Conversion of cysteine to formylglycine: A protein modification in the endoplasmic reticulum

In sulfatases a Cα-formylglycine residue is found at a position where their cDNA sequences predict a cysteine residue. In multiple sulfatase deficiency, an inherited lysosomal storage disorder, catalytically inactive sulfatases are synthesized which retain the cysteine residue, indicating that the Cα-formylglycine residue is required for sulfatase activity. Using in vitro translation in the absence or presence of transport competent microsomes we found that newly synthesized sulfatase polypeptides carry a cysteine residue and that the oxidation of its thiol group to an aldehyde is catalyzed in the endoplasmic reticulum. A linear sequence of 16 residues surrounding the Cys-69 in arylsulfatase A is sufficient to direct the oxidation. This novel protein modification occurs after or at a late stage of cotranslational protein translocation into the endoplasmic reticulum when the polypeptide is not yet folded to its native structure.

Guanine nucleotide exchange factor GEF115 specifically mediates activation of Rho and serum response factor by the G protein α subunit Gα13

Signal transduction pathways that mediate activation of serum response factor (SRF) by heterotrimeric G protein α subunits were characterized in transfection systems. Gαq, Gα12, and Gα13, but not Gαi, activate SRF through RhoA. When Gαq, α12, or α13 were coexpressed with a Rho-specific guanine nucleotide exchange factor GEF115, Gα13, but not Gαq or Gα12, showed synergistic activation of SRF with GEF115. The synergy between Gα13 and GEF115 depends on the N-terminal part of GEF115, and there was no synergistic effect between Gα13 and another Rho-specific exchange factor Lbc. In addition, the Dbl-homology (DH)-domain-deletion mutant of GEF115 inhibited Gα13- and Gα12-induced, but not GEF115 itself- or Gαq-induced, SRF activation. The DH-domain-deletion mutant also suppressed thrombin- and lysophosphatidic acid-induced SRF activation in NIH 3T3 cells, probably by inhibition of Gα12/13. The N-terminal part of GEF115 contains a sequence motif that is homologous to the regulator of G protein signaling (RGS) domain of RGS12. RGS12 can inhibit both Gα12 and Gα13. Thus, the inhibition of Gα12/13 by the DH-deletion mutant may be due to the RGS activity of the mutant. The synergism between Gα13 and GEF115 indicates that GEF115 mediates Gα13-induced activation of Rho and SRF.

Influenza C virus CM2 integral membrane glycoprotein is produced from a polypeptide precursor by cleavage of an internal signal sequence

The influenza C virus CM2 protein is a small glycosylated integral membrane protein (115 residues) that spans the membrane once and contains a cleavable signal sequence at its N terminus. The coding region for CM2 (CM2 ORF) is located at the C terminus of the 342-amino acid (aa) ORF of a colinear mRNA transcript derived from influenza C virus RNA segment 6. Splicing of the colinear transcript introduces a translational stop codon into the ORF and the spliced mRNA encodes the viral matrix protein (CM1) (242 aa). The mechanism of CM2 translation was investigated by using in vitro and in vivo translation of RNA transcripts. It was found that the colinear mRNA derived from influenza C virus RNA segment 6 serves as the mRNA for CM2. Furthermore, CM2 translation does not depend on any of the three in-frame methionine residues located at the beginning of CM2 ORF. Rather, CM2 is a proteolytic cleavage product of the p42 protein product encoded by the colinear mRNA: a cleavage event that involves the recognition and cleavage of an internal signal peptide presumably by signal peptidase resident in the endoplasmic reticulum. Alteration of the predicted signal peptidase cleavage site by mutagenesis blocked generation of CM2. The other polypeptide species resulting from the cleavage of p42, designated p31, contains the CM1 coding region and an additional C-terminal 17 aa (formerly the CM2 signal peptide). Protein p31, in comparison to CM1, displays characteristics of an integral membrane protein.

The neuronal RNA-binding protein Nova-2 is implicated as the autoantigen targeted in POMA patients with dementia

