD is distinguished from matrix-soluble acyl-CoA dehydrogenases by its unique C-terminal domain, homodimeric structure, and localization to the inner mitochondrial membrane. We have for the first time expressed and purified VLCAD using a bacterial system. Recombinant VLCAD had similar biochemical properties to those reported for native VLCAD and the bacterial system was used to study six previously described disease-causing missense mutations including the two most common mild mutations (T220M, V243A), a mutation leading to the severe disease phenotype (R429W), and three mutations in the C-terminal domain (A450P, L462P, and R573W). Of particular interest was the finding that the A450P and L462P bacterial extracts had normal or increased amounts of VLCAD antigen and activity. In the pure form L462P had roughly 30% of wild-type activity while A450P was normal. Using computer modeling both mutations were mapped to a predicted charged surface of VLCAD that we postulate interacts with the mitochondrial membrane. In a membrane pull down assay both mutants showed greatly reduced mitochondrial membrane association, suggesting a mechanism for the disease in these patients. In summary, the bacterial expression system developed here will significantly advance our understanding of both the clinical aspects of VLCAD deficiency and the basic biochemistry of the enzyme.

In Vivo Characterization of Tandem C‐Terminal Thioesterase Domains in Arthrofactin Synthetase

Macrocyclization of a peptide or a lipopeptide occurs at the last step of synthesis and is usually catalyzed by a single C-terminal thioesterase (Te) domain. Arthrofactin synthetase (Arf) from Pseudomonas sp. MIS38 represents a novel type of nonribosomal peptide synthetase that contains unique tandem C-terminal Te domains, ArfC_Te1 and ArfC_Te2. In order to analyze their function in vivo, site-directed mutagenesis was introduced at the putative active-site residues in ArfC_Te1 and ArfC_Te2. It was found that both Te domains were functional. Peaks corresponding to arthrofactin and its derivatives were absent in ArfC_Te1:S89A, ArfC_Te1:S89T, and ArfC_Te1:E26G/F27A mutants, and the production of arthrofactin by ArfC_Te2:S92A, ArfC_Te2:S92A/D118A, and ArfCDeltaTe2 was reduced by 95 % without an alteration of the cyclic lipoundecapeptide structure. These results suggest that Ser89 in ArfC_Te1 is essential for the completion of macrocyclization and the release of product. Glu26 and Phe27 residues are also part of the active site of ArfC_Te1. ArfC_Te2 might have been added during the evolution of Arf in order to improve macrocyclization efficiency.

The crystal structure of DR2241 from Deinococcus radiodurans at 1.9 Å resolution reveals a multi-domain protein with structural similarity to chelatases but also with two additional novel domains

A unique family of proteins have been identified in the Deinococcus genus with an N-terminal cobalamin (vitamin B 12) chelatase domain denoted CbiX and an additional unique C-terminal domain with unknown function. Here we report the first crystal structure from this new family of proteins with the structure of Deinococcus radiodurans protein DR2241. The structure reveals a multi-domain protein where domains A (residues 1–132) has the same fold as the small CbiX (CbiX S), domains A and B (residues 1–272) follow the chelatase super-family fold and the two additional unique domains C and D have no structural homologues. Domain D harbours the sequence motifs CxxC and CxxxC, in which DR2241 gives the first evidence that these motifs bind a [4Fe–4S] iron–sulphur cluster. In solution there are indications of multimeric forms, and in the crystallographic asymmetric unit a tetramer is found where domains C and D are involved in stabilising the tetrameric assembly.

Structural insights into the first step of RNA-dependent cysteine biosynthesis in archaea

Cysteine is ligated to tRNA(Cys) by cysteinyl-tRNA synthetase in most organisms. However, in methanogenic archaea lacking cysteinyl-tRNA synthetase, O-phosphoserine is ligated to tRNA(Cys) by O-phosphoseryl-tRNA synthetase (SepRS), and the phosphoseryl-tRNA(Cys) is converted to cysteinyl-tRNA(Cys). In this study, we determined the crystal structure of the SepRS tetramer in complex with tRNA(Cys) and O-phosphoserine at 2.6-A resolution. The catalytic domain of SepRS recognizes the negatively charged side chain of O-phosphoserine at a noncanonical site, using the dipole moment of a conserved alpha-helix. The unique C-terminal domain specifically recognizes the anticodon GCA of tRNA(Cys). On the basis of the structure, we engineered SepRS to recognize tRNA(Cys) mutants with the anticodons UCA and CUA and clarified the anticodon recognition mechanism by crystallography. The mutant SepRS-tRNA pairs may be useful for translational incorporation of O-phosphoserine into proteins in response to the stop codons UGA and UAG.

