scholarly journals Protein features for assembly of the RNA editing helicase 2 subcomplex (REH2C) in Trypanosome holo-editosomes

2019 ◽  
Author(s):  
Vikas Kumar ◽  
Pawan K. Doharey ◽  
Shelly Gulati ◽  
Joshua Meehan ◽  
Mary G. Martinez ◽  
...  
Keyword(s):  
Amino Acids ◽  
Rna Editing ◽  
Zinc Finger Protein ◽  
Rna Helicases ◽  
Guide Rnas ◽  
C Terminus ◽  
Rnp Complex ◽  
Ob Fold ◽  
The Stability

AbstractUridylate insertion/deletion RNA editing inTrypanosoma bruceiis a complex system that is not found in humans, so there is interest in targeting this system for drug development. This system uses hundreds of small non-coding guide RNAs (gRNAs) to modify the mitochondrial mRNA transcriptome. This process occurs in holo-editosomes that assemble several macromoleculartransfactors around mRNA including the RNA-free RNA editing core complex (RECC) and auxiliary ribonucleoprotein (RNP) complexes. Yet, the regulatory mechanisms of editing remain obscure. The enzymatic accessory RNP complex, termed the REH2C, includes mRNA substrates and products, the multi-domain 240 kDa RNA Editing Helicase 2 (REH2) and an intriguing 8-zinc finger protein termed REH2-Associated Factor 1 (H2F1). Both of these proteins are essential in editing. REH2 is a member of the DExH/RHA subfamily of RNA helicases with a conserved C-terminus that includes a regulatory OB-fold domain. In trypanosomes,H2F1 recruits REH2 to the editing apparatus, andH2F1 downregulation causes REH2 fragmentation. Our systematic mutagenesis dissected determinants in REH2 andH2F1 for the assembly of REH2C, the stability of REH2, and the RNA-mediated association of REH2C with other editingtransfactors. We identified functional OB-fold amino acids in eukaryotic DExH/RHA helicases that are conserved in REH2 and that impact the assembly and interactions of REH2C.H2F1 upregulation stabilized REH2in vivo. Mutation of the core cysteines or basic amino acids in individual zinc fingers affected the stabilizing property ofH2F1 but not its interactions with other examined editing components. This result suggests that most, if not all, fingers may contribute to REH2 stabilization. Finally, a recombinant REH2 (240 kDa) established that the full-length protein is abona fideRNA helicase with ATP-dependent unwinding activity. REH2 is the only DExH/RHA-type helicase in kinetoplastid holo-editosomes.

scholarly journals The C-Terminal Flexible Domain of the Heme Chaperone CcmE Is Important but Not Essential for Its Function

2003 ◽  
Vol 185 (13) ◽  
pp. 3821-3827 ◽  
Author(s):  
Elisabeth Enggist ◽  
Linda Thöny-Meyer
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Heme Binding ◽  
Membrane Anchor ◽  
Terminal Domain ◽  
Conserved Domain ◽  
C Terminus ◽  
Helical Turn ◽  
Low Activity

ABSTRACT CcmE is a heme chaperone active in the cytochrome c maturation pathway of Escherichia coli. It first binds heme covalently to strictly conserved histidine H130 and subsequently delivers it to apo-cytochrome c. The recently solved structure of soluble CcmE revealed a compact core consisting of a β-barrel and a flexible C-terminal domain with a short α-helical turn. In order to elucidate the function of this poorly conserved domain, CcmE was truncated stepwise from the C terminus. Removal of all 29 amino acids up to crucial histidine 130 did not abolish heme binding completely. For detectable transfer of heme to type c cytochromes, only one additional residue, D131, was required, and for efficient cytochrome c maturation, the seven-residue sequence 131DENYTPP137 was required. When soluble forms of CcmE were expressed in the periplasm, the C-terminal domain had to be slightly longer to allow detection of holo-CcmE. Soluble full-length CcmE had low activity in cytochrome c maturation, indicating the importance of the N-terminal membrane anchor for the in vivo function of CcmE.

