The last five amino acid residues at the C-terminus of PRK1/PKN is essential for full lipid responsiveness

Biochemical and physiological studies of Synechococcus cyanobacteria have indicated the presence of a low-Mr heavy-metal-binding protein with marked similarity to eukaryotic metallothioneins (MTs). We report here the characterization of a Synechococcus prokaryotic MT isolated by gel-permeation and reverse-phase chromatography. The large number of variants of this molecule found during chromatographic separation could not be attributed to the presence of major isoproteins as assessed by amino acid analysis and amino acid sequencing of isoforms. Two of the latter were shown to have identical primary structures that differed substantially from the well-described eukaryotic MTs. In addition to six long-chain aliphatic residues, two aromatic residues were found adjacent to one another near the centre of the molecule, making this the most hydrophobic MT to be described. Other unusual features included a pair of histidine residues located in repeating Gly-His-Thr-Gly sequences near the C-terminus and a complete lack of association of hydroxylated residues with cysteine residues, as is commonly found in eukaryotes. Similarly, aside from a single lysine residue, no basic amino acid residues were found adjacent to cysteine residues in the sequence. Most importantly, sequence alignment analyses with mammalian, invertebrate and fungal MT sequences showed no statistically significant homology aside from the presence of Cys-Xaa-Cys structures common to all MTs. On the other hand, like other MTs, the prokaryotic molecule appears to be free of alpha-helical structure but has a considerable amount of beta-structure, as predicted by both c.d. measurements and the Chou & Fasman empirical relations. Considered together, these data suggested that some similarity between the metal-thiolate clusters of the prokaryote and eukaryote MTs may exist.

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Deletion of the Actin-Associated Tropomyosin Tpm3 Leads to Reduced Cell Complexity in Cultured Hippocampal Neurons—New Insights into the Role of the C-Terminal Region of Tpm3.1

Cells ◽

10.3390/cells10030715 ◽

2021 ◽

Vol 10 (3) ◽

pp. 715

Author(s):

Tamara Tomanić ◽

Claire Martin ◽

Holly Stefen ◽

Esmeralda Parić ◽

Peter Gunning ◽

...

Keyword(s):

Amino Acid ◽

Hippocampal Neurons ◽

Molecular Mechanisms ◽

Growth Cones ◽

Amino Acid Residues ◽

Terminal Amino Acid ◽

C Terminus ◽

Primary Mouse ◽

Terminal Amino

Tropomyosins (Tpms) have been described as master regulators of actin, with Tpm3 products shown to be involved in early developmental processes, and the Tpm3 isoform Tpm3.1 controlling changes in the size of neuronal growth cones and neurite growth. Here, we used primary mouse hippocampal neurons of C57/Bl6 wild type and Bl6Tpm3flox transgenic mice to carry out morphometric analyses in response to the absence of Tpm3 products, as well as to investigate the effect of C-terminal truncation on the ability of Tpm3.1 to modulate neuronal morphogenesis. We found that the knock-out of Tpm3 leads to decreased neurite length and complexity, and that the deletion of two amino acid residues at the C-terminus of Tpm3.1 leads to more detrimental changes in neurite morphology than the deletion of six amino acid residues. We also found that Tpm3.1 that lacks the 6 C-terminal amino acid residues does not associate with stress fibres, does not segregate to the tips of neurites, and does not impact the amount of the filamentous actin pool at the axonal growth cones, as opposed to Tpm3.1, which lacks the two C-terminal amino acid residues. Our study provides further insight into the role of both Tpm3 products and the C-terminus of Tpm3.1, and it forms the basis for future studies that aim to identify the molecular mechanisms underlying Tpm3.1 targeting to different subcellular compartments.

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Peroxisome targeting signal of rat liver acyl-coenzyme A oxidase resides at the carboxy terminus

Molecular and Cellular Biology ◽

10.1128/mcb.9.1.83-91.1989 ◽

1989 ◽

Vol 9 (1) ◽

pp. 83-91

Author(s):

S Miyazawa ◽

T Osumi ◽

T Hashimoto ◽

K Ohno ◽

S Miura ◽

...