X-ray Crystallographic and Steady State Fluorescence Characterization of the Protein Dynamics of Yeast Polyadenylate Polymerase

Polyadenylate polymerase (PAP) catalyzes the synthesis of poly(A) tails on the 3′-end of pre-mRNA. PAP is composed of three domains: an N-terminal nucleotide-binding domain (homologous to the palm domain of DNA and RNA polymerases), a middle domain (containing other conserved, catalytically important residues), and a unique C-terminal domain (involved in protein–protein interactions required for 3′-end formation). Previous X-ray crystallographic studies have shown that the domains are arranged in a V-shape such that they form a central cleft with the active site located at the base of the cleft at the interface between the N-terminal and middle domains. In the previous studies, the nucleotides were bound directly to the N-terminal domain and exhibited a conspicuous lack of adenine-specific interactions that would constitute nucleotide recognition. Furthermore, it was postulated that base-specific contacts with residues in the middle domain could occur either as a result of a change in the conformation of the nucleotide or domain movement. To address these issues and to better characterize the structural basis of substrate recognition and catalysis, we report two new crystal structures of yeast PAP. A comparison of these structures reveals that the N-terminal and C-terminal domains of PAP move independently as rigid bodies along two well defined axes of rotation. Modeling of the nucleotide into the most closed state allows us to deduce specific nucleotide interactions involving residues in the middle domain (K215, Y224 and N226) that are proposed to be involved in substrate binding and specificity. To further investigate the nature of PAP domain flexibility, 2-aminopurine labeled molecular probes were employed in steady state fluorescence and acrylamide quenching experiments. The results suggest that the closed domain conformation is stabilized upon recognition of the correct subtrate, MgATP, in an enzyme-substrate ternary complex. The implications of these results on the enzyme mechanism of PAP and the possible role for domain motion in an induced fit mechanism are discussed.

A New Class of Signal Transducer in His-Asp Phosphorelay Systems

Nitrate transport activity of the LtnT permease of the cyanobacterium Synechococcus elongatus is activated when LtnA, a response regulator without an effector domain, is phosphorylated by LtnB, a hybrid histidine kinase. We identified a protein (LtnC) that is required for activation of LtnT. LtnC consists of an N-terminal histidine-containing phosphoacceptor (HisKA) domain, a receiver domain, and a unique C-terminal domain found in some cyanobacterial proteins. Because LtnC lacks an ATP-binding kinase domain of a histidine kinase, it is incapable of autophosphorylation, but LtnC is phosphorylated by LtnA. The histidine residue in the HisKA domain but not the aspartate residue in the receiver domain is essential for phosphorylation of LtnC and activation of LtnT. LtnC phosphorylation leads to oligomerization of the protein. Fusion of the C-terminal domain of LtnC to glutathione S-transferase, which forms oligomers, also activates LtnT, suggesting that oligomerization of the LtnC C-terminal domain causes LtnT activation. These results indicate that the C-terminal domain of LtnC acts as an effector domain that directs the output of the signal from the phosphorelay system. The two-step (His-Asp-His) phosphorelay system, composed of the LtnB, LtnA, and LtnC proteins, is distinct from the known phosphorelay systems, namely, the typical two-component system (His-Asp) and the multistep phosphorelay system (His-Asp-His-Asp), because the HisKA domain of LtnC is the terminal phosphoacceptor that determines the signal output. LtnC is a new class of signal transducer in His-Asp phosphorelay systems that contains a HisKA domain and an effector domain.

Functional Divergence of a Unique C-terminal Domain of Leucyl-tRNA Synthetase to Accommodate Its Splicing and Aminoacylation Roles

Leucyl-tRNA synthetase (LeuRS) performs dual essential roles in group I intron RNA splicing as well as protein synthesis within the yeast mitochondria. Deletions of the C terminus differentially impact the two functions of the enzyme in splicing and aminoacylation in vivo. Herein, we determined that a fiveamino acid C-terminal deletion of LeuRS, which does not complement a null strain, can form a ternary complex with the bI4 intron and its maturase splicing partner. However, the complex fails to stimulate splicing activity. The x-ray co-crystal structure of LeuRS showed that a C-terminal extension of about 60 amino acids forms a discrete domain, which is unique among the LeuRSs and interacts with the corner of the L-shaped tRNALeu. Interestingly, deletion of the entire yeast mitochondrial LeuRS C-terminal domain enhanced its aminoacylation and amino acid editing activities. In striking contrast, deletion of the corresponding C-terminal domain of Escherichia coli LeuRS abolished aminoacylation of tRNALeu and also amino acid editing of mischarged tRNA molecules. These results suggest that the role of the leucine-specific C-terminal domain in tRNA recognition for aminoacylation and amino acid editing has adapted differentially and with surprisingly opposite effects. We propose that the secondary role of yeast mitochondrial LeuRS in RNA splicing has impacted the functional evolution of this critical C-terminal domain.

