Forward Orientation Research Articles

ALU Repeats in Promoters Are Position-Dependent Co-Response Elements (coRE) that Enhance or Repress Transcription by Dimeric and Monomeric Progesterone Receptors

We have conducted an in silico analysis of progesterone response elements (PRE) in progesterone receptor (PR) up-regulated promoters. Imperfect inverted repeats, direct repeats, and half-site PRE are widespread, not only in PR-regulated, but also in non-PR-regulated and random promoters. Few resemble the commonly used palindromic PRE with three nucleotide (nt) spacers. We speculated that PRE may be necessary but insufficient to control endogenous PR-dependent transcription. A search for PRE partners identified a highly conserved 234-nt sequence invariably located within 1-2 kb of transcription start sites. It resembles ALU repeats and contains binding sites for 11 transcription factors. The 234-nt sequence of the PR-regulated 8-oxoguanine DNA glycosylase promoter was cloned in the forward or reverse orientation in front of zero, one, or two inverted repeat PRE, and one or tandem PRE half-sites, driving luciferase. Under these conditions the 234-nt sequence functions as a co-response element (coRE). From the PRE or tandem half-sites, the reverse coRE is a strong activator of PR and glucocorticoid receptor-dependent transcription. The forward coRE is a powerful repressor. The prevalence of PRE half-sites in natural promoters suggested that PR monomers regulate transcription. Indeed, dimerization-domain mutant PR monomers were stronger transactivators than wild-type PR on PRE or tandem half-sites. This was repressed by the forward coRE. We propose that in natural promoters the coRE functions as a composite response element with imperfect PRE and half-sites to present variable, orientation-dependent transcription factors for interaction with nearby PR.

The Roles of Selected Arginine and Lysine Residues of TAFI (Pro-CPU) in Its Activation to TAFIa by the Thrombin-Thrombomodulin Complex

Thrombomodulin (TM) increases the catalytic efficiency of thrombin (IIa)-mediated activation of thrombin-activable fibrinolysis inhibitor (TAFI) 1250-fold. Negatively charged residues of the C-loop of TM-EGF-like domain 3 are required for TAFI activation. Molecular models suggested several positively charged residues of TAFI with which the C-loop residues could interact. Seven TAFI mutants were constructed to determine if these residues are required for efficient TAFI activation. TAFI wild-type or mutants were activated in the presence or absence of TM and the kinetic parameters of TAFI activation were determined. When the three consecutive lysine residues in the activation peptide of TAFI were substituted with alanine (K42/43/44A), the catalytic efficiencies for TAFI activation with TM decreased 8-fold. When other positively charged surface residues of TAFI (Lys-133, Lys-211, Lys-212, Arg-220, Lys-240, or Arg-275) were mutated to alanine, the catalytic efficiencies for TAFI activation with TM decreased by 1.7-2.7-fold. All decreases were highly statistically significant. In the absence of TM, catalytic efficiencies ranged from 2.8-fold lower to 1.24-fold higher than wild-type. None of these, except the 2.8-fold lower value, was statistically significant. The average half-life of the TAFIa mutants was 8.1+/-0.6 min, and that of wild type was 8.4+/-0.3 min at 37 degrees C. Our data show that these residues are important in the activation of TAFI by IIa, especially in the presence of TM. Whether the mutated residues promote a TAFI-TM or TAFI-IIa interaction remains to be determined. In addition, these residues do not influence spontaneous inactivation of TAFIa.

Issues about Innovation of Present Theory of Accounting

With the method of theoretical analysis, this thesis, systematically analyzed the limitations of the present theory of accounting in recycle economy and the reasons for that, analyzed the characteristics of accounting in recycle economy, and, according to the internal demand of recycle economy, put forward brand new development orientation of theory of accounting that would be suitable for recycle economy development. It specially discussed four suggestions, including constructing brand new accounting information disclosure model in recycle economy, bringing resource assets into accounting system, introducing new accounting method, and improving the control theory for accounting cost (including safety cost). This thesis will be a reference for the development of theory of accounting in Chinese recycle economy.

