Skip Navigation


Human Molecular Genetics Advance Access originally published online on July 29, 2003
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
12/18/2359    most recent
ddg253v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (14)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Ganesh, S.
Right arrow Articles by Yamakawa, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ganesh, S.
Right arrow Articles by Yamakawa, K.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Human Molecular Genetics, 2003, Vol. 12, No. 18 2359-2368
DOI: 10.1093/hmg/ddg253
© 2003 Oxford University Press

The Lafora disease gene product laforin interacts with HIRIP5, a phylogenetically conserved protein containing a NifU-like domain

Subramaniam Ganesh1,2,{dagger},*, Naomi Tsurutani1,{dagger}, Toshimitsu Suzuki1, Kazunori Ueda3, Kishan Lal Agarwala1, Hiroyuki Osada3, Antonio V. Delgado-Escueta4 and Kazuhiro Yamakawa1

1Laboratory for Neurogenetics, RIKEN Brain Science Institute, Wako-shi, Japan, 2Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur, India, 3Antibiotics Laboratory, RIKEN, Wako-shi, Japan and 4Epilepsy Genetics/Genomics Laboratories, Comprehensive Epilepsy Program, UCLA School of Medicine and VA GLAHS West Los Angeles Medical Center, Los Angeles, CA, USA

Received May 24, 2003; Accepted July 20, 2003

DDBJ/EMBL/GenBank accession nos AY335194 and AY335195


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Lafora disease is an autosomal recessive type of progressive myoclonus epilepsy caused by mutations in the EPM2A gene. The EPM2A gene-encoded protein laforin is a dual-specificity phosphatase that associates with polyribosomes. Because the cellular functions of laforin are largely unknown, we used the yeast-two hybrid system to screen for protein(s) that interact with laforin. We found that laforin interacts with a phylogenetically conserved protein HIRIP5 that harbors a NifU-like domain. Both in vitro and in vivo assay have shown that the interaction is specific and that laforin probably uses its N-terminal CBD-4 domain to interact with the C-terminal NifU-like domain of the HIRIP5 protein. HIRIP5 encodes a cytosolic protein and is expressed ubiquitously, perhaps reflecting a house-keeping function. The presence of a NifU-like domain in the HIRIP5 protein raises an interesting possibility that it may be involved in iron homeostasis. Although the significance of the interaction between HIRIP5 and laforin proteins is not yet fully known, because laforin dephosphorylated HIRIP5 in vitro, HIRIP5 promises to be an interesting laforin-binding partner and would contribute to the understanding of the molecular pathology of Lafora disease.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Lafora disease (LD) is one of the severe types of progressive myoclonus epilepsies characterized by the presence of periodic acid–Schiff positive (PAS+) intracellular inclusion bodies (1). LD starts at around 15 years with stimulus-sensitive grand mal tonic–clonic, absence, visual and myoclonic seizures. Progressive dementia, psychosis, cerebellar ataxia, dysarthria, muscle wasting and respiratory failure lead to death within a decade. Mutations in the EPM2A gene at 6q24 encoding a dual-specificity phosphatase (laforin) have been found to be responsible for up to 80% of LD cases and about 36 or more mutations have been reported thus far. Identified mutations include homozygous deletions, frame shift, nonsense and missense mutations, suggesting inactivation of both copies leads to the development of LD (2–7). While these are spread throughout the coding region, exon 1 mutations appear to be associated with an early-onset cognitive deficit subphenotype of LD (7).

The EPM2A gene is composed of at least four exons spanning around 500 kb region, and encodes a 331-amino acid protein termed laforin. While laforin shows no homology to any known protein, it contains a consensus motif for protein–tyrosine phosphatase at its carboxyl terminal. Functional studies revealed that laforin is an active dual-specificity phosphatase (DSPD) and that it is a cytoplasmic protein associating with polyribosome (8). Differential splicing of a minor transcript produces a laforin variant that targets nucleus (9). Laforin contains a functional carbohydrate-binding domain (CBD-4) at its amino terminal (5,10,11). The EPM2A gene is expressed ubiquitously, and in mouse brain Epm2a transcripts were detected in a regionally restricted manner (2,10,12). Similar to humans, null mutants for the Epm2a gene in mice develop Lafora inclusion bodies, neurodegeneration, ataxia, myoclonus epilepsy and impaired behavioral response (13). Although these results suggest that LD is a primary neurodegenerative disorder and provide evidence for laforin's critical role in neuronal survival, very little has been discovered about the cellular cascade of functions of laforin. In an initial attempt to clarify the cellular mechanisms of laforin's function, we searched for proteins interacting with laforin by means of yeast-two hybrid screening. Here, we report that a novel phylogenetically conserved NifU-family protein, HIRIP5, interacts specifically with laforin.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of HIRIP5 gene by yeast two-hybrid screening
In order to search for laforin-interacting proteins, a yeast two-hybrid screen was carried out. A human fetal brain cDNA library was screened using human full-length laforin as bait (pEG202-LDH). The screen revealed 24 distinct cDNAs encoding proteins capable of interacting with laforin and did not interact with negative controls used in the screen. Sequence analysis revealed that clone YTH134, which carried an 867 bp cDNA insert, was isolated six times. An additional clone (YTH74) that had a 435 bp insert, shared sequence identity with the 3' end of the clone YTH134. The clone YTH134 revealed an open reading frame (ORF) for 232 amino acids, lacked any stop codon in the 5' region and was almost identical to the sequence of the HIRIP5 gene (GenBank accession no. AJ132584) reported during the course of this study (14). A major difference was noted, however, between the two cDNA sequences; YTH134 lacked a stretch of 90 bases, between nucleotides 10 and 12, when compared with the HIRIP5 and showed an in-frame coding sequence 5' to the initiation codon reported by Lorain et al. (14). To confirm whether this deletion represented a possible isoform of HIRIP5 transcript or was a cloning artifact, a human fetal brain cDNA library was screened and seven independent human cDNA clones were isolated. Sequence analysis revealed that five of the seven clones had an insert of 1460 bp and the other two were partial clones containing 520 bp inserts. The larger cDNA clones were identical to YTH134 in having the 90 bp deletion and showed additional sequence at the 3' and 5' ends (GenBank accession no. AY335194). An analysis using the ATGpr program (www.hri.co.jp/atgpr/) identified a longest ORF of 762 bp encoding a protein of 254 amino acids (Fig. 1). There are five potential initiation codons in-frame; at nucleotide positions 200, 374, 455, 623 and 713 from the 5' end (Fig. 1). With regard to the Kozak consensus, ATG-200 showed a higher reliability score (0.47) and an in-frame stop codon (TAG) is located a short distance upstream of this ATG (Fig. 1). Thus the larger cDNA clones isolated in the present study extend the coding region and provide the 5' (199 bp) and 3' (499 bp) untranslated regions for the HIRIP5 transcript. We also isolated a cDNA clone (M24A; GenBank accession no. AY335195) for mouse Hirip5 gene (Fig. 1B). This partial clone predicts an ORF for 245 amino acids and extends the homology to the N-terminal sequence of human HIRIP5 protein presented in this study. Overall the human and mouse HIRIP5 genes share 85% identity in the ORF and 90% identity and 93% similarity at protein level (Fig. 1B).



