Gly-rich Domain Research Articles

Dissection of FUS domains involved in regulation of SnRNP70 gene expression.

FUS is one of the causative factors of amyotrophic lateral sclerosis. Loss and/or gain of its physiological functions has been believed to be linked to the pathogenesis of this condition. However, its functions remain incompletely understood. This study dissected the domains of FUS regulating the expression of SnRNP70, which functions in mRNA splicing. Biochemical analysis revealed that all FUS domains except for RGG1 contribute to determining Snrnp70 transcript abundance and thus its protein abundance. RNA-Seq analysis using the Gly-rich domain-deleted mutant coupled with snRNP70 knockdown revealed that FUS has a potential to regulate gene expression in both snRNP70-dependent and snRNP70-independent manners through the Gly-rich domain. These results provide insight into molecular details of the regulation of gene expression by FUS.

Molecular dissection of ALS-linked TDP-43 - involvement of the Gly-rich domain in interaction with G-quadruplex mRNA.

TDP-43 is the major pathogenic protein of amyotrophic lateral sclerosis (ALS). Previously, we identified that TDP-43 interacts with G-quadruplex (G4)-containing RNA and is involved in their long-distance transport in neurons. For the molecular dissection of the TDP-43 and G4-RNA interaction, we analyzed it here invitro and in cultured cells using a set of 10 mutant TDP-43 proteins from familial and sporadic ALS patients as well as using the TDP-43 C-terminal Gly-rich domain alone. Our results altogether indicate the involvement of the Gly-rich region of TDP-43 in the initial recognition and binding of G4-RNA, which then induces tight binding of TDP-43 with target RNAs, supposedly in conjunction with its RNA recognition motifs.

Behavioral defects in a DCTN1G71A transgenic mouse model of Perry syndrome

Perry syndrome is a rare neurodegenerative disease characterized by parkinsonism, depression/apathy, weight loss, and central hypoventilation. Our previously-conducted genome-wide association scan and subsequent studies identified nine mutations in DCTN1, the largest protein subunit of the dynactin complex, in patients with Perry syndrome. These included G71A in the microtubule-binding cytoskeleton-associated protein Gly-rich domain of p150Glued. The dynactin complex is essential for function of the microtubule-based cytoplasmic retrograde motor dynein.To test the hypothesis that the G71A mutation in the DCTN1 gene is sufficient to cause Perry syndrome, we generated DCTN1G71A transgenic mice. These mice initially developed normally, but young animals showed decreased exploratory activity and aged animals showed impaired motor coordination. These behavioral defects parallel apathy-like symptoms and parkinsonism encountered in Perry syndrome. TDP-43 aggregates were not detected in the substantia nigra and cerebral cortex of the transgenic mice, although pathological aggregates of TDP-43 have been considered a major neuropathological feature of Perry syndrome. Our study reveals that a single mutation in the DCTN1 gene recapitulates symptoms of Perry syndrome patients, and provides evidence that DCTN1G71A transgenic mice represent a novel rodent model of Perry syndrome.

RNA Recognition Motif-Containing Protein ORRM4 Broadly Affects Mitochondrial RNA Editing and Impacts Plant Development and Flowering.

Plant RNA editosomes modify cytidines (C) to uridines (U) at specific sites in plastid and mitochondrial transcripts. Members of the RNA-editing factor interacting protein (RIP) family and Organelle RNA Recognition Motif-containing (ORRM) family are essential components of the Arabidopsis (Arabidopsis thaliana) editosome. ORRM2 and ORRM3 have been recently identified as minor mitochondrial editing factors whose silencing reduces editing efficiency at ∼6% of the mitochondrial C targets. Here we report the identification of ORRM4 (for organelle RRM protein 4) as a novel, major mitochondrial editing factor that controls ∼44% of the mitochondrial editing sites. C-to-U conversion is reduced, but not eliminated completely, at the affected sites. The orrm4 mutant exhibits slower growth and delayed flowering time. ORRM4 affects editing in a site-specific way, though orrm4 mutation affects editing of the entire transcript of certain genes. ORRM4 contains an RRM domain at the N terminus and a Gly-rich domain at the C terminus. The RRM domain provides the editing activity of ORRM4, whereas the Gly-rich domain is required for its interaction with ORRM3 and with itself. The presence of ORRM4 in the editosome is further supported by its interaction with RIP1 in a bimolecular fluorescence complementation assay. The identification of ORRM4 as a major mitochondrial editing factor further expands our knowledge of the composition of the RNA editosome and reveals that adequate mitochondrial editing is necessary for normal plant development.