Protein domain organisation: adding order

BackgroundDomains are the building blocks of proteins. During evolution, they have been duplicated, fused and recombined, to produce proteins with novel structures and functions. Structural and genome-scale studies have shown that pairs or groups of domains observed together in a protein are almost always found in only one N to C terminal order and are the result of a single recombination event that has been propagated by duplication of the multi-domain unit.Previous studies of domain organisation have used graph theory to represent the co-occurrence of domains within proteins. We build on this approach by adding directionality to the graphs and connecting nodes based on their relative order in the protein. Most of the time, the linear order of domains is conserved. However, using the directed graph representation we have identified non-linear features of domain organization that are over-represented in genomes. Recognising these patterns and unravelling how they have arisen may allow us to understand the functional relationships between domains and understand how the protein repertoire has evolved.ResultsWe identify groups of domains that are not linearly conserved, but instead have been shuffled during evolution so that they occur in multiple different orders. We consider 192 genomes across all three kingdoms of life and use domain and protein annotation to understand their functional significance.To identify these features and assess their statistical significance, we represent the linear order of domains in proteins as a directed graph and apply graph theoretical methods. We describe two higher-order patterns of domain organisation: clusters and bi-directionally associated domain pairs and explore their functional importance and phylogenetic conservation.ConclusionTaking into account the order of domains, we have derived a novel picture of global protein organization. We found that all genomes have a higher than expected degree of clustering and more domain pairs in forward and reverse orientation in different proteins relative to random graphs with identical degree distributions. While these features were statistically over-represented, they are still fairly rare. Looking in detail at the proteins involved, we found strong functional relationships within each cluster. In addition, the domains tended to be involved in protein-protein interaction and are able to function as independent structural units. A particularly striking example was the human Jak-STAT signalling pathway which makes use of a set of domains in a range of orders and orientations to provide nuanced signaling functionality. This illustrated the importance of functional and structural constraints (or lack thereof) on domain organisation.

The A312L 5′-UTR of Chlorella virus PBCV-1 is a translational enhancer in Arabidopsis thaliana

PBCV-1 ( Paramecium bursaria Chlorella virus) is a large double stranded DNA virus that replicates in certain eukaryotic chlorella like green algae. The PBCV-1 A312L gene encodes a 33-kDa protein whose function currently is unknown. The 5′-UTR of the A312L mRNA is 153 nucleotides, longer than the 5′-UTR in any other PBCV-1 gene. The sequence 5′-AAAC was repeated 17 times within 156 bp 5′ to the A312L gene start codon and this sequence was repeated 13 times continuously in the 5′-UTR of the mRNA. Recombinant genes were constructed in vector pBI121 that contained the A312L 5′-UTR, in both the forward and inverse-complement orientations, fused to the GUS gene under the control of the CaMV 35S promoter. These constructs were introduced into Arabidopsis thaliana and the results indicated that the A312L 5′-UTR functions as a translational enhancer only in the forward orientation. Overall, the ratio of GUS enzyme activity to GUS mRNA was 15-fold higher in constructs derived from the A312L 5′-UTR in the forward orientation as compared to constructs containing the 5′-UTR in the inverse-complement orientation or those lacking the A312L 5′-UTR.

Reduced Genotoxicity of Avian Sarcoma Leukosis Virus Vectors in Rhesus Long-term Repopulating Cells Compared to Standard Murine Retrovirus Vectors

Insertional mutagenesis continues to be a major concern in hematopoietic stem-cell gene therapy. Nonconventional gene transfer vectors with more favorable integration features in comparison with conventional retrovirus and lentivirus vectors are being developed and optimized. In this study, we report for the first time a systematic analysis of 198 avian sarcoma leukosis virus (ASLV) insertion sites identified in rhesus long-term repopulating cells, and a comparison of ASLV insertion patterns to Moloney murine leukemia virus (MLV) (n = 396) and simian immunodeficiency virus (SIV) (n = 289) using the newly released rhesus genome databank. Despite a weak preference toward gene-coding regions, ASLV integration is nonclustered, does not favor gene-rich regions, transcription start sites, or CpG islands. There was no propensity for ASLV insertions within or near proto-oncogenes, and most importantly, no insertions close to or within the Mds1-Evi1 locus, which is in contrast to the significant over-representation of this insertion site for MLV vectors in the same transplantation model. Furthermore, ASLV long terminal repeats (LTRs) do not have detectable promoter and enhancer activity in a quantitative luciferase assay to measure neighboring gene activation. The combination of these features is unique for ASLV and suggests that optimized vectors based on this virus could be useful and safe for gene transfer to hematopoietic stem cells and progenitor cells.