Paraneoplastic opsoclonus myoclonus ataxia (POMA) is a neurologic disorder thought to be mediated by an autoimmune attack against onconeural disease antigens that are expressed by gynecologic or lung tumors and by neurons. One POMA disease antigen, termed Nova-1, has been identified as a neuron-specific KH-type RNA-binding protein. Nova-1 expression is restricted to specific regions of the central nervous system, primarily the hindbrain and ventral spinal cord, which correlate with the predominantly motor symptoms in POMA. However, POMA antisera recognize antigens that are widely expressed in both caudal and rostral regions of the central nervous system, and some patients develop cognitive symptoms. We have used POMA antisera to clone a cDNA encoding a second POMA disease antigen termed Nova-2. Nova-2 is closely related to Nova-1, and is expressed at high levels in neurons during development and in adulthood, and at lower levels in the adult lung. In the postnatal mouse brain, Nova-2 is expressed in a pattern that is largely reciprocal with Nova-1, including high levels of Nova-2 expression in the neocortex and hippocampus. Functional characterization of Nova-2 in RNA selection and nitrocellulose filter-binding assays reveals that Nova-2 binds RNA with high affinity and with sequence specificity that differs from Nova-1. Our results demonstrate that the immune response in POMA targets a family of highly related sequence-specific neuronal RNA-binding proteins. The expression pattern of the Nova-2 protein is likely to underlie the development of cognitive deficits in some POMA patients.

The regulators of G protein signaling (RGS) domains of RGS4, RGS10, and GAIP retain GTPase activating protein activity in vitro

Regulators of G protein signaling (RGS) proteins accelerate GTP hydrolysis by Gi but not by Gs class α-subunits. All RGS proteins share a conserved 120-amino acid sequence termed the RGS domain. We have demonstrated that the RGS domains of RGS4, RGS10, and GAIP retain GTPase accelerating activity with the Gi class substrates Giα1, Goα, and Gzα in vitro. No regulatory activity of the RGS domains was detected for Gsα. Short deletions within the RGS domain of RGS4 destroyed GTPase activating protein activity and Giα1 substrate binding. Comparable protein–protein interactions between Giα1–GDP–AlF4− and the RGS domain or full-length RGS4 were detected using surface plasmon resonance.

Gene cloning, sequence analysis, and expression of 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase

The gene encoding 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase (MHPCO; EC 1.14.12.4) was cloned by using an oligonucleotide probe corresponding to the N terminus of the enzyme to screen a DNA library of Pseudomonas sp. MA-1. The gene encodes for a protein of 379 amino acid residues corresponding to a molecular mass of 41.7 kDa, the same as that previously estimated for MHPCO. MHPCO was expressed in Escherichia coli and found to have the same properties as the native enzyme from Pseudomonas sp. MA-1. This study shows that MHPCO is a homotetrameric protein with one flavin adenine dinucleotide bound per subunit. Sequence comparison of the enzyme with other hydroxylases reveals regions that are conserved among aromatic flavoprotein hydroxylases.

Gene discovery in the wood-forming tissues of poplar: Analysis of 5,692 expressed sequence tags

A rapidly growing area of genome research is the generation of expressed sequence tags (ESTs) in which large numbers of randomly selected cDNA clones are partially sequenced. The collection of ESTs reflects the level and complexity of gene expression in the sampled tissue. To date, the majority of plant ESTs are from nonwoody plants such as Arabidopsis, Brassica, maize, and rice. Here, we present a large-scale production of ESTs from the wood-forming tissues of two poplars, Populus tremula L. × tremuloides Michx. and Populus trichocarpa ‘Trichobel.’ The 5,692 ESTs analyzed represented a total of 3,719 unique transcripts for the two cDNA libraries. Putative functions could be assigned to 2,245 of these transcripts that corresponded to 820 protein functions. Of specific interest to forest biotechnology are the 4% of ESTs involved in various processes of cell wall formation, such as lignin and cellulose synthesis, 5% similar to developmental regulators and members of known signal transduction pathways, and 2% involved in hormone biosynthesis. An additional 12% of the ESTs showed no significant similarity to any other DNA or protein sequences in existing databases. The absence of these sequences from public databases may indicate a specific role for these proteins in wood formation. The cDNA libraries and the accompanying database are valuable resources for forest research directed toward understanding the genetic control of wood formation and future endeavors to modify wood and fiber properties for industrial use.

Multiprotein bridging factor 1 (MBF1) is an evolutionarily conserved transcriptional coactivator that connects a regulatory factor and TATA element-binding protein