CLUSTER guide RNAs enable precise and efficient RNA editing with endogenous ADAR enzymes in vivo

2022 ◽  
Author(s):  
Philipp Reautschnig ◽  
Nicolai Wahn ◽  
Jacqueline Wettengel ◽  
Annika E. Schulz ◽  
Ngadhnjim Latifi ◽  
...  
Keyword(s):  
Rna Editing ◽  
Guide Rnas

scholarly journals Positive and Negative Regulation of Poly(A) Nuclease

2004 ◽  
Vol 24 (12) ◽  
pp. 5521-5533 ◽  
Author(s):  
David A. Mangus ◽  
Matthew C. Evans ◽  
Nathan S. Agrin ◽  
Mandy Smith ◽  
Preetam Gongidi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Interactions ◽  
Binding Pocket ◽  
Negative Regulation ◽  
Single Amino Acid ◽  
Carboxy Terminus ◽  
Protein Protein Interactions ◽  
C Terminus

ABSTRACT PAN, a yeast poly(A) nuclease, plays an important nuclear role in the posttranscriptional maturation of mRNA poly(A) tails. The activity of this enzyme is dependent on its Pan2p and Pan3p subunits, as well as the presence of poly(A)-binding protein (Pab1p). We have identified and characterized the associated network of factors controlling the maturation of mRNA poly(A) tails in yeast and defined its relevant protein-protein interactions. Pan3p, a positive regulator of PAN activity, interacts with Pab1p, thus providing substrate specificity for this nuclease. Pab1p also regulates poly(A) tail trimming by interacting with Pbp1p, a factor that appears to negatively regulate PAN. Pan3p and Pbp1p both interact with themselves and with the C terminus of Pab1p. However, the domains required for Pan3p and Pbp1p binding on Pab1p are distinct. Single amino acid changes that disrupt Pan3p interaction with Pab1p have been identified and define a binding pocket in helices 2 and 3 of Pab1p's carboxy terminus. The importance of these amino acids for Pab1p-Pan3p interaction, and poly(A) tail regulation, is underscored by experiments demonstrating that strains harboring substitutions in these residues accumulate mRNAs with long poly(A) tails in vivo.

scholarly journals Evidence for a Holin-Like Protein Gene Fully Embedded Out of Frame in the Endolysin Gene of Staphylococcus aureusBacteriophage 187

1999 ◽  
Vol 181 (15) ◽  
pp. 4452-4460 ◽  
Author(s):  
Martin J. Loessner ◽  
Susanne Gaeng ◽  
Siegfried Scherer
Keyword(s):  
Amino Acids ◽  
Cytoplasmic Membrane ◽  
Transmembrane Domain ◽  
De Novo ◽  
Atp Synthesis ◽  
Large Cell ◽  
Lytic Enzyme ◽  
Reading Frame ◽  
C Terminus

ABSTRACT We have cloned, sequenced, and characterized the genes encoding the lytic system of the unique Staphylococcus aureus phage 187. The endolysin gene ply187 encodes a large cell wall-lytic enzyme (71.6 kDa). The catalytic site, responsible for the hydrolysis of staphylococcal peptidoglycan, was mapped to the N-terminal domain of the protein by the expression of defined ply187 domains. This enzymatically active N terminus showed convincing amino acid sequence homology to anN-acetylmuramoyl-l-alanine amidase, whereas the C-terminal part, whose function is unknown, revealed striking relatedness to major staphylococcal autolysins. An additional reading frame was identified entirely embedded out of frame (+1) within the 5′ region of ply187 and was shown to encode a small, hydrophobic protein of holin-like function. The hol187 gene features a dual-start motif, possibly enabling the synthesis of two products of different lengths (57 and 55 amino acids, respectively). Overproduction of Hol187 in Escherichia coli resulted in growth retardation, leakiness of the cytoplasmic membrane, and loss of de novo ATP synthesis. Compared to other holins identified to date, Hol187 completely lacks the highly charged C terminus. The secondary structure of the polypeptide is predicted to consist of two small, antiparallel, hydrophobic, transmembrane helices. These are supposed to be essential for integration into the membrane, since site-specific introduction of negatively charged amino acids into the first transmembrane domain (V7D G8D) completely abolished the function of the Hol187 polypeptide. With antibodies raised against a synthetic 18-mer peptide representing a central part of the protein, it was possible to detect Hol187 in the cytoplasmic membrane of phage-infected S. aureus cells. An important indication that the protein actually functions as a holin in vivo was that the gene (but not the V7D G8D mutation) was able to complement a phage λ Sam mutation in a nonsuppressing E. coli HB101 background. Plaque formation by λgt11::hol187 indicated that both phage genes have analogous functions. The data presented here indicate that a putative holin is encoded on a different reading frame within the enzymatically active domain of ply187 and that the holin is synthesized during the late stage of phage infection and found in the cytoplasmic membrane, where it causes membrane lesions which are thought to enable access of Ply187 to the peptidoglycan of phage-infected Staphylococcus cells.