Cooperative exonization of MaLR and AluJo elements contributed an alternative promoter and novel splice variants of RNF19

The RNF19 protein, which contains RING-finger and IBR motifs, acts as an E3 ubiquitin ligase localized to Lewy bodies. RNF19 is located on human chromosome 8q22.2, has a 4.4-kb transcript, and is ubiquitously expressed in various tissues. Here, we identified an alternative RNF19 promoter region and alternative RNF19 transcripts derived from MaLR (mammalian apparent LTR-retrotransposon) and AluJo elements. Comparative analyses indicated human-specific expression of the MaLR- and AluJo-related transcripts. From the expression analysis of 72 tissue samples including human normal, tumor, and primate tissues, three different isoforms (V1, V2, and V3) of MaLR-derived transcripts were identified. Quantitative RT-PCR analysis showed a dominant expression pattern of the V2 MaLR-derived transcript. A reporter gene assay for MaLR element promoter activity indicated that pGL2-RNF19/MaLR in the forward orientation is capable of driving luciferase gene expression in Cos7 and HCT116 cells. These findings suggest that RNF19 has acquired a new promoter and alternative exons via continuous retrotransposition events of MaLR and AluJo elements during mammalian and primate evolution, respectively.

Recent Developments in 5‘-Terminal Cap Analogs: Synthesis and Biological Ramifications.

The 5‘-terminus of eukaryotic messenger RNA (mRNA) molecules contains a unique ‘cap’ structure: 7- methylguanosine (m7G) linked by a 5‘-5‘-triphosphate bridge to the first nucleoside in the standard polymer chain. This 5‘- cap is recognized by numerous enzymes involved in the transport and translation of mRNA, as well as its processing, both in terms of generating mature mRNA from the initial transcript and its natural degradation. In vitro generation of capped RNAs uses a cap analog in which two nucleosides are joined in a 5‘-5‘ triphosphate linkage, allowing incorporation of the cap along with the first nucleotide in the polymer chain. The conventional cap analog, m7G[5‘]ppp[5‘]G, has a 3‘-OH on both nucleoside moieties, resulting in the incorporation of the dinucleotide in either orientation. This results in one-third to one-half of the mRNAs containing a non-methylated cap and a methylated initial nucleotide. These ‘reverse cap’ structures bind poorly to elF4E and other cap binding proteins, greatly reducing the overall translational efficiency in vitro and biological activity in vivo. This drawback of the conventional cap analog has been addressed by creating an “anti-reverse cap analog” (ARCA), m2 7, 3-OG[5‘]ppp[5‘]G, in which the 3‘-OH of the ribose connected to the m7Guo moiety is replaced with a 3‘-O-methyl group, allowing incorporation only in the forward orientation due to the presence of the single extendable 3‘-OH group. In addition to the chemical modification at the 3‘-OH group of the ribose connected to the m7G moiety, modifications at the 2‘-OH group also preclude attachment of a second nucleotide by polymerase, again forcing the forward orientation. This mini-review focuses on recent developments in the synthesis of novel cap analogs, highlighting modifications at several positions including; the heterocyclic bases, the sugars, and the α-, β-, and γ-phosphates in the triphosphate chain, and assessing their impact on biological activity.

Genetic analysis of the variable region genes encoding a monospecific human natural anti-DNA antibody.