Efficient expression and purification of recombinant human μ-calpain using anEscherichia coliexpression system

Calpains comprise a superfamily of Ca(2+) -regulated cysteine proteases that are indispensable for the regulation of various cellular functions. Of these, the mammalian μ- and m-calpains are the best characterized isoforms. They are ubiquitously expressed and form heterodimers consisting of a distinct 80-kDa catalytic subunit (CAPN1 for μ-calpain and CAPN2 for m-calpain) and a common 30-kDa regulatory subunit (CAPNS1). To date, various expression systems have been developed for producing recombinant calpains for structural and functional studies; however, no low-cost, simple and efficient bacterial expression system for μ-calpain has been available, because the protein forms aggregates. Here, we established an efficient method for producing active recombinant human μ-calpain using an Escherichia coli expression system. This was achieved by co-expressing CAPN1 and CAPNS1 lacking the N-terminal Gly-rich domain (CAPNS1ΔGR) in the SoluBL21 strain. From 1 L of E. coli culture, over 2 and 6 mg, respectively, of μ-calpain and its active-site mutant μ-calpain:C115S (CAPN1:C115S+CAPNS1ΔGR) were purified by two successive column chromatographies. Compared to the native enzyme, the purified μ-calpain showed almost identical properties, demonstrating its suitability for use in structural and functional studies. This is the first report of the bacterial expression and the simple and efficient purification of active recombinant μ-calpain.

The backbone dynamics of the amyloid precursor protein transmembrane helix provides a rationale for the sequential cleavage mechanism of γ-secretase.

The etiology of Alzheimer's disease depends on the relative abundance of different amyloid-β (Aβ) peptide species. These peptides are produced by sequential proteolytic cleavage within the transmembrane helix of the 99 residue C-terminal fragment of the amyloid precursor protein (C99) by the intramembrane protease γ-secretase. Intramembrane proteolysis is thought to require local unfolding of the substrate helix, which has been proposed to be cleaved as a homodimer. Here, we investigated the backbone dynamics of the substrate helix. Amide exchange experiments of monomeric recombinant C99 and of synthetic transmembrane domain peptides reveal that the N-terminal Gly-rich homodimerization domain exchanges much faster than the C-terminal cleavage region. MD simulations corroborate the differential backbone dynamics, indicate a bending motion at a diglycine motif connecting dimerization and cleavage regions, and detect significantly different H-bond stabilities at the initial cleavage sites. Our results are consistent with the following hypotheses about cleavage of the substrate: First, the GlyGly hinge may precisely position the substrate within γ-secretase such that its catalytic center must start proteolysis at the known initial cleavage sites. Second, the ratio of cleavage products formed by subsequent sequential proteolysis could be influenced by differential extents of solvation and by the stabilities of H-bonds at alternate initial sites. Third, the flexibility of the Gly-rich domain may facilitate substrate movement within the enzyme during sequential proteolysis. Fourth, dimerization may affect substrate processing by decreasing the dynamics of the dimerization region and by increasing that of the C-terminal part of the cleavage region.

The p150Glued CAP-Gly Domain Regulates Initiation of Retrograde Transport at Synaptic Termini

p150(Glued) is the major subunit of dynactin, a complex that functions with dynein in minus-end-directed microtubule transport. Mutations within the p150(Glued) CAP-Gly microtubule-binding domain cause neurodegenerative diseases through an unclear mechanism. A p150(Glued) motor neuron degenerative disease-associated mutation introduced into the Drosophila Glued locus generates a partial loss-of-function allele (Gl(G38S)) with impaired neurotransmitter release and adult-onset locomotor dysfunction. Disruption of the p150(Glued) CAP-Gly domain in neurons causes a specific disruption of vesicle trafficking atterminal boutons (TBs), the distal-most ends of synapses. Gl(G38S) larvae accumulate endosomes along with dynein and kinesin motor proteins within swollen TBs, and genetic analyses show that kinesin and p150(Glued) function cooperatively at TBs to coordinate transport. Therefore, the p150(Glued) CAP-Gly domain regulates dynein-mediated retrograde transport at synaptic termini, and this function of dynactin is disrupted by a mutation that causes motorneuron disease.