The N‐terminal ATPase AT‐hook‐containing region of the Arabidopsis chromatin‐remodeling protein SPLAYED is sufficient for biological activity

The SNF2-like chromatin-remodeling ATPase SPLAYED (SYD) was identified as a co-activator of floral homeotic gene expression in Arabidopsis. SYD is also required for meristem maintenance and regulates flowering under a non-inductive photoperiod. SNF2 ATPases are structurally and functionally conserved from yeast to humans. In addition to the conserved protein features, SYD has a large unique C-terminal domain. We show here that SYD is present as two forms in the nucleus, full-length and truncated, with the latter apparently lacking the C-terminal domain. The ratio of the two forms of endogenous SYD differs in juvenile and in adult tissues. Furthermore, an SYD variant lacking the C-terminal domain (SYDDeltaC) rescues the syd null mutant, indicating that the N-terminal ATPase AT-hook-containing region of SYD is sufficient for biological activity. Plants expressing SYDDeltaC show molecular and morphological phenotypes opposite to those of the null mutant, suggesting that the construct results in increased activity. This increased activity is at least in part due to elevated SYD protein levels in these lines. We propose that the C-terminal domain may control SYD accumulation and/or specific activity in the context of the full-length protein. The presence of the C-terminal domain in rice SYD suggests that its role is probably conserved in the two classes of flowering plants.

Biochemical Characterization and Crystal Structure of Synechocystis Arogenate Dehydrogenase Provide Insights into Catalytic Reaction

The extreme diversity in substrate specificity, and in the regulation mechanism of arogenate/prephenate dehydrogenase enzymes in nature, makes a comparative structural study of these enzymes of great interest. We report here on the biochemical and structural characterization of arogenate dehydrogenase from Synechocystis sp. (TyrAsy). This work paves the way for the understanding of the structural determinants leading to diversity in substrate specificity, and of the regulation mechanisms of arogenate/prephenate dehydrogenases. The overall structure of TyrAsy in complex with NADP was refined to 1.6 A. The asymmetric unit contains two TyrAsy homodimers, with each monomer consisting of a nucleotide binding N-terminal domain and a particularly unique alpha-helical C-terminal dimerization domain. The substrate arogenate was modeled into the active site. The model of the ternary complex enzyme-NADP-arogenate nicely reveals at the atomic level the concerted mechanism of the arogenate/prephenate dehydrogenase reaction.

A potential role for the Src‐like adapter protein SLAP‐2 in signaling by the colony stimulating factor‐1 receptor

The development of macrophages from myeloid progenitor cells is primarily controlled by the growth factor colony stimulating factor-1 (CSF-1) and its cognate receptor, a transmembrane tyrosine kinase encoded by the c-Fms proto-oncogene. The CSF-1 receptor exerts its biological effects on cells via a range of signaling proteins including Erk1/2 and Akt. Here we have investigated the potential involvement of the Src-like adapter protein (SLAP-2) in signaling by the CSF-1 receptor in mouse bone marrow-derived macrophages. RT-PCR analysis revealed constitutive expression of the SLAP-2 gene in bone marrow macrophages. Surprisingly, co-immunoprecipitation and GST binding experiments demonstrated that the CSF-1 receptor could bind to SLAP-2 in a ligand-independent manner. Furthermore, the binding of SLAP-2 to the CSF-1 receptor involved multiple domains of SLAP-2. SLAP-2 also bound c-Cbl, with the interaction being mediated, at least in part, by the unique C-terminal domain of SLAP-2. Overexpression of SLAP-2 in bone marrow macrophages partially suppressed the CSF-1-induced tyrosine phosphorylation and/or expression level of a approximately 80 kDa protein without affecting CSF-1-induced global tyrosine phosphorylation, or activation of Akt or Erk1/2. Significantly, CSF-1 stimulation induced serine phosphorylation of SLAP-2. Pharmacologic inhibition of specific protein kinases revealed that CSF-1-induced phosphorylation of SLAP-2 was dependent on JNK activity. Taken together, our results suggest that SLAP-2 could potentially be involved in signaling by the CSF-1 receptor.