Recent evidence suggests that natural autoantibodies may play an integral role in the development of the normal immune repertoire. To explore the genetic origins of these antibodies, we have isolated and sequenced the variable (V) region genes encoding both the heavy (H) and light (L) chains of a natural anti-DNA antibody, Kim11.4. The genes appear to be derived from the VH4.18 (subgroup VHIV), JH5, Hum1L1 (subgroup V lambda I) and J lambda 3 germline genes. The origin of the H chain diversity gene is more obscure, being potentially derived from one or more of several germline genes, arranged in either the forward or reverse orientations. Both the Kim11.4 VH and VL genes share significant degrees of similarity with those utilized in other autoantibodies, indicating that at least some degree of V restriction may exist in human autoreactive B cells. The pattern of nucleotide differences between the Kim11.4 VH and VL genes and their putative germline counterparts suggests that the Kim11.4 genes may have undergone somatic mutation and arisen as a result of antigen selection.

Phosphorylation Regulates Tau Interactions with Src Homology 3 Domains of Phosphatidylinositol 3-Kinase, Phospholipase Cγ1, Grb2, and Src Family Kinases

The microtubule-associated protein tau can associate with various other proteins in addition to tubulin, including the SH3 domains of Src family tyrosine kinases. Tau is well known to aggregate to form hyperphosphorylated filamentous deposits in several neurodegenerative diseases (tauopathies) including Alzheimer disease. We now report that tau can bind to SH3 domains derived from the p85alpha subunit of phosphatidylinositol 3-kinase, phospholipase Cgamma1, and the N-terminal (but not the C-terminal) SH3 of Grb2 as well as to the kinases Fyn, cSrc, and Fgr. However, the short inserts found in neuron-specific isoforms of Src prevented the binding of tau. The experimentally determined binding of tau peptides is well accounted for when modeled into the peptide binding cleft in the SH3 domain of Fyn. After phosphorylation in vitro or in transfected cells, tau showed reduced binding to SH3 domains; no binding was detected with hyperphosphorylated tau isolated from Alzheimer brain, but SH3 binding was restored by phosphatase treatment. Tau mutants with serines and threonines replaced by glutamate, to mimic phosphorylation, showed reduced SH3 binding. These results strongly suggest that tau has a potential role in cell signaling in addition to its accepted role in cytoskeletal assembly, with regulation by phosphorylation that may be disrupted in the tauopathies including Alzheimer disease.

Effects of blade tip modifications on wind turbine performance using vortex model

The effects of blade tip modifications on a wind turbine blade are studied with the design code developed previously, by taking into account the curving of the blade axis in or out of the plane of rotation. This is an area of interest for manufacturers of wind turbines to improve the aerodynamic performance, as has been done with airplane wings and also to use the swept tips to unload the blades during wind gusts by changing the local incidences with a nose-down torsional moment. The vortex model, based on Goldstein approach, treats each blade as a lifting line generating a helicoidal vortex sheet, supporting the trailed vorticity along prescribed helices whose pitch is determined to satisfy the wake equilibrium condition. As the lifting line is given sweep in the plane of rotation or dihedral in the plane containing the blade and the rotor axis, the induced velocities by the bound vortex at the lifting line are no longer zero and the blade flow is also affected by the modified vortex sheet geometry, according to the Biot–Savart formula. The study is performed with a two-bladed rotor with the NREL blade as point of reference. A series of tests is carried out with the design code, comparing the design of a rotor blade with straight axis or with a ±10% (forward or backward) sweep, dihedral or winglet. Results indicate that the aerodynamic performance are in general enhanced with these tip modifications, although the trends differ between forward and backward orientations, with some nonlinear effects associated with the wake geometry.

The second intron of AGAMOUS drives carpel- and stamen-specific expression sufficient to induce complete sterility in Arabidopsis