Multiprotein bridging factor 1 (MBF1) is a transcriptional cofactor that bridges between the TATA box-binding protein (TBP) and the Drosophila melanogaster nuclear hormone receptor FTZ-F1 or its silkworm counterpart BmFTZ-F1. A cDNA clone encoding MBF1 was isolated from the silkworm Bombyx mori whose sequence predicts a basic protein consisting of 146 amino acids. Bacterially expressed recombinant MBF1 is functional in interactions with TBP and a positive cofactor MBF2. The recombinant MBF1 also makes a direct contact with FTZ-F1 through the C-terminal region of the FTZ-F1 DNA-binding domain and stimulates the FTZ-F1 binding to its recognition site. The central region of MBF1 (residues 35–113) is essential for the binding of FTZ-F1, MBF2, and TBP. When the recombinant MBF1 was added to a HeLa cell nuclear extract in the presence of MBF2 and FTZ622 bearing the FTZ-F1 DNA-binding domain, it supported selective transcriptional activation of the fushi tarazu gene as natural MBF1 did. Mutations disrupting the binding of FTZ622 to DNA or MBF1, or a MBF2 mutation disrupting the binding to MBF1, all abolished the selective activation of transcription. These results suggest that tethering of the positive cofactor MBF2 to a FTZ-F1-binding site through FTZ-F1 and MBF1 is essential for the binding site-dependent activation of transcription. A homology search in the databases revealed that the deduced amino acid sequence of MBF1 is conserved across species from yeast to human.

Prion protein NMR structure and species barrier for prion diseases

The structural basis of species specificity of transmissible spongiform encephalopathies, such as bovine spongiform encephalopathy or “mad cow disease” and Creutzfeldt–Jakob disease in humans, has been investigated using the refined NMR structure of the C-terminal domain of the mouse prion protein with residues 121–231. A database search for mammalian prion proteins yielded 23 different sequences for the fragment 124–226, which display a high degree of sequence identity and show relevant amino acid substitutions in only 18 of the 103 positions. Except for a unique isolated negative surface charge in the bovine protein, the amino acid differences are clustered in three distinct regions of the three-dimensional structure of the cellular form of the prion protein. Two of these regions represent potential species-dependent surface recognition sites for protein–protein interactions, which have independently been implicated from in vitro and in vivo studies of prion protein transformation. The third region consists of a cluster of interior hydrophobic side chains that may affect prion protein transformation at later stages, after initial conformational changes in the cellular protein.

Identification of an early endosomal protein regulated by phosphatidylinositol 3-kinase

Phosphatidylinositol 3-kinases (PI 3-kinases) have been implicated in membrane trafficking in the secretory and endocytic pathways of yeast and mammalian cells, but the molecular mechanisms by which these lipid kinases operate are not known. Here we identify a protein of 170 kDa that is rapidly released from cell membranes in response to wortmannin, a potent inhibitor of mammalian PI 3-kinases. The amino acid sequence of peptides from p170 reveal its identity to early endosomal antigen (EEA) 1, an endosomal antigen with homology to several yeast proteins genetically implicated in membrane trafficking. Immunofluorescence analysis of 3T3-L1 adipocytes with antisera against p170/EEA1 reveal a punctate peripheral pattern that becomes diffuse in response to wortmannin. In vitro, p170/EEA1 binds specifically to liposomes containing PIns(3)P, suggesting that the effect of wortmannin on cells is due to inhibition of PIns(3)P production. Thus, p170/EEA1 may define a family of proteins that mediate the regulatory effects of 3′-phosphoinositides on membrane trafficking in yeast and mammalian cells.

Cloning of a novel T-cell protein FYB that binds FYN and SH2-domain-containing leukocyte protein 76 and modulates interleukin 2 production

T cell receptor ζ (TcRζ)/CD3 ligation initiates a signaling cascade that involves src kinases p56lck and ζ-associated protein 70, leading to the phosphorylation of substrates such as TcRζ, Vav, SH2-domain-containing leukocyte protein 76 (SLP-76), cbl, and p120/130. FYN binding protein (FYB or p120/130) associates with p59fyn, the TcRζ/CD3 complex, and becomes tyrosine-phosphorylated in response to receptor ligation. In this study, we report the cDNA cloning of human and murine FYB and show that it is restricted in expression to T cells and myeloid cells and possesses an overall unique hydrophilic sequence with several tyrosine-based motifs, proline-based type I and type II SH3 domain binding motifs, several putative lysine/glutamic acid-rich nuclear localization motifs, and a SH3-like domain. In addition to binding the src kinase p59fyn, FYB binds specifically to the hematopoietic signaling protein SLP-76, an interaction mediated by the SLP-76 SH2 domain. In keeping with this, expression of FYB augmented interleukin 2 secretion from a T cell hybridoma, DC27.10, in response to TcRζ/CD3 ligation. FYB is therefore a novel hematopoietic protein that acts as a component of the FYN and SLP-76 signaling cascades in T cells.