Mutagenesis analysis of the nsp4 main proteinase reveals determinants of arterivirus replicase polyprotein autoprocessing.

Nonstructural protein 4 (nsp4; 204 amino acids) is the chymotrypsin-like serine main proteinase of the arterivirus Equine arteritis virus (order Nidovirales), which controls the maturation of the replicase complex. nsp4 includes a unique C-terminal domain (CTD) connected to the catalytic two-beta-barrel structure by the poorly conserved residues 155 and 156. This dipeptide might be part of a hinge region (HR) that facilitates interdomain movements and thereby regulates (in time and space) autoprocessing of replicase polyproteins pp1a and pp1ab at eight sites that are conserved in arteriviruses. To test this hypothesis, we characterized nsp4 proteinase mutants carrying either point mutations in the putative HR domain or a large deletion in the CTD. When tested in a reverse genetics system, three groups of mutants were recognized (wild-type-like, debilitated, and dead), which was in line with the expected impact of mutations on HR flexibility. When tested in a transient expression system, the effects of the mutations on the production and turnover of replicase proteins varied widely. They were cleavage product specific and revealed a pronounced modulating effect of moieties derived from the nsp1-3 region of pp1a. Mutations that were lethal affected the efficiency of polyprotein autoprocessing most strongly. These mutants may be impaired in the accumulation of nsp5-7 and/or suffer from delayed or otherwise perturbed processing at the nsp5/6 and nsp6/7 junctions. On average, the production of nsp7-8 seems to be the most resistant to debilitating nsp4 mutations. Our results further prove that the CTD is essential for a vital nsp4 property other than catalysis.

RECQ5/QE DNA Helicase Interacts with Retrotransposon mdg3 gag, an HIV Nucleocapsid-Related Protein

Three hereditary human disorders, Werner’s syndrome, Bloom’s syndrome and a subset of Rothmund-Thomson syndrome, are associated with the loss of function of the respective RecQ homologues BLM, WRN, and RTS. These RecQ homologues are composed of a conserved helicase domain flanking C- and N-terminal domains. In contrast to BLM, WRN, and RTS, another RecQ homologue, RECQ5, possesses only short N-terminal region preceding the helicase domain and a long unique C-terminal domain. Although no disease has yet been genetically linked to a mutation in RECQ5, the prominent roles of RecQ helicase in the maintenance of genome stability suggest that RECQ5 helicase is likely to be important in vivo. To acquire a better understanding of RECQ5 function, we investigated protein interaction with the C-terminal domain of Drosophila melanogaster RECQ5/QE. A portion of Drosophila melanogaster retrotransposon mdg3, which corresponds to a nucleocapsid protein (gag-NC), was identified by use of the yeast two-hybrid system as interacting specifically with it. Glutathione S-transferase (GST) pull down experiments indicated that the mdg3 gag-NC bound mainly to an acidic region in the C-terminal domain of RECQ5/ QE, which is adjacent to the RecQ helicase domain. The helicase activity of RECQ5/QE was stimulated by mdg3 gag-NC protein in vitro. These data suggest that RECQ5/QE helicase interacts physically and functionally with mdg3 gag-NC through the acidic region and that RecQ homologue might be involved in retrotransposition and genomic stability.

Cloning and genomic characterization of sytdep, a new synaptotagmin XIV-related gene

We have identified a new human gene coined sytdep ( synapto tagmin XIV- derived protein) in human neutrophils. Sytdep encodes a 188-amino acid sequence with a 21.435 kDa deduced molecular mass, showing 75% identity to human synaptotagmin (syt) XIV. Human neutrophils express sytdep, but not syt XIV. Sytdep was upregulated during HL-60 neutrophil differentiation. Sytdep gene is located in human chromosome 4 and contains a unique exon, whereas syt XIV gene, located in chromosome 1, comprises 10 exons with 9 introns. Mouse genome did not contain sytdep. The N-terminal region of sytdep shows no homology with any known protein and, unlike synaptotagmin XIV isoforms, sytdep shows a unique C-terminal C2B domain. Polyclonal antibodies against the C2B domain of syt XIV recognized sytdep as a 27-kDa protein in human neutrophils. Genomic analyses suggest that human sytdep could derive from a retrotranslocation of a syt XIV transcript into chromosome 4.