Cellular Model of TAR DNA-binding Protein 43 (TDP-43) Aggregation Based on Its C-terminal Gln/Asn-rich Region

TDP-43 is one of the major components of the neuronal and glial inclusions observed in several neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. These characteristic aggregates are a "landmark" of the disease, but their role in the pathogenesis is still obscure. In previous works, we have shown that the C-terminal Gln/Asn-rich region (residues 321-366) of TDP-43 is involved in the interaction of this protein with other members of the heterogeneous nuclear ribonucleoprotein protein family. Furthermore, we have shown that the interaction through this region is important for TDP-43 splicing inhibition of cystic fibrosis transmembrane regulator exon 9, and there were indications that it was involved in the aggregation process. Our experiments show that in cell lines and primary rat neuronal cultures, the introduction of tandem repeats carrying the 331-369-residue Gln/Asn region from TDP-43 can trigger the formation of phosphorylated and ubiquitinated aggregates that recapitulate many but not all the characteristics observed in patients. These results establish a much needed cell-based TDP-43 aggregation model useful to investigate the mechanisms involved in the formation of inclusions and the gain- and loss-of-function consequences of TDP-43 aggregation within cells. In addition, it will be a powerful tool to test novel therapeutic strategies/effectors aimed at preventing/reducing this phenomenon.

A novel type III crustin (Crus Es2) identified from Chinese mitten crab Eriocheir sinensis

Antimicrobial peptides are important effectors in the host innate immune response against microbial invasion. In the present study, the cDNA encoding a crustin (designated Crus Es2) was cloned from Chinese mitten crab Eriocheir sinensis by using EST analysis and rapid amplification of cDNA ends (RACE) approach. The full-length cDNA of Crus Es2 was of 1237 bp, containing a 5′ untranslated region (UTR) of 12 bp, a 3′ UTR of 886 bp with a poly (A) tail, and an open reading frame (ORF) of 339 bp encoding a polypeptide of 112 amino acids with a signal peptide of 19 amino acids. The Crus Es2 contained a typical WAP domain, but lacked the Gly-rich domain of the type II crustin and the Cys-rich region present in both type I and type II crustin, suggesting that Crus Es2 should be classified as a type III crustin. The mRNA transcripts of Crus Es2 could be detected in haemocytes and gill, and its expression level in haemocytes was up-regulated after Listonella anguillarum challenge, while decreased after Micrococcus luteus challenge. The mature peptide coding region of Crus Es2 was cloned into pET-21a (+) and expressed in Escherichia coli. The purified recombinant Crus Es2 inhibited the growth of Gram-positive bacteria at MIC of 0.093–0.37 μM. The results indicated that Crus Es2 was involved in immune response of E. sinensis against bacterial challenge.

Molecular architecture and engineering of spider dragline silk protein

Abstract Spider dragline silk, which is produced in spider major ampullate gland, is composite proteinacious fiber with highly repetitive Ala-Gly-rich domain. The unique combination of both high tensile strength and high elasticity makes spider dragline silk superior to almost any other natural or synthetic fibers. Cloning of the genes reveals that the silk is composed of at least two major proteins. Each protein component contains multiple repeats of modular structures that alternate between Ala-rich domains and Gly-rich domains. Molecular engineering not only opens a door to the production of spidroins but also provides a valuable experimental system to test and further establish the relationship between modular structures and mechanical properties. Here, based on our own studies, we review the latest progress of the modular structure and genetic engineering and outline the future prospects.