View larger version (51K):
[in this window]
[in a new window]
  		Figure 1. Nucleotide sequence and predicted amino acid sequence of human and murine HIRIP5 gene. (A) Nueclotide sequence of human HIRIP5 full-length cDNA clone, Lip2 (GenBank accession no. AY335194). Uppercase letters indicate the coding region and lower case letters designate the 5'- and 3'-UTRs. The positions of the five probable start codons are shown within boxes. The arrow defines the region that showed insertion of 90 bases in the cDNA reported by Lorain et al. (14). Arrow heads and vertical bars define the 5' and 3' end boundaries of the YTH134 and YTH74 cDNA clones, respectively. The positions of the putative MurD ligase domain and NifU-like domain are given in bold fonts and the underlined letters, respectively. (B) Amino acid sequence of the HIRIP5 gene of human and mouse. Vertical lines represent identities and dotted lines denote the conservative substitutions.

 
A sequence comparison using the conserved domain database revealed that HIIRIP5 protein contains a NifU-like domain (pfam01106; Figs 1A and 2A). The NifU-like domain is the only common region known between the NifU protein from nitrogen-fixing bacteria and rhodobacterial species (15). Comparison of the NifU domain of the HIRIP5 with sequences of other known NifU (cyanobacteria) and NifU-like (yeast and C. elegans) proteins demonstrated striking evolutionary conservation (Fig. 2A). HIRIP5 also appears to contain a weak consensus sequence for the catalytic motif for MurD-ligase domain (pfam01225), which partly overlaps the NifU-like sequence (Figs 1A and 2B). Mur family ligase contains a number of related ligase enzymes that catalyze consecutive steps in the synthesis of peptidoglycan, a cross-linked polysaccharide peptide complex of indefinite size found in the inner cell wall of all bacteria (16).



View larger version (90K):
[in this window]
[in a new window]
  		Figure 2. Sequence alignment of HIRIP5 with various structurally defined NifU-like domain (A) and MurD ligase domain containing proteins (B), as identified in the conserved domain data base (www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Numbers of the left indicate the respective GenBank accession numbers of the NifU domain or the MurD domain containing proteins from various species (AAA93019, Anabaena variabilis; AAC3331, Cyanothece Sp PCC8801; CAB15212, Bacillus subtilis; BAA18665, Synechocystis Sp. PCC6803; CAA94609, C. elegans; P32860, S. cerevisiae; P23121, Azotobacter chroococcum; 1FGS, Latobecillus casei; P3773, E. coli; 031211, Staphylococcus aureus; 083361, Treponema pallidum; Q03522, Bacillus subtilis; O06222, Mycobacterium tuberculosis; O33804, Streptomyces toyocaensis; O86109, Anabaena variabilis). Dense shading indicates identical amino acids and light shading shows conservative substitutions. Numbers given on either side of the HIRIP5 sequence indicate the positions of the respective domains.

 
A human genome BLAST analysis identified at least eight exons for the HIRIP5, spanning nearly 42 kb on chromosome 2p12. HIRIP5 sequence also showed significant homology (92% identity) to a chromosome 3 genomic contig (Hs3_5655) spanning nearly 770 bases. Since it lacked any intronic sequence, this could probably represent a non-functional pseudogene for HIRIP5.

HIRIP5 is a member of a highly conserved protein family
To examine the HIRIP5 sequence for relationship to other genes and proteins, BLAST comparisons were performed. Significant sequence homology almost throughout the HIRIP5 sequence was found in fly (GenBank accession no. AAF1622.1), worm (NP501917.1), yeast (T40430), Arabidopsis (NP_566673.1), Neurospora (CAD21196.1), and amongst many free living and parasitic bacteria [Brucella melitensis (NP_540725.1) and Rickettsia prowazekii (NP_221029.1)], suggesting that HIRIP5 contains numerous functionally and/or structurally indispensable domains that are phylogenetically conserved.

Expression profile of human and mouse HIRIP5
Lorain et al. (14) documented the expression profile of HIRIP5 gene in a multiple tissue northern blot. We extended this analysis to developmental stages, sub-regions of brain and compared it with the expression pattern in mouse. In both species, HIRIP5 showed ubiquitous expression profile and was found to express during embryonic development (Fig. 3A). In human adult brain, a single transcript of 1.4 kb size appeared in all regions tested (Fig. 3B). Likewise, Hirip5 gene in mouse was ubiquitously expressed and transcripts were detected during embryonic development (Fig. 3C and D). We further documented the overall expression of the Hirip5 gene in mouse brain. In situ hybridization in 300 µm parasagittal sections of adult mouse brain indicates that Hirip5 was expressed in a regionally restricted manner (Fig. 4A). The strongest signal was detected in cerebellum and hippocampus. Moderate level expression was detected in the cerebral cortex, olfactory bulb, striatum and thalamus. Overall, the expression pattern of the Hirip5 gene overlaps that of the Epm2a gene (11,12).



View larger version (36K):
[in this window]
[in a new window]
  		Figure 3. Expression profile of human and mouse HIRIP5 transcripts: northern blots representing multiple human fetal tissues (A), parts of adult human brain tissues (B), multiple adult mouse tissues (C) and total embryonic tissues from various developmental stages (D). The size of the transcript in both human and mouse was ~1.4 kb.