Preparation and Characterization of a Monoclonal Antibody Against CKLF1 Using DNA Immunization withIn VivoElectroporation

Chemokine-like factor 1 (CKLF1) is a newly cloned human cytokine from PHA-stimulated U937 cells in our laboratory, which belongs to a novel gene family and has at least three alternative RNA splicing forms: CKLF2, CKLF3, and CKLF4. Former studies demonstrated that CKLF1 has a chemotaxis effect on different leukocytes both in vitro and in vivo. It can also stimulate the proliferation of mouse skeletal muscle cells and bone marrow cells. This study was designed to generate CKLF1-specific monoclonal antibody (MAb) for further exploration of its structure and function. Mice were immunized intramuscularly with naked plasmid DNA encoding CKLF1 gene with in vivo electroporation. Hybridomas were generated by the fusion of the spleenocytes to Sp2/0 myeloma cells. One hybridoma cell line designated as M4, which is stable in secreting anti- CKLF1 MAb, was generated. It belongs to the IgG1 isotype and is specific for the unique C-terminal domain of CKLFs protein. The specificity of this antibody has been assessed by enzyme-linked immunosorbent assay (ELISA) and Western blot. Our results demonstrated that intramuscular injection of naked DNA encoding CKLF1 gene combined with in vivo electroporation is an effective and simple method to raise MAbs that can be used for basic and clinical research of CKLF1 protein.

Photochemistry and Photobiology of Cryptochrome Blue‐light Photopigments: The Search for a Photocycle

Cryptochromes are flavoproteins that exhibit high sequence and structural similarity to the light-dependent DNA-repair enzyme, photolyase. Cryptochromes have lost the ability to repair DNA; instead, they use the energy from near-UV/blue light to regulate a variety of growth and adaptive processes in organisms ranging from bacteria to humans. The photocycle of cryptochrome is not yet known, although it is hypothesized that it may share some similarity to that of photolyase, which utilizes light-driven electron transfer from the catalytic flavin chromophore. In this review, we present genetic evidence for the photoreceptive role of cryptochromes and discuss recent biochemical studies that have furthered our understanding of the cryptochrome photocycle. In particular, the role of the unique C-terminal domain in cryptochrome phototransduction is discussed.

Regulation and splicing of scavenger receptor class B type I in human macrophages and atherosclerotic plaques

BackgroundThe protective role of high-density lipoprotein (HDL) in the cardiovascular system is related to its role in the reverse transport of cholesterol from the arterial wall to the liver for subsequent excretion via the bile. Scavenger receptor class B type I (SR-BI) binds HDL and mediates selective uptake of cholesterol ester and cellular efflux of cholesterol to HDL. The role of SR-BI in atherosclerosis has been well established in murine models but it remains unclear whether SR-BI plays an equally important role in atherosclerosis in humans. The aim of this study was to investigate the expression of SR-BI and its isoforms in human macrophages and atherosclerotic plaques.MethodsThe effect of hypoxia and minimally modified low-density lipoprotein (mmLDL), two proatherogenic stimuli, on SR-BI expression was studied in human monocyte-derived macrophages from healthy subjects using real-time PCR. In addition, SR-BI expression was determined in macrophages obtained from subjects with atherosclerosis (n = 15) and healthy controls (n = 15). Expression of SR-BI isoforms was characterized in human atherosclerotic plaques and macrophages using RT-PCR and DNA sequencing.ResultsSR-BI expression was decreased in macrophages after hypoxia (p < 0.005). In contrast, SR-BI expression was increased by exposure to mmLDL (p < 0.05). There was no difference in SR-BI expression in macrophages from patients with atherosclerosis compared to controls. In both groups, SR-BI expression was increased by exposure to mmLDL (p < 0.05). Transcripts corresponding to SR-BI and SR-BII were detected in macrophages. In addition, a third isoform, referred to as SR-BIII, was discovered. All three isoforms were also expressed in human atherosclerotic plaque. Compared to the other isoforms, the novel SR-BIII isoform was predicted to have a unique intracellular C-terminal domain containing 53 amino acids.ConclusionWe conclude that SR-BI is regulated by proatherogenic stimuli in humans. However, we found no differences between subjects with atherosclerosis and healthy controls. This indicates that altered SR-BI expression is not a common cause of atherosclerosis. In addition, we identified SR-BIII as a novel isoform expressed in human macrophages and in human atherosclerotic plaques.