The nucleocapsid protein of SARS coronavirus has a high binding affinity to the human cellular heterogeneous nuclear ribonucleoprotein A1

The nucleocapsid (N) protein of SARS coronavirus (SARS_CoV) is a major structural component of virions, which appears to be a multifunctional protein involved in viral RNA replication and translation. Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is related to the pre-mRNA splicing in the nucleus and translation regulation in the cytoplasm. In this report, based on the relevant biophysical and biochemical assays, the nucleocapsid protein of SARS_CoV (SARS_N) was discovered to exhibit high binding affinity to human hnRNP A1. GST pull-down results clearly demonstrated that SARS_N protein could directly and specifically bind to human hnRNP A1 in vitro. Yeast two-hybrid assays further indicated in vivo that such binding relates to the fragment (aa 161–210) of SARS_N and the Gly-rich domain (aa 203–320) of hnRNP A1. Moreover, kinetic analyses by surface plasmon resonance (SPR) technology revealed that SARS_N protein has a specific binding affinity against human hnRNP A1 with K D at 0.35 ± 0.02 μM ( k on = 5.83 ± 0.42 × 10 3 M −1 s −1 and k off = 2.06 ± 0.12 × 10 −3 s −1). It is suggested that both SARS_N and hnRNP A1 proteins are possibly within the SARS_CoV replication/transcription complex and SARS_N/human hnRNP A1 interaction might function in the regulation of SARS_CoV RNA synthesis. In addition, the determined results showed that SARS_N protein has only one binding domain for interacting with human hnRNP A1, which is different from the mouse hepatitis virus (MHV) binding case where the nucleocapsid protein of MHV (MHV_N) was found to have two binding domains involved in the MHV_N/hnRNP A1 interaction, thereby suggesting that SARS_N protein might carry out a different binding mode to bind to human hnRNP A1 for its further function performance in comparison with MHV_N.

Solution structure, backbone dynamics and chitin binding of the anti-fungal protein from Streptomyces tendae TÜ901

AFP1 is a recently discovered anti-fungal, chitin-binding protein from Streptomyces tendae Tü901. Mature AFP1 comprises 86 residues and exhibits limited sequence similarity to the cellulose-binding domains of bacterial cellulases and xylanases. No similarity to the Cys and Gly-rich domains of plant chitin-binding proteins (e.g. agglutinins, lectins, hevein) is observed. AFP1 is the first chitin-binding protein from a bacterium for which anti-fungal activity was shown. Here, we report the three-dimensional solution structure of AFP1, determined by nuclear magnetic resonance spectroscopy. The protein contains two antiparallel β-sheets (five and four β-strands each), that pack against each other in a parallel β-sandwich. This type of architecture is conserved in the functionally related family II of cellulose-binding domains, albeit with different connectivity. A similar fold is also observed in other unrelated proteins (spore coat protein from Myxococcus xanthus, β-B2 and γ-B crystallins from Bos taurus, canavalin from Jack bean). AFP1 is therefore classified as a new member of the βγ-crystallin superfamily. The dynamics of the protein was characterized by NMR using amide 15N relaxation and solvent exchange data. We demonstrate that the protein exhibits an axially symmetric (oblate-like) rotational diffusion tensor whose principal axis coincides to within 15° with that of the inertial tensor. After completion of the present structure of AFP1, an identical fold was reported for a Streptomyces killer toxin-like protein. Based on sequence comparisons and clustering of conserved residues on the protein surface for different cellulose and chitin-binding proteins, we postulate a putative sugar-binding site for AFP1. The inability of the protein to bind short chitin fragments suggests that certain particular architectural features of the solid chitin surface are crucial for the interaction.

Site-directed Mutagenesis of Diphosphoinositol Polyphosphate Phosphohydrolase, a Dual Specificity NUDT Enzyme That Attacks Diadenosine Polyphosphates and Diphosphoinositol Polyphosphates