 


View larger version (84K):
[in this window]
[in a new window]
  		Figure 4. (A) Tissue in situ hybridization analyses of HIRIP5 in mouse. A parasagittal section of adult mouse brain was hybridized with a DIG-labeled Hirip5 antisense RNA probe. Hirip5 was expresses extensively in the cerebellum, hippocampus and olfactory bulb. Cb, cerebellum; Dc, distal cortex; Fc, frontal cortex; Hc, hippocampus; Ob, olfactory bulb; St, striatum; Th, thalamus. (B) Co-localization of HIRIP5 protein with laforin. HeLa cells that had been co-transfected with pEGFP-LD1 (laforin) and pNMYC-HIRIP5 (HIRIP5) constructs were used for immunofluorescence staining. Anti-Myc antibody was used for the detection of HIRIP5 protein, and the green fluorescence of the GFP was used to visualize laforin localization. The merged image on the right reveals an almost complete co-localization for the laforin and HIRIP5 proteins. Scale bar, 10 µM. (C) Co-localization of HIRIP5 protein with a marker for polyribosome. HeLa cell transiently transfected with pNMYC-HIRIP5 construct was double stained for the HIRIP5 protein and endogenous QM protein, a ribosomal resident. The merged image reveal the complete co-localization.

 
Subcellular localization of HIRIP5
We studied the subcellular distribution of HIRIP5 and whether it co-localizes with laforin. Immunofluorescence staining carried out on HeLa cells, expressing Myc-tagged HIRIP5 and GFP-tagged laforin showed that HIRIP5 is cytosolic protein and is found in the same cellular compartment as laforin, which allows for a physical association (Fig. 4B). A similar distribution of HIRIP5 protein has been observed in cells overexpressing HIRIP5 alone, indicating that its subcellular localization is not determined by interaction with laforin (Fig. 4C). There was no difference in the subcellular localization of HIRIP5 having the C-terminal or N-terminal Myc-tags (data not shown). In order to ascertain whether HIRIP5 associates with polyribosomes, transfected cells were fractionated by differential centrifugation. Immunoblotting of the fractionated samples revealed that HIRIP5 protein partitioned into the soluble fraction suggesting that HIRIP5 do not associate with ribosomes (Fig. 5B). The fractionation property of the HIRIP5 protein did not change when co-expressed with laforin (data not shown).



View larger version (41K):
[in this window]
[in a new window]
  		Figure 5. Expression of full-length HIRIP5 in HeLa cells. (A) Proteins extracted from HeLa cells transiently transfected with the expression construct pNMYC-HIRIP5 and immuno-blotted with anti-Myc antibody, resulting in the detection of a single band around 30 kDa range in the transfected cells and not in the untransfacted control cells. (B) Western blot analysis of subcellular fractions derived from HeLa cells transfected with pNMYC-HIRIP5 construct. Differential centrifugation was performed to obtain nuclear, heavy membrane, light membrane and soluble fractions. An ~30 kDa band was enriched in the soluble fraction confirming that HRIP5 is a cytoplasmic soluble protein. The CBD domain of laforin interacts with HIRIP5 protein: (C) 35S-labeled HIRIP5 protein binds to GST-laforin and not to GST immobilized on glutathione beads. The input lane was loaded with one tenth of the volume of radioactively labeled HIRIP5 protein used in this in vitro interaction assay. (D) HIRIP5 interaction with laforin in vivo by co-immunoprecipitation analysis. HeLa cells co-transfected with pEGFP or pEGFP-LDH1 and pNMYC-HIRIP5 consteructs were collected 48 h after transfection. The supernatants of cells were immunoprecipitated with anti-GFP antibody and immunoblot analyses were done using HRP-conjugated anti-Myc antibody. HIRIP5 was co-immunoprecipitated by anti-GFP antibody in HeLa cells that express GFP-lafroin and HIRIP5 proteins but in cells that express the GFP and HIRIP5. (E) HeLa cells co-transfected with pEGFP, pEGFP-LDH1, or pEGFP-LDH1-CBD and pNMYC-HIRIP5 constructs were collected 48 h after transfection. The supernatants of cells were immunoprecipitated with anti-myc antibody and immunoblot analyses were done using anti-GFP antibody. HIRIP5 was co-immunoprecipitated by anti-myc antibody in cells that express laforin-GFP or the truncated laforin containing only the CBD domain and GFP (arrow heads) but not in cells that express only the GFP. Immunoreactive Ig heavy chain (~50 kDa) and light chain (~25 kDa) are indicated by closed circles. The weaker signal obtained for laforin-CBD-GFP protein is due to its lower expression level when compared laforin-GFP. (F) Schematic diagram of the deletion constructs that express laforin as GFP-fusion proteins.

 
In vitro pull-down assays
To confirm the interaction observed between HIRIP5 and laforin proteins observed in yeast two-hybrid system, we used the in vitro pull-down assays. For the assay, GST-tagged laforin was produced as described previously (8). The pNMyc-HRIRP5 construct was used for the production of HIRIP5 protein by using an in vitro transcription and translation system in the presence of 35S-labeled methionine (Fig. 5C). The pNMyc-HIRIP5 construct produced two polypeptides of ~30 and 24 kDa (Fig. 5C). The 30 kDa protein corresponds to the expected size of Myc-tagged HIRIP5 whereas the 24 kDa polypeptide is most likely to have originated from the ATG-455. Purified recombinant GST-tagged laforin full-length protein and GST were incubated with 35S-labeled in vitro translated HIRIP5. Both the polypeptides of the HIRIP5 protein were retained by GST-laforin beads, whereas no non-specific binding to GST or to the agarose beads alone was observed (Fig. 5C).

In vivo interaction
To further characterize the in vivo interaction of HIRIP5 and laforin proteins, we co-transfected HeLa cells with pEGFP-LDH vectors expressing GFP-laforin fusion protein and pNMyc-HIRIP5 construct expressing Myc-tagged HRIRP5 protein. When GFP-laforin was immunoprecipitated from cell lysates with anti-GFP antibody, co-transfected HIRIP5 protein was co-immunoprecipitated (Fig. 5D). However no HIRIP5 was co-immunoprecipitated by anti-GFP antibody in HeLa cells co-transfected with vectors expressing GFP and HIRIP5 proteins (Fig. 5D). Conversely, anti-Myc antibody also co-immunoprecipated the GFP-laforin protein from the co-transfected cells; however anti-Myc antibody did not co-immunoprecipitate the GFP protein suggesting that the interaction is indeed between laforin and HIRIP5 (Fig. 5E). To further determine which domain in the laforin protein is required for the interaction with HIRIP5, we generated N- and C-terminal deletion mutants of the laforin protein in vector pGEFP-LDH1 (Fig. 5F). These deletion constructs were co-transfected with pNMyc-HIRIP5 vector and the cell lysates were co-immunoprecipitated with anti-Myc antibody. The CBD domain of laforin was sufficient for the interaction with HIRIP5 (Fig. 5E). The EGFP vector that had the DSPD domain did not express in the HeLa cells despite several attempts. Therefore its interaction with the HIRIP5 could not be evaluated.

