Role of N-terminal 28-amino-acid region of Rhizopus oryzae lipase in directing proteins to secretory pathway of Aspergillus oryzae

2008 ◽  
Vol 79 (6) ◽  
Author(s):  
Shinji Hama ◽  
Sriappareddy Tamalampudi ◽  
Naoki Shindo ◽  
Takao Numata ◽  
Hideki Yamaji ◽  
...  
Keyword(s):  
Amino Acid ◽  
Aspergillus Oryzae ◽  
Secretory Pathway ◽  
Rhizopus Oryzae ◽  
Rhizopus Oryzae Lipase ◽  
Amino Acid Region ◽  
Acid Region

Amino Acid Region 1000–1008 of Coagulation Factor V Is a Dynamic Regulator for the Emergence of Procoagulant Activity

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2221-2221
Author(s):  
Joesph R Wiencek ◽  
Jamila Hirbawi ◽  
Mahesheema Na ◽  
Michael Kalafatis
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Coagulation Factor ◽  
Procoagulant Activity ◽  
Factor V ◽  
Prothrombinase Complex ◽  
Amino Acid Region ◽  
Coagulation Factor V ◽  
Acid Region

Abstract Abstract 2221 The intricate process of hemostasis is a highly regulated mechanism which implements the conversion of prothrombin to thrombin and the crucial formation of a fibrin clot. The direct progression of hemostasis is pivotal to the prevention of various clotting disorders associated to hypercoagulation and excess bleeding. Upon vascular injury, the proteolytic conversion of prothrombin to thrombin compatible to rates of survival is catalyzed by the prothrombinase complex composed of the enzyme, factor Xa (fXa), the cofactor, factor Va (fVa), assembled on a phospholipid membrane in the presence of divalent metal ions. Coagulation factor V (fV) is synthesized as a multi-domain (A1-A2-B-A3-C1-C2) quiescent procofactor with nominal procoagulant activity. Following the three sequential catalytic cleavages by a-thrombin at Arg709, Arg1018 and Arg1545 amino acids 710–1545 of the B-domain are liberated to generate the noncovalently associated light and heavy chains of fVa. The cleavage at Arg1545 is crucial for full procoagulant activity. The heterodimer of fVa is composed of a heavy chain associated with the 2 A domains (residues 1–303 and 317–656) and a light chain composed of one A domain (1546-1877) and two C domains (residues 1878–2036 and 2037–2196). Since single chain fV does not bind fXa, the proper removal of the B-domain is vital to generate procoagulant activity. The incorporation of fVa into the prothrombinase complex results in a 300,000-fold increase in the catalytic efficiency of fXa for thrombin generation. Appropriate binding of fVa to fXa during prothrombinase function is essential to the proper activation of the substrate, prothrombin. Previous studies have determined the heavy and light chains of fVa to have fXa interactive sites. A highly basic region of amino acids in the B-domain suggests a potential sheathing of either the heavy or light chain fXa interface sites. To verify this hypothesis we investigated the role of amino acid region 1000–1008 that contains seven basic amino acid residues. To ascertain the role of this region we have constructed a recombinant mutant fV molecule with all activation cleavage sites (R709/R1018/R1545) mutated to glutamine (fV*T3Q), a mutant fV molecule with region 1000–1008 deleted (fVΔ1000-1008), and a mutant fV molecule containing the same deletion with all activation cleavage sites changed to glutamine (fVΔ1000-1008/*T3Q). The recombinant molecules along with wild type fV (fVWT) were transiently expressed in COS7L cells, purified to homogeneity, and assessed for their capability to bind fXa within prothrombinase prior (fV) and after incubation with thrombin (fVa). The data showed that fV*T3Q and fVa*T3Q were unable to interact with fXa. In contrast, the Kd values for fVΔ1000-1008 (0.9 nM), fVaΔ1000-1008 (0.4 nM), fVΔ1000-1008*T3Q (0.7 nM) and fVaΔ1000-1008*T3Q (0.5 nM), were similar to the affinity of fVaWT for fXa (0.22 nM). Two-stage clotting assays revealed that while fVa*T3Q was practically devoid of clotting activity, the mutant molecules fVaΔ1000-1008, and fVaD1000-1008*T3Q had clotting activities comparable to fVaWT. Thus, unactivated fVΔ1000-1008*T3Q has an affinity for fXa that is similar to the affinity of fVaWT for the enzyme. In addition, fVΔ1000-1008*T3Q that cannot be cleaved and activated by thrombin or activated during the course of the clotting assay, has similar clotting activity as fVaWT (∼3110 U/mg). The data presented in this study provide an important insight into one of the possible roles of the B domain of factor V, explicitly the fXa interactive sites on fVa are covered/inhibited by amino acids 1000–1008 of the fV B-domain. These data strongly suggest that amino acid region 1000–1008 of fV contains a regulatory sequence protecting the organisms from spontaneous binding of the procofactor to fXa and unnecessary prothrombinase complex formation which will result in catastrophic physiological consequences. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Role of the Acidic Hirudin-like COOH-Terminal Amino Acid Region of Factor Va Heavy Chain in the Enhanced Function of Prothrombinase†‡