Diphosphoinositol polyphosphate phosphohydrolase (DIPP) hydrolyzes diadenosine 5',5"'-P(1),P(6)-hexaphosphate (Ap(6)A), a Nudix (nucleoside diphosphate attached-moiety "x") substrate, and two non-Nudix compounds: diphosphoinositol pentakisphosphate (PP-InsP(5)) and bis-diphosphoinositol tetrakisphosphate ((PP)(2)-InsP(4)). Guided by multiple sequence alignments, we used site-directed mutagenesis to obtain new information concerning catalytically essential amino acid residues in DIPP. Mutagenesis of either of two conserved glutamate residues (Glu(66) and Glu(70)) within the Nudt (Nudix-type) catalytic motif impaired hydrolysis of Ap(6)A, PP-InsP(5), and (PP)(2)-InsP(4) >95%; thus, all three substrates are hydrolyzed at the same active site. Two Gly-rich domains (glycine-rich regions 1 and 2 (GR1 and GR2)) flank the Nudt motif with potential sites for cation coordination and substrate binding. GR1 comprises a GGG tripeptide, while GR2 is identified as a new functional motif (GX(2)GX(6)G) that is conserved in yeast homologues of DIPP. Mutagenesis of any of these Gly residues in GR1 and GR2 reduced catalytic activity toward all three substrates by up to 95%. More distal to the Nudt motif, H91L and F84Y mutations substantially decreased the rate of Ap(6)A and (PP)(2)-InsP(4) metabolism (by 71 and 96%), yet PP-InsP(5) hydrolysis was only mildly reduced (by 30%); these results indicate substrate-specific roles for His(91) and Phe(84). This new information helps define DIPP's structural, functional, and evolutionary relationships to Nudix hydrolases.

Cloning and characterization of human CIRP (cold-inducible RNA-binding protein) cDNA and chromosomal assignment of the gene.

Cold stress induces in microorganisms the synthesis of several proteins that are involved in various cellular processes such as transcription, translation and recombination. Recently, the cold-inducible RNA-binding protein (Cirp) was found to be induced in rodent cells by mild cold stress (32 degrees C). Cirp consists of an N-terminal RNA-binding domain and a C-terminal Gly-rich domain, and plays an essential role in cold-induced suppression of cell proliferation. We report here the cloning of a cDNA encoding an 18-kDa protein with 95.3% identity in an amino-acid sequence to that of mouse Cirp. The human CIRP gene has been mapped to the chromosomal locus 19p13.3 by fluorescence in-situ hybridization. CIRP mRNA is constitutively expressed in all cell lines examined, including K562, HepG2, NC65, HeLa, T24, and NEC8 cells. In all of them, the levels of CIRP mRNA and protein were increased within 12 h after a temperature down-shift from 37 degrees C to 32 degrees C. These results demonstrated that CIRP is a cold-shock protein in human cells. Identification of CIRP may provide a clue to the regulatory mechanisms of cold responses in human cells.

HnRNP A1 Selectively Interacts Through its Gly-rich Domain with Different RNA-binding Proteins

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are abundant nuclear polypeptides, most likely involved in different steps of pre-mRNA processing. Protein A1 (34 kDa), a prominent member of the hnRNP family, seems to act by modulating the RNA secondary structure and by antagonizing some splicing factors (SR proteins) in splice-site selection and exon skipping/inclusion. A role of A1 in the nucleo-cytoplasmic transport of RNA has also been proposed. These activities might depend not only on the RNA-binding properties of the protein but also on specific protein-protein interactions. Here we report that A1 can indeed selectively interact, in vitro, both with itself and with other hnRNP basic “core” proteins. Such selective binding is mediated exclusively by the Gly-rich C-terminal domain, where a novel protein-binding motif constituted by hydrophobic repeats can be envisaged. The same domain is necessary and sufficient to promote specific interaction in vivo, as assayed by the yeast two-hybrid assay. Moreover, an in vitrointeraction with some SR proteins was also observed. These observations suggest that diverse and specific protein-protein interactions might contribute to the different functions of the hnRNP A1 protein in mRNA maturation.

Nucleotide sequence of the gene presumably encoding the adsorption protein of filamentous phage φLf

A 1170-nucleotide fragment of φLf DNA was sequenced. This fragment contains an open reading frame, ORF367, encoding a protein of 367 amino acids (aa) (36710 Da). ORF367 is located downstream from the gene encoding the major coat protein (gVIIIp) and a Rho-independent termination signal. Sequence analysis revealed that the gene product has a Gly-rich domain (70 aa) at the center and a hydrophobic region (26 aa) at the C terminus. These structural features suggest that ORF367 may encode the adsorption protein of φLf.