Laforin dephosphorylates HIRIP5 in vitro
To clarify whether HIRIP5 could be a substrate for laforin, phosphoserine/threonine and phosphotyrosine phosphatase activities of laforin were determined by measuring the release of inorganic phosphate from the [32P]phosphorylated HIRIP5 protein. As illustrated in Figure 6, laforin displayed detectable level of phosphatase activities whereas the phosphatase inactive laforin mutant, C226S, showed negligible levels of phosphate release. HIRIP5 therefore could be a potential substrate for laforin in vivo.



View larger version (16K):
[in this window]
[in a new window]
  		Figure 6. GST-laforin fusion protein (10 µg) was reacted with [32P]Ser/Thr-labeled HIRIP5 or [32P]Tyr-labeled HIRIP5 under identical conditions and the labeled inorganic phosphate released from the substrate was measured after trichloroacetic acid precipitation. ‘Total’ refers to the total count present in the substrate before dephosphorylation, and ‘laforin’ refers the labeled inorganic phosphate released from the substrate after dephosphorylation by GST-laforin. ‘C266S’ refers sto the dephosphorylation reaction done with the catalytically inactive laforin mutant.

 
MurD ligase assay
The E. coli expressed GST-HIRIP5 fusion protein was used for an ATP-dependent MurD ligase activity by monitoring the formation of product ADP using the pyruvate kinase and lactate dehydrogenase coupled enzyme assay. Under the conditions used, no MurD ligase activity was detected for the recombinant GST-HIRIP5 fusion protein whereas the controls worked (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Our studies have identified the protein HIRIP5 that associates with the Lafora disease gene product laforin. HIRIP5 was isolated through a yeast two-hybrid screen and when sequenced was found to contain a NiFU domain. Although the sequence of HIRIP5 provided no immediate clues to its function or how it might affect that of laforin, it is still of interest as its interaction with laforin was shown to be specific. HIRIP5 not only binds to laforin in yeast two-hybrid system but also in vitro as shown by GST pull-down experiments and in vivo as shown by co-immunoprecipatation from transfected cells expressing both proteins.

The full-length HIRIP5 cDNA clones isolated in the present study corrected and extended the known HIRIP5 gene open reading frame by adding 25 amino acids to the potentially functionally important N-terminal region of the protein. All three independently isolated human cDNA clones and a mouse clone shared this newly defined N-terminal region, suggesting that this form represents the abundant transcript of the HIRIP5 gene. The HIRIP5 gene maps to chromosomes 2p12, the region that has been associated with several neurological syndromes including bipolar disorder (17), dyslexia (18), Alstrom syndrome (19) and Miyoshi myopathy (20). It would be of interest to investigate whether HIRIP5 is indeed associated with these diseases. The deduced HIRIP5 protein contains a NifU-like domain at its carboxyl terminal. The NifU domain, present at the carboxyl terminal, is the only region that is conserved amongst nitrogen fixing proteins and is involved in nitrogen fixation in several prokaryotes. With the exception of the essential genes coding for and regulating the expression of the nitrogenase subunits, the functions of other NifU genes appear to be associated with cofactor biosynthesis, amino acid inter-conversion, and regulation of ammonia storage (15). However, not all biochemical activities of the NifU gene products have been identified yet (21). Some of these proteins might represent specialized versions of general metabolic enzymes. Thus their presence cannot be precluded from non-nitrogen-fixing organisms. The HIRIP5 protein also shows consensus sequence for a potential MurD ligase domain. The MurD ligases (UDP-N-acetylmuramoyl-L-alanine-D-glutamate ligase) are cytoplasmic enzymes that catalyze the addition of D-glutamate to the nucleotide precursor UDP-N-acetylmuramoyl-L-alanine in the murein biosynthetic pathway. The product of the reaction is UDP-N-acetylmuramoyl-L-alanine-D-glutamate (22). However, in our in vitro assay, the recombinant human HIRIP5 protein failed to show detectable levels of ligase activity. Therefore the specific roles of the HIRIP5 gene in the cellular physiology are yet to be understood.

The expression of HIRIP5 in human and mice was examined and compared with the expression pattern previously documented for the EPM2A gene. Northern analysis showed that HIRIP5 is ubiquitously expressed in both human and mouse and the expression pattern of HIRIP5 largely overlaps with the EPM2A gene (2,10,12). Owing to its ubiquitous expression profile, we argue that HIRIP5 may have a house-keeping role. Although we have not analyzed yet in greater detail the cell type-specific expression of the HIRIP5 gene, at least in mouse brain sections the Hirip5 staining intensity correlates roughly with neuronal density, therefore expression of Hirip5 appears to be restricted to neurons. It is of interest to note that a very similar expression pattern was documented for Epm2a in mouse (10). Thus it is likely that in cells that express EPM2A, the function of HIRIP5 may be altered due to its interaction with laforin, and/or HIRIP5 may in turn affect the function of laforin.

The subcellular localization of HIRIP5 was also studied to investigate whether HIRIP5 and laforin are in the same cellular compartment. Immunofluorescence studies carried out on transiently transfected HeLa cells revealed that, like laforin, HIRIP5 is a cytosolic protein. Both HIRIP5 and laforin seem to be present diffusely throughout the cytoplasm and the staining pattern did not vary between cells that express both HIRIP5 and laforin proteins and those that express exclusively either one of them. However, unlike laforin, HIRIP5 did not show strong association with ribosomes. Curiously, HIRIP5 did not show nuclear localization either. This observation questions the relevance of the report by Lorain et al. (14), wherein an in vitro interaction between HIRIP5 and HIRA proteins has been shown. The HIRA gene, one of the candidates for the Di-George syndrome (23), encodes a protein that is predominantly nuclear in distribution (24). HIRA protein is a transcriptional co-repressor that regulates cell cycle-dependent histone gene transcription, possibly by remodeling local chromatin structure (25). Therefore additional experiments will be necessary to determine whether HIRA and HIRIP5 proteins also interact and to explore the functional relevance of this in vivo interaction with regard to laforin's cellular functions.