Biochemistry ◽  
2008 ◽  
Vol 47 (30) ◽  
pp. 7963-7974 ◽  
Author(s):  
Jamila Hirbawi ◽  
Michael A. Bukys ◽  
Melissa A. Barhoover ◽  
Evrim Erdogan ◽  
Michael Kalafatis
Keyword(s):  
Amino Acid ◽  
Heavy Chain ◽  
Terminal Amino Acid ◽  
Amino Acid Region ◽  
Factor Va ◽  
Terminal Amino ◽  
Acid Region

The Role of Amino Acids 659-663 of Factor Va Heavy Chain During Prothrombin Activation by Prothrombinase.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2131-2131
Author(s):  
Jamila Hirbawi ◽  
Michael Kalafatis
Keyword(s):  
Amino Acid ◽  
Heavy Chain ◽  
Anion Binding ◽  
Prothrombinase Complex ◽  
Amino Acid Region ◽  
Factor Va ◽  
Cofactor Activity ◽  
Prothrombin Activation ◽  
Acid Region

Abstract Abstract 2131 Poster Board II-108 The proteolytic conversion of prothrombin to thrombin is catalyzed by the prothrombinase complex composed of the enzyme, factor Xa (fXa), and the cofactor, factor Va (fVa), assembled on a membrane surface in the presence of Ca2+. FXa alone can activate prothrombin following sequential cleavages at Arg271 and Arg320 yielding the transient inactive intermediate prethrombin 2. However, the interaction of fVa with fXa on a membrane/cell surface in the presence of divalent metal ions and formation of the prothrombinase complex results in the reversal of the order of cleavages and a 300,000-fold increase in the catalytic efficiency of fXa for thrombin generation. A first cleavage of prothrombin by prothrombinase at Arg320 produces the active intermediate meizothrombin, while the second cleavage at Arg271 produces thrombin. Thrombin and prothrombin contain two positively charged binding regions (anion binding exosite I, ABE-I and anion binding exosite II, ABE II), that are crucial for protein function. Initial cleavage of prothrombin at Arg320 by prothrombinase which is absolutely factor Va dependent, entirely exposes (pro)exosite I. FVa is required for the specific recognition of prothrombinase by (pro)exosite I of prothrombin. The COOH-terminal region of the heavy chain of fVa contains acidic amino acid clusters that are important for cofactor activity. We have investigated the role of amino acid region 659-663 that contains five consecutive acidic amino acid residues. To ascertain the function of this region, site-directed mutagenesis was performed. We have constructed a mutant molecule with this region deleted (fVD659-663) and a mutant molecule in which all five residues were mutated to lysine (fV5K, charge reversal). The recombinant molecules along with wild type fV (fVWT) were transiently expressed in COS7L cells, purified to homogeneity, and assessed for cofactor activity. Two-stage clotting assays revealed that the mutant molecules had reduced clotting activities compared to fVaWT. Kinetic analyses studying prothrombinase assembled with the mutant molecules demonstrated diminished kcat values, while the affinity of all mutant molecules for factor plasma-derived fXa was similar to fVaWT. Gel electrophoresis analyzing plasma-derived and recombinant mutant prothrombin activation demonstrated delayed cleavage of prothrombin at both Arg320 and Arg271 by prothrombinase assembled with the mutant molecules. Using recombinant prothrombin molecules we determined that cleavage at Arg271 by prothrombinase assembled with either fVaD659-663 or fVa5K was severely impaired compared to cleavage at Arg320 by prothrombinase assembled with the same recombinant cofactor molecules, resulting in lingering of meizothrombin throughout the activation process. To ascertain the effect of the mutations of the fVa heavy chain on the cleavage at Arg271 alone following the transition that occurs after cleavage at Arg320, we compared the rate of cleavage of active-site blocked meizothrombin (FPR-meizothrombin) by prothrombinase assembled with either fVaWT or fVaD659-663. The data demonstrate a delay for cleavage of FPR-meizothrombin at Arg271 by prothrombinase assembled with fVaD659-663 as compared to the same reaction catalyzed by prothrombinase assembled with fVaWT. Quantitative scanning densitometry of fragment 1•2-A demonstrated a ∼4-fold delay in cleavage of FPR-meizothrombin at Arg271 by prothrombinase assembled with fVaD659-663, compared to cleavage at Arg271 by prothrombinase assembled with fVaWT. Direct comparison between the rates of cleavage of FPR-meizothrombin by membrane-bound fXa alone or by prothrombinase assembled with fVaRVVD659-663 do not show any significant differences. Thus, deletion of amino acid region 659-663 virtually eliminates the acceleration in the rate of cleavage at Arg271 of meizothrombin attributed to the interaction of fVa with fXa. These data demonstrate that amino acid sequence 659DDDED663 from the factor Va heavy chain, regulates meizothrombin concentration during activation of prothrombin by prothrombinase. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Atg1 kinase in fission yeast is activated by Atg11-mediated dimerization and cis-autophosphorylation