Keyword(s):

Amino Acid ◽

Rat Liver ◽

Coenzyme A ◽

Terminal Region ◽

Proteinase K ◽

Amino Acid Residues ◽

C Terminus ◽

Targeting Signal ◽

Acyl Coenzyme A

To identify the topogenic signal of peroxisomal acyl-coenzyme A oxidase (AOX) of rat liver, we carried out in vitro import experiments with mutant polypeptides of the enzyme. Full-length AOX and polypeptides that were truncated at the N-terminal region were efficiently imported into peroxisomes, as determined by resistance to externally added proteinase K. Polypeptides carrying internal deletions in the C-terminal region exhibited much lower import activities. Polypeptides that were truncated or mutated at the extreme C terminus were totally import negative. When the five amino acid residues at the extreme C terminus were attached to some of the import-negative polypeptides, the import activities were rescued. Moreover, the C-terminal 199 and 70 amino acid residues of AOX directed fusion proteins with two bacterial enzymes to peroxisomes. These results are interpreted to mean that the peroxisome targeting signal of AOX residues at the C terminus and the five or fewer residues at the extreme terminus have an obligatory function in targeting. The C-terminal internal region also has an important role for efficient import, possibly through a conformational effect.

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Differentiation of propeptide residues regulating the compartmentalization, maturation and activity of the broad-range phospholipase C of Listeria monocytogenes

Biochemical Journal ◽

10.1042/bj20100557 ◽

2010 ◽

Vol 432 (3) ◽

pp. 557-566 ◽

Cited By ~ 10

Author(s):

Emily R. Slepkov ◽

Alan Pavinski Bitar ◽

Hélène Marquis

Keyword(s):

Cell Wall ◽

Listeria Monocytogenes ◽

Amino Acid ◽

Enzymatic Activity ◽

Phospholipase C ◽

Bacterial Pathogen ◽

Amino Acid Residues ◽

C Terminus ◽

N Terminus ◽

Vacuolar Acidification

The intracellular bacterial pathogen Listeria monocytogenes secretes a broad-range phospholipase C enzyme called PC-PLC (phosphatidylcholine phospholipase C) whose compartmentalization and enzymatic activity is regulated by a 24-amino-acid propeptide (Cys28–Ser51). During intracytosolic multiplication, bacteria accumulate the proform of PC-PLC at their membrane–cell-wall interface, whereas during cell-to-cell spread vacuolar acidification leads to maturation and rapid translocation of PC-PLC across the cell wall in a manner that is dependent on Mpl, the metalloprotease of Listeria. In the present study, we generated a series of propeptide mutants to determine the minimal requirement to prevent PC-PLC enzymatic activity and to identify residues regulating compartmentalization and maturation. We found that a single residue at position P1 (Ser51) of the cleavage site is sufficient to prevent enzymatic activity, which is consistent with P1′ (Trp52) being located within the active-site pocket. We observed that mutants with deletions at the N-terminus, but not the C-terminus, of the propeptide are translocated across the cell wall more effectively than wild-type PC-PLC at a physiological pH, and that individual amino acid residues within the N-terminus influence Mpl-mediated maturation of PC-PLC at acidic pH. However, deletion of more than 75% of the propeptide was required to completely prevent Mpl-mediated maturation of PC-PLC. These results indicate that the N-terminus of the propeptide regulates PC-PLC compartmentalization and that specific residues within the N-terminus influence the ability of Mpl to mediate PC-PLC maturation, although a six-residue propeptide is sufficient for Mpl to mediate PC-PLC maturation.

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Noncharged amino acid residues at the solvent-exposed positions in the middle and at the C terminus of the α-helix have the same helical propensity

Protein Science ◽

10.1110/ps.0304303 ◽

2003 ◽

Vol 12 (6) ◽

pp. 1169-1176 ◽

Cited By ~ 20

Author(s):

Dmitri N. Ermolenko ◽

John M. Richardson ◽

George I. Makhatadze

Keyword(s):

Amino Acid ◽

Amino Acid Residues ◽

C Terminus ◽

Α Helix

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The Acinetobacter baylyi hfq Gene Encodes a Large Protein with an Unusual C Terminus

Journal of Bacteriology ◽

10.1128/jb.00490-09 ◽

2009 ◽

Vol 191 (17) ◽

pp. 5553-5562 ◽

Cited By ~ 20

Author(s):

Dominik Schilling ◽

Ulrike Gerischer

Keyword(s):