In the present study we overexpressed the laforin and HIRIP5 proteins and provided evidence for their in vivo interaction. Furthermore we show that a truncated laforin having only the CBD region was able to pull down the HIRIP5 protein. While this experiment did not rule out the possibility that the C-terminal DSPD region does not interact with HIRIP5 (because the DSPD truncated construct did not express in HeLa cells for unknown reasons), the present set of results is clear enough to suggest that CBD region alone is sufficient for the interaction with HIRIP5. Likewise, we are yet to formally localize the region of HIRIP5 responsible for its ability to interact with laforin, but the initial yeast two-hybrid identification of the partial HIRIP5 cDNA clone, YTH74, encoding a portion of the C-terminus of the HIRIP5 (encompassing the NifU domain) indicates that this region is sufficient for the interaction with laforin. Thus, it appears that the C-terminal segment of the HIRIP5 protein interacts with the N-terminal half of laforin.

The presence of CBD in the laforin and the potential of HIRIP5 in peptidoglycan biosynthesis raise an interesting possibility that HIRIP5 and laforin interact through an intermediary carbohydrate moiety that also happens to be present in the in vitro transcription/translation extracts used to prepare the radiolabeled partner for the GST-pull-downs. Alternatively, this interaction may serve to localize one or the other protein to glycan synthesis suspected to be defective in LD (8,11,13). This raises the question of whether HIRIP5 could be a substrate for laforin. The deduced HIRIP5 amino acid sequence contains several potential targets for dephosphorylation. Here we demonstrated that at least some of the phosphorylated residues (both Ser/Thr and Tyr) in the HIRIP5 proteins can be dephosphorylated with laforin in vitro, although understanding its physiological relevance merits further study and should help to clarify the cellular roles of HIRIP5.

The HIRIP5 ortholog in yeast is a mitochondrial matrix protein and is involved iron metabolism, especially in Fe–S cluster assembly (26). Not all Fe–S cluster proteins are mitochondrial in localization. In mammals, at least one of them has been shown to be cytosolic in nature (27). In humans, alternative splicing of a common pre-mRNA of the IscU gene results in synthesis of two NifU domain-containing proteins that differ at the N-terminus and localize either to the cytosol or to the mitochondria. Further, both isoforms were found in protein complexes from mitochondria and cytosol, implying that Fe–S cluster assembly takes place in multiple subcellular compartments in mammalian cells (27). This raises interesting possibilities that HIRIP5 may also be involved in iron homeostasis. The complexity and importance of iron metabolism are underscored by recent studies on the neurodegenerative disease, Friedreich's ataxia (28,29). Curiously, in Epm2a knockout mouse neurodegenerative changes involve several mitochondrial abnormalities (13). Given the interaction between HIRIP5 and laforin, it would be of interest now to explore the possible disturbance in the iron homeostasis in the Epm2a mutant mouse. Therefore our future research is directed at determining the potential relationship between laforin and the function(s) of HIRIP5 protein in iron metabolism.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmid constructs
A cDNA insert from the clone LDH1 (8) containing the complete coding region of the EMP2A gene was cloned in-frame into the BamHI and NotI sites of the pEG202 vector (OriGene Technologies Inc, Rockville, MD, USA). The clone was named pEG202-LDH and used as bait for the yeast two-hybrid screening. Vector pGEX-LDH, expressing GST-laforin fusion protein, and vector pEGFP-LDH, expressing GFP-laforin fusion protein, were reported in a previous study (8). A new construct was created in which the exon 1 coding region of EPM2A was PCR amplified and cloned in-frame into pEGFP-N1 vector (Clontech Laboratories Inc., Palo Alto, CA, USA) to create the pEGFP-LDH1-CBD expression construct. On transfection, pEGFP-LDH1-CBD construct would express a truncated laforin containing only the CBD-4 domain as a fusion protein to the N-terminus of EGFP (Fig. 5F). Another construct (pEGFP-LDH1-DSPD) was created in which the coding region representing exons 2, 3 and 4 of EPM2A gene was cloned in-frame into the pEGFP-N1 vector. A Kozak consensus translation signal sequence (aaggcaATGGACCTC) was added in frame to 5' end of the coding sequence. Construct pEGFP-LDH1-DSPD would produce an N-terminal truncated laforin-EGFP chimeric protein (Fig. 5F). An HIRIP5 expression construct was created in which the coding region of HIRIP5 gene was placed in frame to 3' end of c-myc epitope of the pcDNA3-MycN vector (Invitrogen Inc., Carlsbad, CA, USA). This construct, which would express N-terminal myc-tagged HIRIP5 protein, was named as pNMYC-HIRIP5. Similarly, the c-myc epitope was placed at the 3' end of HIRIP5 by cloning the HIRIP5 coding sequence into pcDNA3.1/Myc-His (+)A vector (Invitrogen Inc., Carlsbad, CA, USA). This construct, which would express a C-terminal myc-tagged HIRIP5 protein, was named as pCMYC-HIRIP5. For all expression experiments, the pNMyc-HIRIP5 construct was used. For immunofluorescence studies both constructs were used. Finally, a bacterial expression construct was created by fusing in-frame the HIRIP5 coding sequence into the pGEX6P2 GST protein expression vector to encode GST-HIRIP5 fusion protein. This vector was called pGEX-HIRIP5. ORFs of all constructs were verified by sequencing.

Yeast two-hybrid screen
The Duplex-A yeast two-hybrid system (OriGene Technologies Inc., Rockville, MD, USA) was used in this experiment. All experiments were performed in yeast strain EGY48. Bait vector pEG202-LDH was transformed into EGY48 cells using the lithium acetate method as recommended and the transformants then tested for the absence of autoactivation of the lacZ reporter gene. For the library screening, a single colony of EGY48 cells transformed with pEG202-LDH was grown overnight and then transformed with a human fetal brain cDNA library constructed in vector pJG4-5 (OriGene). Approximately 2x106 transformants were plated on YNB(gal)-his-ura-trp-leu selective plates. After incubation at 30°C for 3–5 days, positive clones were further tested for galactose growth dependence and lacZ expression. Plasmid DNAs were isolated from ß-gal-positive clones by growth in YNB(gal)-his-ura-trp-leu plates followed by transformation into E. coli KC8.