Gene containment technologies that prevent transgene dispersal through pollen, fruit and seed are in immediate demand to address concerns of gene flow from transgenic crops into wild species or close relatives. In this study, we isolated the enhancer element of Arabidopsis AGAMOUS that drives gene expression specifically in stamens and carpels. By fusing this AG enhancer to a minimal 35S promoter fragment, two tissue-specific promoters, fAGIP and rAGIP in forward and reverse orientations, respectively, were created and fused to the GUS reporter. Transgenic Arabidopsis plants harboring either fAGIP::GUS or rAGIP::GUS displayed similar GUS expression specifically in carpel and stamen tissues and their primordial cells. To test their utility for engineering sterility, the promoters were fused to the Diphtheria toxin A (DT-A) gene coding for a ribosome inactivating protein as well as the Barnase gene coding for an extracellular ribonuclease, and tested for tissue-specific ablation. Over 89% of AGIP::DT-A and 68% of AGIP::Barnase transgenic plants displayed specific and precise ablation of stamens and carpels and are completely sterile. These transgenic plants showed normal vegetative development with prolonged vegetative growth. To evaluate the stability of the sterile phenotype, 16 AGIP::DT-A lines underwent two consecutive cutback generations and showed no reversion of the floral phenotype. This study demonstrates a simple, precise and efficient approach to achieve absolute sterility through irreversible ablation of both male and female floral organs. This approach should have a practical application for transgene containment in ornamental, landscaping, and woody species, whose seeds and fruits are of no economic value.

Hepatocyte nuclear factor 1-alpha mutation in normal glucose-tolerant subjects and early-onset type 2 diabetic patients

Background/AimsThe prevalence of diabetes in Korea is reported to be approximately 10%, but cases of maturity-onset diabetes of the young (MODY) are rare in Korea. A diagnostic technique for autosomal dominant MODY is being actively sought. In this regard, we used a DNA chip to investigate the frequency of mutations of the MODY3 gene (hepatocyte nuclear factor-1α) in Korean patients with early-onset type 2 diabetes.MethodsThe genomic DNA of 30 normal individuals [age, 24.9±8.6 years] and 25 patients with early-onset type 2 diabetes (age, 27±5.9 years) was extracted, and the MODY3 gene was amplified. The amplified DNA was hybridized onto a MODY3 chip, which has oligonucleotides of 15-25 bases, representing wild-type and mutant MODY3 sequences in both forward and reverse orientations, immobilized on its surface.ResultsAmong the normal subjects, there was no mutation of MODY3. Among those with early-onset type 2 diabetes, there was one case of MODY3 mutation.ConclusionsOur results indicate that MODY3 mutations are not rare in Korean early-onset type 2 diabetes patients in Korea and suggest that MODY3 mutations in patients with early-onset type 2 diabetes need to be further evaluated.

An Iron Enhancer Element in the FTN-1 Gene Directs Iron-dependent Expression in Caenorhabditis elegans Intestine

Ferritin is a ubiquitous protein that sequesters iron and protects cells from iron toxicity. Caenorhabditis elegans express two ferritins, FTN-1 and FTN-2, which are transcriptionally regulated by iron. To identify the cis-acting sequences and proteins required for iron-dependent regulation of ftn-1 and ftn-2 expression, we generated transcriptional GFP reporters corresponding to 5 '-upstream sequences of the ftn-1 and ftn-2 genes. We identified a conserved 63-bp sequence, the iron-dependent element (IDE), that is required for iron-dependent regulation of a ftn-1 GFP reporter in intestine. The IDE contains two GATA-binding motifs and three octameric direct repeats. Site-directed mutagenesis of the GATA sequences, singly or in combination, reduces ftn-1 GFP reporter expression in the intestine. In vitro DNA mobility shift assays show that the intestine-specific GATA protein ELT-2 binds to both GATA sequences. Inhibition of ELT-2 function by RNA interference blocks ftn-1 GFP reporter expression in vivo. Insertion of the IDE into the promoter region of a heterologous reporter activates iron-dependent transcription in intestine. These data demonstrate that the activation of ftn-1 and ftn-2 transcription by iron requires ELT-2 and that the IDE functions as an iron-dependent enhancer in intestine.

Distinctive Integration Profile of Avian Sarcoma Leukosis Virus Vectors in Rhesus Long-Term Repopulating Cells.