Function of conserved domains of hnRNP A1 and other hnRNP A/B proteins.

hnRNP A1 is a pre-mRNA binding protein that antagonizes the alternative splicing activity of splicing factors SF2/ASF or SC35, causing activation of distal 5' splice sites. The structural requirements for hnRNP A1 function were determined by mutagenesis of recombinant human hnRNP A1. Two conserved Phe residues in the RNP-1 submotif of each of two RNA recognition motifs appear to be involved in specific RNA-protein interactions and are essential for modulating alternative splicing. These residues are not required for general pre-mRNA binding or RNA annealing activity. The C-terminal Gly-rich domain is necessary for alternative splicing activity, for stable RNA binding and for optimal RNA annealing activity. hnRNP A1B, which is an alternatively spliced isoform of hnRNP A1 with a longer Gly-rich domain, binds more strongly to pre-mRNA but has only limited alternative splicing activity. In contrast, hnRNP A2 and B1, which have 68% amino acid identity with hnRNP A1, bind more weakly to pre-mRNA and have stronger splice site switching activities than hnRNP A1. We propose that specific combinations of antagonistic hnRNP A/B and SR proteins are involved in regulating alternative splicing of distinct subsets of cellular premRNAs.

Two homologous genes, originated by duplication, encode the human hnRNP proteins A2 and A1.

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 belongs, with A1, B1 and B2, to the basic protein subset of the hnRNP complex in mammalian cells. All these proteins share a modular structure consisting of two conserved RNA binding domains linked to less conserved Gly-rich domains (2xRBD-Gly). In the framework of our studies on the genetic basis of hnRNP proteins structure and diversity we have isolated and sequenced the A2 gene and compared it to the previously described A1 gene. The A2 gene, which exists in a single copy on Ch. 7 band p15, is split in 12 exons including an alternatively spliced 36 nt mini exon specific for the human hnRNP protein B1. In this work we show that the intron/exon organisation of the A2 gene is identical to that of the A1 gene over the entire length, indicating a common origin by gene duplication. Moreover the comparison of corresponding exons evidences significant conservation also in the apparently divergent Gly-rich domains that could define previously unenvisaged structural and/or functional motifs. The A2 gene promoter is also analysed in comparison to that of the A1 gene.

Structure and genomic organization of the ipiB and ipiO gene clusters of Phytophthora infestans

Two in planta-induced ( ipi) genes, designated ipiB and ipiO, of the potato late blight fungus, Phytophthora infestans (Mont.) de Bary, were isolated from a genomic library by a differential hybridization procedure [Pieterse et al., Physiol. Mol. Plant Pathol. (1993a) in press]. Both genes are expressed at high levels in the early phases of the pathogenic interaction of P. infestans with its host plant potato, suggesting that their gene products have a function in the early stages of the infection process. Here, we describe the nucleotide (nt) sequence and genomic organization of ipiB and ipiO. The ipiB gene belongs to a small gene family consisting of at least three genes, designated ipiB1, ipiB2 and ipiB3, which are clustered in a head-to-tail arrangement. The three ipiB genes are highly homologous throughout the coding regions and 5′ and 3′ flanking regions. The P. infestans genome contains two very similar ipiO genes, ipiO1 and ipi02, which are closely linked and arranged in an inverted orientation. The ipiB genes encode three novel, highly similar Glyrich proteins of 301, 343 and 347 amino acids (aa), respectively. The Gly-rich domains of the IPI-B proteins are predominantly composed of two repeats with the core sequences, A/V-G-A-G-L-Y-G-R and G-A-G-Y/V-G-G. The ipiO genes code for two almost identical 152-aa proteins which do not have any homology with sequences present in data libraries. IPI-B, as well as IPI-O, contains putative signal peptides of 20 and 21 aa, respectively, suggesting that they are transported out of the cytoplasm. In the promoter regions of ipiB and ipiO, a 16-nt sequence motif, matching the core sequence, GCTCATTYYNCAWTTT (where N = A or C or G or T; W = A or T; Y = C or T), was found. This sequence motif appears to be present around the transcription start point ( tsp) of seven out of eight oomycetous genes for which the tsp have been determined, suggesting that oomycetes have a sequence preference for transcription initiation.