Amino Acid ◽

Gene Localization ◽

Amino Acid Residues ◽

Acinetobacter Baylyi ◽

Reading Frame ◽

Rich Domain ◽

C Terminus ◽

The Difference ◽

Cell Phenotypes

ABSTRACT In gammaproteobacteria the Hfq protein shows a great variation in size, especially in its C-terminal part. Extremely large Hfq proteins consisting of almost 200 amino acid residues and more are found within the gammaproteobacterial family Moraxellaceae. The difference in size compared to other Hfq proteins is due to a glycine-rich domain near the C-terminal end of the protein. Acinetobacter baylyi, a nonpathogenic soil bacterium and member of the Moraxellaceae encodes a large 174-amino-acid Hfq homologue containing the unique and repetitive amino acid pattern GGGFGGQ within the glycine-rich domain. Despite the presence of the C-terminal extension, A. baylyi Hfq complemented an Escherichia coli hfq mutant in vivo. By using polyclonal anti-Hfq antibodies, we detected the large A. baylyi Hfq that corresponds to its annotated size indicating the expression and stability of the full protein. Deletion of the complete A. baylyi hfq open reading frame resulted in severe reduction of growth. In addition, a deletion or overexpression of Hfq was accompanied by the loss of cell chain assembly. The glycine-rich domain was not responsible for growth and cell phenotypes. hfq gene localization in A. baylyi is strictly conserved within the mutL-miaA-hfq operon, and we show that hfq expression starts within the preceding miaA gene or further upstream.

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Polymorphism of cow colostrum proteinase inhibitor

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19810807 ◽

1981 ◽

Vol 46 (3) ◽

pp. 807-816 ◽

Cited By ~ 2

Author(s):

Věra Jonáková ◽

Dana Čechová ◽

Otakar Mach

Keyword(s):

Amino Acid ◽

Primary Structure ◽

Proteinase Inhibitor ◽

Protein Structures ◽

Amino Acid Residues ◽

Net Charge ◽

C Terminus ◽

Isoelectric Points ◽

The Individual ◽

Protein Moiety

Cow colostrum contains three isoinhibitors A, B, and C, which are glycoproteins. In this study isoinhibitor A was isolated and characterized and the structure of its protein moiety compared with the known protein structures of isoinhibitors B and C. It was found that the primary structure of isoinhibitor A is identical with the primary structure of isoinhibitor B except that the C-terminus of its molecule is shorter by five amino acid residues. Four discrete chromatographic forms (I-IV) with different isoelectric points (pI 3.8 - I, 4.0 - II, 4.3 - III, and 4.5 - IV) were isolated by chromatography on SE-Sephadex, Form I is identical with isoinhibitor A. Forms II, III, and IV are represented by mixtures of isoinhibitors A, B, and C with a heterogeneous carbohydrate moiety which affects the total net charge of the individual inhibitor forms.

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Neuropeptide FF (FLFQPQRF-NH2) and its Fragments Bind to α2δ Subunit of Voltage-Gated Calcium Channels

Journal of Pharmacy & Pharmaceutical Sciences ◽

10.18433/jpps30358 ◽

2019 ◽

Vol 22 ◽

pp. 292-300

Author(s):

Hanna Skubatz

Keyword(s):

Cerebral Cortex ◽

Amino Acid ◽

Cho Cells ◽

New Drugs ◽

Rat Cerebral Cortex ◽

Amino Acid Residues ◽

C Terminus ◽

Neuropeptide Ff ◽

Α2δ Subunit ◽

Ca2 Channels

Purpose: Gabapentin, a drug for neuropathic pain, exerts its therapeutic effect via inhibition of the a2d subunit of N-type Ca2+ channels. Thus, finding peptides that specifically displace gabapentin from its binding site may lead to the development of new drugs. Methods: Displacement of bound [3H]-gabapentin in membrane preparations of rat cerebral cortex and of human Cav2.2/β3/α2δ1 expressed in CHO cell line. Results: Neuropeptide FLFQPQRF-NH2 specifically displaced bound [3H]-gabapentin in membrane preparations from rats and CHO cells. Truncation of the C-terminus of FLFQPQRF-NH2 by three amino acid residues to produce FLFQP-NH2 improved the displacement of gabapentin. FLFQP-NH2 displaced bound [3H]-gabapentin with IC50 and Ki values of 2.7 µM and 1.7 µM, respectively. Deletion of two amino acid residues (FQ) in the middle of the FLFQP-NH2 sequence yielded FLP-NH2 that displaced bound [3H]-gabapentin with a lower affinity. IC50 and Ki values were 11.9 µM and 7.8 µM, respectively. Neutral binding cooperativity existed when of FLFQP-NH2, FLP- NH2 and gabapentin when incubated together. FLFQPQRF-NH2 but not FLFQP-NH2 displaced bound [3H]-gabapentin to membrane preparations of human Cav2.2/b3/a2d1 expressed in CHO cells. Conclusion: FLFQPQRF-NH2, FLFQP-NH2 and FLP-NH2 displace bound gabapentin in membrane preparations of rat cerebral cortex. Binding cooperativity was detected when GBP/FLFQP-NH2/FLP-NH2 were incubated together. These novel binding sites may provide new approaches to modulate L-type Ca2+ channels.