Insertional mutagenesis continues to be a major concern in hematopoietic stem cell gene therapy. Non-conventional gene transfer vectors with more favorable integration features in comparison to conventional retrovirus and lentivirus vectors are being pursued. The avian sarcoma leukosis virus (ASLV) has been reported to have an unbiased integration pattern in cell lines, but this vector system has not previously been investigated for transduction of repopulating hematopoietic cells. We investigated the efficiency and integration profile of this vector in the clinically-relevant rhesus macaque autologous transplantation model. Using an ASLV derived RCAS (Replication Competent, ALV LTR with A Splice acceptor) vector, we could obtain transduction efficiencies of up to 33% ex vivo in rhesus CD34+ cells. We have been able to transplant two rhesus macaques with ASLV-transduced autologous CD34+ cells and achieve long-term gene marking levels of 1–3% as far as 18 months post-transplant. Using an optimized linear-amplification mediated PCR (LAM-PCR), we have been able to identify so far close to 300 unique insertion sites in the two animals. Here we reported for the first time a systematic analysis of 239 ASLV insertion sites identified in rhesus long-term repopulating cells. Out of 239 unique insertions identified in 4–18 months post-transplant granulocytes and lymphocytes, 99 (41.1%) have landed within RefSeq gene coding regions. 14 (5.8%) have landed within 5kb upstream of RefSeq genes, indicating no preference of inserting into transcription start site. 10 (4.2%) have landed within 5kb downstream of RefSeq genes, which is comparable to random insertions. No insertion into the Mds1-Evi1 locus has been identified to date, at any time point, which is in striking contrast to significant overrepresentation of this insertion site for MLV vectors in the same transplantation model. No preference into CpG islands was found. We further employed a rapid and quantitative assay for measuring neighboring gene activation by vector provirus LTR enhancers using a luciferase assay. LTR from vectors are cloned into the pACT5 plasmid, upstream of a minimal promoter-IRES-luciferease cassette, allowing measurement of neighboring gene activation by a simple luciferase assay. RCAS vectors produced no detectable luciferase activation, both in the forward and reverse orientations, suggesting lack of enhancer activity in the LTRs in mammalian cells, in contrast to significant enhancer activity and read-through transcription from MLV vectors measured in the same assay. Our findings indicate that ASLV derived RCAS vectors have a potentially safer integration pattern in comparison to commonly used MLV and HIV derived vectors. Furthermore, ASLV LTRs do not have significant promoter and enhancer activity in mammalian cells, which provide a second safety feature of the system. Therefore, these vectors should be further explored for hematopoietic stem cell gene transfer purposes.

Mandibular Growth Changes and Cervical Vertebral Maturation

To evaluate mandibular dimensional changes and regional remodeling occurring during five intervals of circumpubertal growth. This investigation evaluated a unique sample of subjects in whom tantalum implants were placed into the craniofacial complex during childhood. The sample was obtained from the Mathews and Ware implant study originally conducted at the University of California San Francisco in the 1970s, with longitudinal cephalometric records of 20 subjects (13 female, 7 male) available for evaluation. Cephalograms at six consecutive stages of cervical vertebral maturation (CS1 through CS6) were analyzed. Peak mandibular growth was noted during the interval from CS3 to CS4. Forward rotation of the mandible was due to greater mandibular growth posteriorly than anteriorly. Progressive closure of the condylar-ramus-occlusal (CRO) angle resulted in a forward and upward orientation of the ramus relative to the corpus of the mandible due to increased vertical growth of the condyle. A peak in mandibular growth at puberty was substantiated. Mandibular remodeling and condylar rotation continue to occur after the growth spurt.

Heads or tails, does it make a difference? Capsule endoscope direction in small bowel studies is important

Capsule endoscopy has opened the small bowel for direct inspection. However, the diagnostic sensitivity of capsule endoscopy is not 100 %. We have observed that forward orientation and backward orientation of the camera in the small bowel provide different information on pathological findings. Double-head capsule endoscopy may offer the answer to this problem.

Improved Human β-globin Expression from Self-inactivating Lentiviral Vectors Carrying the Chicken Hypersensitive Site-4 (cHS4) Insulator Element