Isolation of full-length cDNA clones for human and mouse HIRIP5
A human fetal brain cDNA library (UNI Zap vector, Stratagene) and adult mouse brain cDNA library (Lambda ZAP vector, Strategene) were screened using the insert of the yeast two-hybrid screen positive clone YTH134 as a probe. Approximately 2x106 plaques were hybridized overnight and positive clones phages were transformed into plasmids by in vivo excision and the inserts were sequenced.

Northern blotting and whole-mount RNA in situ hybridization
Northern blots for mouse and human tissues were commercially obtained (Clontech, Palo Alto, CA, USA). For hybridization, DNA probes were labeled with [{alpha}32P]dCTP and hybridized overnight in ExpressHyb hybridization solution (Clontech) as recommended by the manufacturer. For whole-mount RNA in situ hybridization, a mouse Hirip5 cDNA clone containing 903 bp of the coding region in the Bluescript vector SK+/- was used for the generation of sense and anti-sense riboprobes using DIG-RNA labeling kit (Boehringer Mannheim). The whole-mount in situ hybridization protocol of Spector et al. (30) was followed for hybridizing vibratome sections (300 µm-thick) of adult mice brains as described earlier (10). Sections were hybridized with anti-sense or sense probe and processed for immunological detection as recommended by the manufacturer.

In vitro translation and in vitro binding assays
The TNT T7 Quick Coupled transcription/translation system (Promega Corporation, Madison) was used for the in vitro synthesis of HIRIP5 protein. Five micrograms of pNMyc-HIRIP5 construct was used for the synthesis in the presence of 35S-labeled methionine. For in vitro binding, an aliquot of GST-fusion protein bound to the glutathione–agarose beads (1 : 1 slurry) (Amersham Pharmacia Biotech) was incubate with the 35S-labeled in vitro translated HIRIP5 protein for 1 h at 4°C. After incubation the beads were washed four times with HNTG buffer (20 mM HEPES-KOH, pH 7.5, 100 mM NaCl, 0.1% Triton X-100 and 10% glycerol) to remove unbound protein. Bound proteins were eluted from the beads by boiling in SDS sample buffer, size separated in SDS–PAGE and detected by autoradiography.

Cell culture and transfection
HeLa cells cultured overnight in Dulbecco's modified medium (DMEM; Life Technologies Inc., Grand Island, NY, USA) containing 10% calf serum and transfected with expression constructs using Lipofectamine Plus reagent (Life Technologies) following the guidelines supplied by the manufacturer. After 48 h, the transfected cells were processed for immunofluorescence staining, immunoblot analysis or immunoprecipitation.

Immunofluorescence microscopy
HeLa cells transfected with pNMYC-HIRIP5 or co-transfected with pEGFP-LDH and pNMYC-HIRIRP5 were fixed and processed for immunofluorescence microscopy as described (8). N-terminal or C-terminal Myc-tag of the HIRIP5 protein was detected with mouse monoclonal anti-Myc antibody (Calbiochem-Novabiochem Corp, San Diego, CA, USA).

Immunoprecipitation
HeLa cells co-transfected with pEGFP-N1 or pEGFP-LDH1 or pEGFP-CBD and pNMYc-HIRIP5 were collected 48 h after transfection. The cells were lysed in lysis buffer (10 mM Tris pH 7.4, 100 mM NaCl, 1 mM EDTA, 20 µg/ml PMSF, 20 µg/ml leupeptin, 100 µM Na3VO4 and 50 mM NaF) containing 0.5% NP-40. Cellular debris was removed by centrifugation at 16 000g for 10 min at 4°C. The supernatants were preincubated with protein G-Sepharose (Amersham Pharmacia Biotech) for 2 h at 4°C and then incubated with monoclonal anti-GFP (Boehringer Mannheim, Germany) or anti-Myc antibody (Calbiochem) for 1 h at 4°C. After incubation, protein G-Sepharose was used for precipitation. The beads were washed with lysis buffer four times and then eluted with SDS sample buffer for immunoblot analysis.

Immunoblotting
Protein samples were run on a 15–25% gradient SDS–polyacrylamide gel and transferred onto a nitrocellulose filter (Schleicher and Schuell, Dassel, CA, USA) as described previously (8). After blocking with 3.5% non-fat dry milk powder, the membranes were processed through sequential incubations with primary antibody followed by secondary antibody. Immunoreactive proteins on the filter were visualized using ECF western blotting kit (Amersham Pharmacia).

Expression of GST fusion protein
Escherchia coli BL21 cells containing pGEX6P, pGEX-HIRIP5, pGEX-LDH or pGEX-LDH-C266S constructs were cultured at 20°C for 16 h and expressed GST or GST-fusion proteins were purified as reported earlier (8).

Phosphatase assay
Phosphatase assay was performed as described by Ganesh et al. (8), using the GST-HRIRP5 fusion protein as the substrate. In brief, 32P-labeled GST-HIRIP5 protein was prepared by in vitro phosphorylation of serine and threonine residues with cAMP-dependent protein kinase (New England Biolabs, Beverly, MA, USA) or phophorylation of tyrosine residues with c-abl (Calbiochem-Novabiochem) in the presence of [{gamma}32P]ATP. The reaction was terminated with the addition of 20% TCA and the pellet was dissolved in substrate solubilization buffer (50 mM Tris pH 8.0, 0.1 mM EDTA, 1 mM DTT and 0.01% CHAPS). The GST-HIRIP5 was purified using GSH sepharose and was treated with prescision protease to cleave the HIRIP5 from the GST tag. The purified HIRIP5 was used as the substrate for dephosphorylation by GST-laforin at 30°C for 10 min. The reaction was terminated by the addition of 100% TCA and chilled on ice. After centrifugation, the clear supernatant was collected and the amount of radioactivity released as 32Pi was counted by a liquid scintillation counter.

MurD ligase assay
MurD ligase assay was performed as described by El-Sherbeini et al. (31), using the GST-HIRIP5 as the enzyme.


   ACKNOWLEDGEMENTS
 
We thank Dr M. El-Sherbeini of Merck Research Laboratories, Rahway (USA), for the gift of MurD Ligase substrate. This work was supported by research grants from the RIKEN Brain Science Institute (S.G. and K.Y.), Wako-shi, and the Indian Institute of Technology, Kanpur (S.G.). AVD-E was supported by NIH grant no. NS42376.