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Characterization of the Phd Repressor-Antitoxin Boundary

Journal of Bacteriology ◽

10.1128/jb.187.2.765-770.2005 ◽

2005 ◽

Vol 187 (2) ◽

pp. 765-770 ◽

Cited By ~ 19

Author(s):

James Estle McKinley ◽

Roy David Magnuson

Keyword(s):

Amino Acid ◽

Point Mutations ◽

Modular Organization ◽

Amino Acid Residues ◽

The Third ◽

Linear Sequence ◽

C Terminus ◽

Overlapping Sets ◽

Repressor Activity

ABSTRACT The P1 plasmid addiction operon (a classic toxin-antitoxin system) encodes Phd, an unstable 73-amino-acid repressor-antitoxin protein, and Doc, a stable toxin. It was previously shown by deletion analysis that the N terminus of Phd was required for repressor activity and that the C terminus was required for antitoxin activity. Since only a quarter of the protein or less was required for both activities, it was hypothesized that Phd might have a modular organization. To further test the modular hypothesis, we constructed and characterized a set of 30 point mutations in the third and fourth quarters of Phd. Four mutations (PhdA36H, V37A, I38A, and F44A) had major defects in repressor activity. Five mutations (PhdD53A, D53R, E55A, F56A, and F60A) had major defects in antitoxin activity. As predicted by the modular hypothesis, point mutations affecting each activity belonged to disjoint, rather than overlapping, sets and were separated rather than interspersed within the linear sequence. A final deletion experiment demonstrated that the C-terminal 24 amino acid residues of Phd (preceded by a methionine) retained full antitoxin activity.

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The ς70 Transcription Factor TyrR Has Zinc-Stimulated Phosphatase Activity That Is Inhibited by ATP and Tyrosine

Journal of Bacteriology ◽

10.1128/jb.182.4.1053-1061.2000 ◽

2000 ◽

Vol 182 (4) ◽

pp. 1053-1061 ◽

Cited By ~ 10

Author(s):

Shimin Zhao ◽

Qin Zhu ◽

Ronald L. Somerville

Keyword(s):

Amino Acid ◽

Phosphatase Activity ◽

Sequence Similarity ◽

Zinc Ion ◽

Amino Acid Residues ◽

Protein Residues ◽

E Coli ◽

C Terminus ◽

Atp Analogues ◽

High Degree

ABSTRACT The TyrR protein of Escherichia coli (513 amino acid residues) is the chief transcriptional regulator of a group of genes that are essential for aromatic amino acid biosynthesis and transport. The TyrR protein can function either as a repressor or as an activator. The central region of the TyrR protein (residues 207 to 425) is similar to corresponding polypeptide segments of the NtrC protein superfamily. Like the NtrC protein, TyrR has intrinsic ATPase activity. Here, we report that TyrR possesses phosphatase activity. This activity is subject to inhibition by l-tyrosine and its analogues and by ATP and ATP analogues. Zinc ion (2 mM) stimulated the phosphatase activity of the TyrR protein by a factor of 57. The phosphatase-active site of TyrR was localized to a 31-kDa domain (residues 191 to 467) of the protein. However, mutational alteration of distant amino acid residues at both the N terminus and the C terminus of TyrR altered the phosphatase activity. Haemophilus influenzae TyrR (318 amino acid residues), a protein with a high degree of sequence similarity to the C terminus of the E. coli TyrR protein, exhibited a phosphatase activity similar to that of E. coliTyrR.

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