Effective gene therapy for beta-thalassemia major (beta-TM) requires consistent, high expression of human beta-globin (hbeta-globin) in red blood cells (RBCs). Several groups have now shown that lentiviral (LV) vectors stably transmit the hbeta/hgamma-globin genes and large elements of the locus control region, resulting in correction of the murine thalassemia intermedia (TI) phenotype and survival of mice with the TM phenotype. However, current LVs show variable hbeta/hgamma-globin expression and require a high number of vector copies/cell for a therapeutic effect. To address this, we designed LVs flanked by the chicken hypersensitive site-4 (cHS4) chromatin insulator element and compared them with their "un-insulated" counterparts. We observed a consistent twofold-higher hbeta expression from insulated vectors in single-copy mouse erythroleukemia cell clones, an increase that resulted from reduced position effect variegation (PEV) and increased probability of expression from individual integrants. This effect was confirmed in vivo: an approximately twofold increase in hbeta expression was seen in the RBC progeny of murine hematopoietic stem cells, with significantly higher numbers of hbeta-expressing cells in individual secondary spleen colony-forming units. In summary, cHS4-insulated hbeta-globin LVs showed distinct chromatin barrier activity, resulting in higher, consistent hbeta expression. These studies have important implications for vector design for clinical trials for gene therapy for hemoglobinopathies.

Functional Characterization of AtATM1, AtATM2, and AtATM3, a Subfamily of Arabidopsis Half-molecule ATP-binding Cassette Transporters Implicated in Iron Homeostasis

The functional capabilities of one of the smallest subfamilies of ATP-binding cassette transporters from Arabidopsis thaliana, the AtATMs, are described. Designated AtATM1, AtAATM2, and AtATM3, these half-molecule ABC proteins are homologous to the yeast mitochondrial membrane protein ATM1 (ScATM1), which is clearly implicated in the export of mitochondrially synthesized iron/sulfur clusters. Yeast ATM1-deficient (atm1) mutants grow very slowly (have a petite phenotype), are respiration-deficient, accumulate toxic levels of iron in their mitochondria, and show enhanced compensatory high affinity iron uptake. Of the three Arabidopsis ATMs, AtATM3 bears the closest functional resemblance to ScATM1. Heterologously expressed AtATM3 is not only able to complement the yeast atm1 petite phenotype but is also able to suppress the constitutively high capacity for high affinity iron uptake associated with loss of the chromosomal copy of ScATM1, abrogate intra-mitochondrial iron hyperaccumulation, and restore mitochondrial respiratory function and cytochrome c levels. By comparison, AtATM1 only weakly suppresses the atm1 phenotype, and AtATM2 exerts little or no suppressive action but instead is toxic when expressed in this system. The differences between AtATM3 and AtATM1 are maintained after exchanging their target peptides, and these proteins as well as AtATM2 colocalize with the mitochondrial fluor MitoTracker Red when expressed in yeast as GFP fusions. Although its toxicity when heterologously expressed in yeast, except when fused with GFP, precludes the functional analysis of native AtATM2, a common function, mitochondrial export of Fe/S clusters or their precursors for the assembly of cytosolic Fe/S proteins, is inferred for AtATM3 and AtATM1.

Structural Basis for Polyproline Recognition by the FE65 WW Domain

The neuronal protein FE65 functions in brain development and amyloid precursor protein (APP) signaling through its interaction with the mammalian enabled (Mena) protein and APP, respectively. The recognition of short polyproline sequences in Mena by the FE65 WW domain has a central role in axon guidance and neuronal positioning in the developing brain. We have determined the crystal structures of the human FE65 WW domain (residues 253–289) in the apo form and bound to the peptides PPPPPPLPP and PPPPPPPPPL, which correspond to human Mena residues 313–321 and 347–356, respectively. The FE65 WW domain contains two parallel ligand-binding grooves, XP (formed by residues Y269 and W280) and XP2 (formed by Y269 and W271). Both Mena peptides adopt a polyproline helical II conformation and bind to the WW domain in a forward (N–C) orientation through selection of the PPPPP motif by the XP and XP2 grooves. This mode of ligand recognition is strikingly similar to polyproline interaction with SH3 domains. Importantly, comparison of the FE65 WW structures in the apo and liganded forms shows that the XP2 groove is formed by an induced-fit mechanism that involves movements of the W271 and Y269 side-chains upon ligand binding. These structures elucidate the molecular determinants underlying polyproline ligand selection by the FE65 WW domain and provide a framework for the design of small molecules that would interfere with FE65 WW–ligand interaction and modulate neuronal development and APP signaling.