   FOOTNOTES
 
* To whom correspondence should be addressed at: Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur 208016, India. Tel: +91 5122597552; Fax: +91 5122597103; Email: sganesh{at}iitk.ac.in

Correspondence may also be addressed to Dr Kazuhiro Yamakawa, Lab. for Neurogenetics, RIKEN Brain Science Institute, 2-1, Hirosawa, Wakoshi 351-0198, Japan. Tel: +81 484679703; Fax: +81 484677095; Email: yamakawa{at}brain.riken.go.jp Back

{dagger} The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Delgado-Escueta, A.V., Ganesh, S. and Yamakawa, K. (2001) Advances in the genetics of progressive myoclonus epilepsy. Am. J. Med. Genet., 106, 129–138.[CrossRef][Web of Science][Medline]

  2. Minassian, B.A., Lee, J.R., Herbrick, J.A., Huizenga, J., Soder, S., Mungall, A.J., Dunham, I., Gardner, R., Fong, C.Y., Carpenter, S. et al. (1998) Mutations in a gene encoding a novel protein tyrosine phosphatase cause progressive myoclonus epilepsy. Nat. Genet., 20, 171–174.[CrossRef][Web of Science][Medline]

  3. Serratosa, J.M., Gomez-Garre, P., Gallardo, M.E., Anta, B., de Bernabe, D.B., Lindhout, D., Augustijn, P.B., Tassinari, C.A., Malafosse, R.M., Topcu, M. et al. (1999) A novel protein tyrosine phosphatase gene is mutated in progressive myoclonus epilepsy of the Lafora type (EPM2). Hum. Mol. Genet., 8, 345–352.[Abstract/Free Full Text]

  4. Minassian, B.A., Ianzano, L., Delgado-Escueta, A.V. and Scherer, S.W. (2000) Identification of new and common mutations in the EPM2A gene in Lafora's disease. Neurology, 54, 488–490.[Abstract/Free Full Text]

  5. Minassian, B.A., Ianzano, L., Meloche, M., Andermann, E., Rouleau, G.A., Delgado-Escueta, A.V. and Scherer, S.W. (2000) Mutation spectrum and predicted function of laforin in Lafora's progressive myoclonus epilepsy. Neurology, 55, 341–346.[Abstract/Free Full Text]

  6. Gomez-Garre, P., Sanz, Y., de Cordoba, S.R. and Serratosa, J.M. (2000) Mutational spectrum of the EPM2A gene in progressive myoclonus epilepsy of Lafora: high degree of allelic heterogeneity and prevalence of deletions. Eur. J. Hum. Genet., 8, 946–954.[CrossRef][Web of Science][Medline]

  7. Ganesh, S., Delgado-Escueta, A.V., Suzuki, T., Francheschetti, S., Riggio, C., Avanzini, G., Rabinowicz, A., Bohlega, S., Bailey, J., Alonso, M.E. et al. (2002) Genotype-phenotype correlations for EPM2A mutations in Lafora's progressive myoclonus epilepsy: exon 1 mutations associate with an early-onset cognitive deficit subphenotype. Hum. Mol. Genet., 11, 1263–1271.[Abstract/Free Full Text]

  8. Ganesh, S., Agarwala, K.L., Ueda, K., Akagi, T., Shoda, K., Usui, T., Hashikawa, T., Osada, H., Delgado-Escueta, A.V. and Yamakawa, K. (2000) Laforin, defective in the progressive myoclonus epilepsy of lafora type, is a dual-specificity phosphatase associated with polyribosomes. Hum. Mol. Genet., 9, 2251–2261.[Abstract/Free Full Text]

  9. Ganesh, S., Suzuki, T. and Yamakawa, K. (2002) Alternate splicing modulates subcellular localization of laforin. Biochem. Biophys. Res. Commun., 291, 1134–1137.[CrossRef][Web of Science][Medline]

  10. Ganesh, S., Agarwala, K.L., Amano, K., Suzuki, T., Delgado-Escueta, A.V. and Yamakawa, K. (2001) Regional and developmental expression of Epm2a gene and its evolutionary conservation. Biochem. Biophys. Res. Commun., 283, 1046–1053.[CrossRef][Web of Science][Medline]

  11. Wang, J., Stuckey, J.A., Wishart, M.J. and Dixon, J.E. (2002) A unique carbohydrate binding domain targets the Lafora disease phosphatase to glycogen. J. Biol. Chem., 277, 2377–2380.[Abstract/Free Full Text]

  12. Ganesh, S., Amano, K., Delgado-Escueta, A.V. and Yamakawa, K. (1999) Isolation and characterization of mouse homologue for human epilepsy gene, EPM2A. Biochem. Biophys. Res. Commun., 257, 24–28.[CrossRef][Web of Science][Medline]

  13. Ganesh, S., Delgado-Escueta, A.V., Sakamoto, T., Avila, M.R., Machado-Salas, J., Hoshii, Y., Akagi, T., Gomi, H., Suzuki, T., Amano, K. et al. (2002) Targeted disruption of the Epm2a gene causes formation of Lafora inclusion bodies, neurodegeneration, ataxia, myoclonus epilepsy and impaired behavioral response in mice. Hum. Mol. Genet., 11, 1251–1262. [Abstract/Free Full Text]

  14. Lorain, S., Lecluse, Y., Scamps, C., Mattei, M.G. and Lipinski, M. (2001) Identification of human and mouse HIRA-interacting protein-5 (HIRIP5), two mammalian representatives in a family of phylogenetically conserved proteins with a role in the biogenesis of Fe/S proteins. Biochim. Biophys. Acta., 1517, 376–383.[Medline]

  15. Ouzounis, C., Bork, P. and Sander, C. (1994) The modular structure of NifU proteins. Trends Biochem. Sci., 19, 199–200.[CrossRef][Web of Science][Medline]

  16. Bertrand, J.A., Auger, G., Fanchon, E., Martin, L., Blanot, D., van Heijenoort, J., and Dideberg, O. (1997) Crystal structure of UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase from Escherichia coli. EMBO J., 16, 3416–3425.[CrossRef][Web of Science][Medline]

  17. McInnis, M.G., Lan, T.H., Willour, V.L., McMahon, F.J., Simpson, S.G., Addington, A.M., MacKinnon, D.F., Potash, J.B., Mahoney, A.T., Chellis, J. et al. (2003) Genome-wide scan of bipolar disorder in 65 pedigrees: supportive evidence for linkage at 8q24, 18q22, 4q32, 2p12, and 13q12. Mol. Psychiat., 8, 288–298.