scholarly journals The C Terminus of Substrates Is Critical but Not Sufficient for Their Degradation by the Pseudomonas aeruginosa CtpA Protease

2020 ◽  
Vol 202 (16) ◽  
Author(s):  
Sammi Chung ◽  
Andrew J. Darwin
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Cell Wall ◽  
Outer Membrane ◽  
Content Type ◽  
C Terminus ◽  
Outer Membrane Lipoprotein ◽  
Membrane Lipoprotein ◽  
Carboxyl Terminal

ABSTRACT Bacterial carboxyl-terminal processing proteases (CTPs) are widely conserved and have been linked to important processes, including signal transduction, cell wall metabolism, and virulence. However, the features that target proteins for CTP-dependent cleavage are unclear. Studies of the Escherichia coli CTP Prc suggested that it cleaves proteins with nonpolar and/or structurally unconstrained C termini, but it is not clear if this applies broadly. Pseudomonas aeruginosa has a divergent CTP, CtpA, which is required for virulence. CtpA works in complex with the outer membrane lipoprotein LbcA to degrade cell wall hydrolases. In this study, we investigated if the C termini of two nonhomologous CtpA substrates are important for their degradation. We determined that these substrates have extended C termini compared to those of their closest E. coli homologs. Removing 7 amino acids from these extensions was sufficient to reduce their degradation by CtpA both in vivo and in vitro. Degradation of one truncated substrate was restored by adding the C terminus from the other but not by adding an unrelated sequence. However, modification of the C termini of nonsubstrates, by adding the C-terminal amino acids from a substrate, did not cause their degradation by CtpA. Therefore, the C termini of CtpA substrates are required but not sufficient for their efficient degradation. Although C-terminal truncated substrates were protected from degradation, they still associated with the LbcA-CtpA complex in vivo. Therefore, degradation of a protein by CtpA requires a C terminus-independent interaction with the LbcA-CtpA complex, followed by C terminus-dependent degradation, perhaps because CtpA normally initiates cleavage at a C-terminal site. IMPORTANCE Carboxyl-terminal processing proteases (CTPs) are found in all three domains of life, but exactly how they work is poorly understood, including how they recognize substrates. Bacterial CTPs have been associated with virulence, including CtpA of Pseudomonas aeruginosa, which works in complex with the outer membrane lipoprotein LbcA to degrade potentially dangerous peptidoglycan hydrolases. We report an important advance by revealing that efficient degradation by CtpA requires at least two separable phenomena and that one of them depends on information encoded in the substrate C terminus. A C terminus-independent association with the LbcA-CtpA complex is followed by C terminus-dependent cleavage by CtpA. Increased understanding of how CTPs target proteins is significant, due to their links to virulence, peptidoglycan remodeling, and other important processes.

scholarly journals Phosphorylation of α-dystrobrevin is essential for αkap accumulation and acetylcholine receptor stability

2020 ◽  
Vol 295 (31) ◽  
pp. 10677-10688
Author(s):  
Po-Ju Chen ◽  
Diego Zelada ◽  
Dina Cheryne Belhasan ◽  
Mohammed Akaaboune
Keyword(s):  
Acetylcholine Receptor ◽  
Muscle Cells ◽  
Underlying Mechanism ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Tyrosine Residues ◽  
C Terminus ◽  
The Stability ◽  
High Level

The maintenance of a high density of the acetylcholine receptor (AChR) is the hallmark of the neuromuscular junction. Muscle-specific anchoring protein (αkap) encoded within the calcium/calmodulin-dependent protein kinase IIα (CAMK2A) gene is essential for the maintenance of AChR clusters both in vivo and in cultured muscle cells. The underlying mechanism by which αkap is maintained and regulated remains unknown. Here, using human cell lines, fluorescence microscopy, and pulldown and immunoblotting assays, we show that α-dystrobrevin (α-dbn), an intracellular component of the dystrophin glycoprotein complex, directly and robustly promotes the stability of αkap in a concentration-dependent manner. Mechanistically, we found that the phosphorylatable tyrosine residues of α-dbn are essential for the stability of α-dbn itself and its interaction with αkap, with substitution of three tyrosine residues in the α-dbn C terminus with phenylalanine compromising the αkap–α-dbn interaction and significantly reducing both αkap and α-dbn accumulation. Moreover, the αkap–α-dbn interaction was critical for αkap accumulation and stability. We also found that the absence of either αkap or α-dbn markedly reduces AChRα accumulation and that overexpression of α-dbn or αkap in cultured muscle cells promotes the formation of large agrin-induced AChR clusters. Collectively, these results indicate that the stability of αkap and α-dbn complex plays an important role in the maintenance of high-level expression of AChRs.