Striking sequence similarity in inter- and intra-specific comparisons of class I SLG alleles from Brassica oleracea and Brassica campestris: Implications for the evolution and recognition mechanism

Self-incompatibility in Brassica is controlled by a single multi-allelic locus (S locus), which contains at least two highly polymorphic genes expressed in the stigma: an S glycoprotein gene (SLG) and an S receptor kinase gene (SRK). The putative ligand-binding domain of SRK exhibits high homology to the secretory protein SLG, and it is believed that SLG and SRK form an active receptor kinase complex with a self-pollen ligand, which leads to the rejection of self-pollen. Here, we report 31 novel SLG sequences of Brassica oleracea and Brassica campestris. Sequence comparisons of a large number of SLG alleles and SLG-related genes revealed the following points. (i) The striking sequence similarity observed in an inter-specific comparison (95.6% identity between SLG14 of B. oleracea and SLG25 of B. campestris in deduced amino acid sequence) suggests that SLG diversification predates speciation. (ii) A perfect match of the sequences in hypervariable regions, which are thought to determine S specificity in an intra-specific comparison (SLG8 and SLG46 of B. campestris) and the observation that the hypervariable regions of SLG and SRK of the same S haplotype were not necessarily highly similar suggests that SLG and SRK bind different sites of the pollen ligand and that they together determine S specificity. (iii) Comparison of the hypervariable regions of SLG alleles suggests that intragenic recombination, together with point mutations, has contributed to the generation of the high level of sequence variation in SLG alleles. Models for the evolution of SLG/SRK are presented.

A maize zinc-finger protein binds the prolamin box in zein gene promoters and interacts with the basic leucine zipper transcriptional activator Opaque2

The prolamin box (P-box) is a highly conserved 7-bp sequence element (5′-TGTAAAG-3′) found in the promoters of many cereal seed storage protein genes. Nuclear factors from maize endosperm specifically interact with the P-box present in maize prolamin genes (zeins). The presence of the P-box in all zein gene promoters suggests that interactions between endosperm DNA binding proteins and the P-box may play an important role in the coordinate activation of zein gene expression during endosperm development. We have cloned an endosperm-specific maize cDNA, named prolamin-box binding factor (PBF), that encodes a member of the recently described Dof class of plant Cys2-Cys2 zinc-finger DNA binding proteins. When tested in gel shift assays, PBF exhibits the same sequence-specific binding to the P-box as factors present in maize endosperm nuclei. Additionally, PBF interacts in vitro with the basic leucine zipper protein Opaque2, a known transcriptional activator of zein gene expression whose target site lies 20 bp downstream of the P-box in the 22-kDa zein gene promoter. The isolation of the PBF gene provides an essential tool to further investigate the functional role of the highly conserved P-box in regulating cereal storage protein gene expression.

From residue matching patterns to protein folding topographies: General model and bovine pancreatic trypsin inhibitor

A coarse-grained model for protein-folding dynamics is introduced based on a discretized representation of torsional modes. The model, based on the Ramachandran map of the local torsional potential surface and the class (hydrophobic/polar/neutral) of each residue, recognizes patterns of both torsional conformations and hydrophobic-polar contacts, with tolerance for imperfect patterns. It incorporates empirical rates for formation of secondary and tertiary structure. The method yields a topological representation of the evolving local torsional configuration of the folding protein, modulo the basins of the Ramachandran map. The folding process is modeled as a sequence of transitions from one contact pattern to another, as the torsional patterns evolve. We test the model by applying it to the folding process of bovine pancreatic trypsin inhibitor, obtaining a kinetic description of the transitions between the contact patterns visited by the protein along the dominant folding pathway. The kinetics and detailed balance make it possible to invert the result to obtain a coarse topographic description of the potential energy surface along the dominant folding pathway, in effect to go backward or forward between a topological representation of the chain conformation and a topographical description of the potential energy surface governing the folding process. As a result, the strong structure-seeking character of bovine pancreatic trypsin inhibitor and the principal features of its folding pathway are reproduced in a reasonably quantitative way.

An ancient family of human endogenous retroviruses encodes a functional homolog of the HIV-1 Rev protein

The human endogenous retrovirus K (HERV-K) family of endogenous retroviruses consists of ≈50 proviral copies per haploid human genome. Herein, the HERV-Ks are shown to encode a sequence-specific nuclear RNA export factor, termed K-Rev, that is functionally analogous to the HIV-1 Rev protein. Like HIV-1 Rev, K-Rev binds to both the Crm1 nuclear export factor and to a cis-acting viral RNA target to activate nuclear export of unspliced RNAs. Surprisingly, this HERV-K RNA sequence, which is encoded within the HERV-K long terminal repeat, is also recognized by HIV-1 Rev. These data provide surprising evidence for an evolutionary link between HIV-1 and a group of endogenous retroviruses that first entered the human genome ≈30 million years ago.