  18. Francks, C., Fisher, S.E., Olson, R.K., Pennington, B.F., Smith, S.D., DeFries, J.C. and Monaco, A.P. (2002) Fine mapping of the chromosome 2p12–16 dyslexia susceptibility locus: quantitative association analysis and positional candidate genes SEMA4F and OTX1. Psychiatr. Genet., 12, 35–41.[CrossRef][Web of Science][Medline]

  19. Macari, F., Lautier, C., Girardet, A., Dadoun, F., Darmon, P., Dutour, A., Renard, E., Bouvagnet, P., Claustres, M., Oliver, C. and Grigorescu, F. (1998) Refinement of genetic localization of the Alstrom syndrome on chromosome 2p12–13 by linkage analysis in a North African family. Hum. Genet., 103, 658–661.[CrossRef][Web of Science][Medline]

  20. Bejaoui, K., Hirabayashi, K., Hentati, F., Haines, J.L., Ben Hamida, C., Belal, S, Miller, R.G., McKenna-Yasek, D. and Weissenbach, J. et al. (1995) Linkage of Miyoshi myopathy (distal autosomal recessive muscular dystrophy) locus to chromosome 2p12–14. Neurology, 45, 768–772.[Abstract/Free Full Text]

  21. Hwang, D.M., Dempsey, A., Tan, K.T. and Liew, C.C. (1996) A modular domain of NifU, a nitrogen fixation cluster protein, is highly conserved in evolution. J. Mol. Evol., 43, 536–540.[Web of Science][Medline]

  22. Bertrand, J.A., Fanchon, E., Martin, L., Chantalat, L., Auger, G., Blanot, D., van Heijenoort, J. and Dideberg, O. (2000) ‘Open’ structures of MurD: domain movements and structural similarities with folylpolyglutamate synthetase. J. Mol. Biol., 301, 1257–1266.[CrossRef][Web of Science][Medline]

  23. Lamour, V., Lecluse, Y., Desmaze, C., Spector, M., Bodescot, M., Aurias, A., Osley, M.A. and Lipinski, M. (1995) A human homolog of the S. cerevisiae HIR1 and HIR2 transcriptional repressors cloned from the DiGeorge syndrome critical region. Hum. Mol. Genet., 4, 791–799.[Abstract/Free Full Text]

  24. Lorain, S., Quivy, J.P., Monier-Gavelle, F., Scamps, C., Lecluse, Y., Almouzni, G. and Lipinski, M. (1998) Core histones and HIRIP3, a novel histone-binding protein, directly interact with WD repeat protein HIRA. Mol. Cell. Biol., 18, 5546–5556.[Abstract/Free Full Text]

  25. Magnaghi, P., Roberts, C., Lorain, S., Lipinski, M. and Scambler, P.J. (1998) HIRA, a mammalian homologue of Saccharomyces cerevisiae transcriptional co-repressors, interacts with Pax3. Nat. Genet., 20, 74–77.[CrossRef][Web of Science][Medline]

  26. Schilke, B., Voisine, C., Beinert, H. and Craig, E. (1999) Evidence for a conserved system for iron metabolism in the mitochondria of Saccharomyces cerevisiae. Proc. Natl Acad. Sci. USA, 96, 10206–10211.[Abstract/Free Full Text]

  27. Tong, W.H. and Rouault, T. (2000) Distinct iron-sulfur cluster assembly complexes exist in the cytosol and mitochondria of human cells. EMBO J., 19, 5692–5700.[CrossRef][Web of Science][Medline]

  28. Campuzano, V., Montermini, L., Molto, M.D., Pianese, L., Cossee, M., Cavalcanti, F., Monros, E., Rodius, F., Duclos, F., Monticelli, A. et al. (1996) Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion. Science, 271, 1423–1427.[Abstract]

  29. Babcock, M., de Silva, D., Oaks, R., Davis-Kaplan, S., Jiralerspong, S., Montermini, L., Pandolfo, M. and Kaplan, J. (1997) Regulation of mitochondrial iron accumulation by Yfh1p, a putative homolog of frataxin. Science, 276, 1709–1172.[Abstract/Free Full Text]

  30. Spector, D.L., Goldman, R.D. and Leinwand, L.A. (1998) Cells. A Laboratory Manual, Vol 3. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

  31. El-Sherbeini, M., Geissler, W.M., Pittman, J., Yuan, X., Wong, K.K., and Pompliano, D.L. (1998) Cloning and expression of Staphylococcus aureus and Treptococcus pyogenes murD genes encoding uridine diphosphate N-acetylmuramoyl-L-alanine:D-glutamate ligases. Gene., 210, 117–125.[CrossRef][Web of Science][Medline]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Hum Mol GenetHome page
M. C. Solaz-Fuster, J. V. Gimeno-Alcaniz, S. Ros, M. E. Fernandez-Sanchez, B. Garcia-Fojeda, O. C. Garcia, D. Vilchez, J. Dominguez, M. Garcia-Rocha, M. Sanchez-Piris, et al.
Regulation of glycogen synthesis by the laforin-malin complex is modulated by the AMP-activated protein kinase pathway
Hum. Mol. Genet., March 1, 2008; 17(5): 667 - 678.
[Abstract] [Full Text] [PDF]

Home page
 NeurologyHome page
E. M. Chan, S. Omer, M. Ahmed, L. R. Bridges, C. Bennett, S. W. Scherer, and B. A. Minassian
Progressive myoclonus epilepsy with polyglucosans (Lafora disease): Evidence for a third locus
Neurology, August 10, 2004; 63(3): 565 - 567.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
E. M. Chan, C. A. Ackerley, H. Lohi, L. Ianzano, M. A. Cortez, P. Shannon, S. W. Scherer, and B. A. Minassian
Laforin preferentially binds the neurotoxic starch-like polyglucosans, which form in its absence in progressive myoclonus epilepsy
Hum. Mol. Genet., June 1, 2004; 13(11): 1117 - 1129.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
12/18/2359    most recent
ddg253v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (14)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Ganesh, S.
Right arrow Articles by Yamakawa, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ganesh, S.
Right arrow Articles by Yamakawa, K.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?