Modification of Trypanosoma brucei mitochondrial rRNA by posttranscriptional 3' polyuridine tail formation

1991 ◽  
Vol 11 (12) ◽  
pp. 5878-5884
Author(s):  
B K Adler ◽  
M E Harris ◽  
K I Bertrand ◽  
S L Hajduk
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Editing ◽  
Total Cell ◽  
Metabolic Labeling ◽  
Mechanism Of Formation ◽  
Guide Rnas ◽  
Mitochondrial Rrna ◽  
Mitochondrial Transcripts ◽  
Reading Frames

Trypanosoma brucei mitochondrial transcripts can be posttranscriptionally processed by uridine addition or deletion. With editing of mRNAs, uridine addition and deletion create precisely altered reading frames. The addition of nonencoded uridines to mitochondrial guide RNAs results in a less precise modification. Although uridines are specifically added to the 3' termini, their number varies, which results in heterogeneous oligo(U) tails on guide RNAs. In this paper, we show that the mitochondrial 9S and 12S rRNAs are also modified by uridine addition. These modifications appear to have aspects in common with both RNA editing and oligo(U) tail formation. Metabolic labeling studies with intact mitochondria and [alpha-32P]UTP, in the absence of transcription, demonstrated the posttranscriptional timing of the event. T1 RNase comparison analyses of cytidine 3',5'-[5'-32P]biphosphate 3'-end-labeled and [alpha-32P]UTP metabolically labeled rRNAs, along with direct RNA sequencing of the 3' termini, identified the site of uridine addition and revealed the creation of an oligo(U) tail for both rRNAs. 12S and 9S rRNAs hybrid selected from total cell RNA exhibited the same modification, demonstrating the presence of this processing in vivo. Moreover, only 3'-poly(U)-tailed 9S and 12S rRNAs were detected in total cellular and mitochondrial RNAs, which suggests that they are the most abundant and probable mature forms. The 12S and 9S rRNA oligo(U) tails differed significantly from each other, with the 12S having a heterogeneous tail of 2 to 17 uridines and the 9S having a tail of precisely 11 uridines. The mechanism of formation and the function of the rRNA poly(U) tails remain to be determined.

scholarly journals Peptide Purification, Complementary Deoxyribonucleic Acid (DNA) and Genomic DNA Cloning, and Functional Characterization of Ghrelin in Rainbow Trout

Endocrinology ◽  
2003 ◽  
Vol 144 (12) ◽  
pp. 5215-5226 ◽  
Author(s):  
Hiroyuki Kaiya ◽  
Masayasu Kojima ◽  
Hiroshi Hosoda ◽  
Shunsuke Moriyama ◽  
Akiyoshi Takahashi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Rainbow Trout ◽  
Functional Characterization ◽  
Somatic Growth ◽  
Decanoic Acid ◽  
C Terminus ◽  
Ghrelin Gene ◽  
High Level

Abstract We have identified ghrelin from the stomach of rainbow trout. Four isoforms of ghrelin peptide were isolated: the C-terminal amidated type of rainbow trout ghrelin (rt ghrelin) composed of 24 amino acids (GSSFLSPSQKPQVRQGKGKPPRV-amide) is a basic form; des-VRQ-rt ghrelin, which deleted three amino acids (V13R14Q15) from rt ghrelin; and further two types of rt ghrelin that retained the glycine residue at the C terminus, rt ghrelin-Gly, and des-VRQ-rt ghrelin-Gly. The third serine residue was modified by octanoic acid, decanoic acid, or the unsaturated form of those fatty acids. In agreement with the isolated peptides, two cDNAs of different lengths were isolated. The rt ghrelin gene has five exons and four introns, and two different mRNA molecules are predicted to be produced by alternative splicing of the gene. A high level of ghrelin mRNA expression was detected in the stomach, and moderate levels were detected in the brain, hypothalamus, and intestinal tracts. Des-VRQ-rt ghrelin stimulated the release of GH in the rat in vivo. Furthermore, des-VRQ-rt ghrelin stimulated the release of GH, but not the release of prolactin and somatolactin in rainbow trout in vivo and in vitro. These results indicate that ghrelin is a novel GH secretagogue in rainbow trout that may affect somatic growth or osmoregulation through GH. Because ghrelin is expressed in various tissues other than stomach, it may play important role(s) in cellular function as a local regulator.

scholarly journals Molecular characterization of the TonB2 protein from the fish pathogen Vibrio anguillarum

2009 ◽  
Vol 418 (1) ◽  
pp. 49-59 ◽  
Author(s):  
Claudia S. López ◽  
R. Sean Peacock ◽  
Jorge H. Crosa ◽  
Hans J. Vogel
Keyword(s):  
Amino Acids ◽  
Solution Structure ◽  
Bacterial Species ◽  
Vibrio Anguillarum ◽  
Fish Pathogen ◽  
E Coli ◽  
Terminal Domain ◽  
C Terminus

In the fish pathogen Vibrio anguillarum the TonB2 protein is essential for the uptake of the indigenous siderophore anguibactin. Here we describe deletion mutants and alanine replacements affecting the final six amino acids of TonB2. Deletions of more than two amino acids of the TonB2 C-terminus abolished ferric-anguibactin transport, whereas replacement of the last three residues resulted in a protein with wild-type transport properties. We have solved the high-resolution solution structure of the TonB2 C-terminal domain by NMR spectroscopy. The core of this domain (residues 121–206) has an αββαβ structure, whereas residues 76–120 are flexible and extended. This overall folding topology is similar to the Escherichia coli TonB C-terminal domain, albeit with two differences: the β4 strand found at the C-terminus of TonB is absent in TonB2, and loop 3 is extended by 9 Å (0.9 nm) in TonB2. By examining several mutants, we determined that a complete loop 3 is not essential for TonB2 activity. Our results indicate that the β4 strand of E. coli TonB is not required for activity of the TonB system across Gram-negative bacterial species. We have also determined, through NMR chemical-shift-perturbation experiments, that the E. coli TonB binds in vitro to the TonB box from the TonB2-dependent outer membrane transporter FatA; moreover, it can substitute in vivo for TonB2 during ferric-anguibactin transport in V. anguillarum. Unexpectedly, TonB2 did not bind in vitro to the FatA TonB-box region, suggesting that additional factors may be required to promote this interaction. Overall our results indicate that TonB2 is a representative of a different class of TonB proteins.

scholarly journals Topogenesis in membranes of the NTB–VPg protein of Tomato ringspot nepovirus: definition of the C-terminal transmembrane domain

2004 ◽  
Vol 85 (2) ◽  
pp. 535-545 ◽  
Author(s):  
Aiming Wang ◽  
Sumin Han ◽  
Hélène Sanfaçon
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Proteinase K ◽  
Hydrophobic Region ◽  
C Terminus ◽  
Microsomal Membranes ◽  
Hydrophobic Amino Acids ◽  
Definition Of

The putative NTP-binding protein (NTB) of Tomato ringspot nepovirus (ToRSV) contains a hydrophobic region at its C terminus consisting of two adjacent stretches of hydrophobic amino acids separated by a few amino acids. In infected plants, the NTB–VPg polyprotein (containing the domain for the genome-linked protein) is associated with endoplasmic reticulum-derived membranes that are active in ToRSV replication. Recent results from proteinase K protection assays suggested a luminal location for the VPg domain in infected plants, providing support for the presence of a transmembrane domain at the C terminus of NTB. In this study, we have shown that NTB–VPg associates with canine microsomal membranes in the absence of other viral proteins in vitro and adopts a topology similar to that observed in vivo in that the VPg is present in the lumen. Truncated proteins containing 60 amino acids at the C terminus of NTB and the entire VPg exhibited a similar topology, confirming that this region of the protein contains a functional transmembrane domain. Deletion of portions of the C-terminal hydrophobic region of NTB by mutagenesis and introduction of glycosylation sites to map the luminal regions of the protein revealed that only the first stretch of hydrophobic amino acids traverses the membrane, while the second stretch of hydrophobic amino acids is located in the lumen. Our results provide additional evidence supporting the hypothesis that the NTB–VPg polyprotein acts as a membrane-anchor for the replication complex.

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