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Zhao-Qian Pan ◽  
Guang-Can Shao ◽  
Xiao-Man Liu ◽  
Quan Chen ◽  
Meng-Qiu Dong ◽  
...  
Keyword(s):  
Amino Acid ◽  
Fission Yeast ◽  
Kinase Activity ◽  
General Mechanism ◽  
Main Role ◽  
Amino Acid Region ◽  
Proteolytic Pathway ◽  
Catalytically Active ◽  
Acid Region

Autophagy is a proteolytic pathway that is conserved from yeasts to mammals. Atg1 kinase is essential for autophagy, but how its activity is controlled remains insufficiently understood. Here, we show that, in the fission yeast Schizosaccharomyces pombe, Atg1 kinase activity requires Atg11, the ortholog of mammalian FIP200/RB1CC1, but does not require Atg13, Atg17, or Atg101. Remarkably, a 62 amino acid region of Atg11 is sufficient for the autophagy function of Atg11 and for supporting the Atg1 kinase activity. This region harbors an Atg1-binding domain and a homodimerization domain. Dimerizing Atg1 is the main role of Atg11, as it can be bypassed by artificially dimerizing Atg1. In an Atg1 dimer, only one Atg1 molecule needs to be catalytically active, suggesting that Atg1 activation can be achieved through cis-autophosphorylation. We propose that mediating Atg1 oligomerization and activation may be a conserved function of Atg11/FIP200 family proteins and cis-autophosphorylation may be a general mechanism of Atg1 activation.

scholarly journals A Carboxy-Terminal 16-Amino-Acid Region of ς38 ofEscherichia coli Is Important for Transcription under High-Salt Conditions and Sigma Activities In Vivo

2000 ◽  
Vol 182 (16) ◽  
pp. 4628-4631 ◽  
Author(s):  
Mio Ohnuma ◽  
Nobuyuki Fujita ◽  
Akira Ishihama ◽  
Kan Tanaka ◽  
Hideo Takahashi
Keyword(s):  
Amino Acid ◽  
Bacterial Species ◽  
High Potassium ◽  
Transcription Analysis ◽  
Amino Acid Region ◽  
Sigma Subunit ◽  
Carboxy Terminal ◽  
Acid Region

ABSTRACT ς38 (or ςS, the rpoS gene product) is a sigma subunit of RNA polymerase in Escherichia coli and directs transcription from a number of stationary-phase promoters as well as osmotically inducible promoters. In this study, we analyzed the function of the carboxy-terminal 16-amino-acid region of ς38 (residues 315 to 330), which is well conserved among the rpoS gene products of enteric bacterial species. Truncation of this region was shown to result in the loss of sigma activity in vivo using promoter-lacZ fusion constructs, but the mutant ς38 retained the binding activity in vivo to the core enzyme. The in vitro transcription analysis revealed that the transcription activity of ς38 holoenzyme under high potassium glutamate concentrations was significantly decreased by the truncation of the carboxy-terminal tail element.

scholarly journals The DNA-binding specificity of the hepatocyte nuclear factor 3/forkhead domain is influenced by amino-acid residues adjacent to the recognition helix.

1994 ◽  
Vol 14 (4) ◽  
pp. 2755-2766 ◽  
Author(s):  
D G Overdier ◽  
A Porcella ◽  
R H Costa
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Hepatocyte Nuclear Factor ◽  
Binding Properties ◽  
Winged Helix ◽  
Amino Acid Region ◽  
Dna Binding Specificity ◽  
Recognition Helix ◽  
Dna Binding Properties ◽  
Acid Region

Three distinct hepatocyte nuclear factor 3 (HNF-3) proteins (HNF-3 alpha, -3 beta, and -3 gamma) are known to regulate the transcription of liver-specific genes. The HNF-3 proteins bind to DNA as a monomer through a modified helix-turn-helix, known as the winged helix motif, which is also utilized by a number of developmental regulators, including the Drosophila homeotic forkhead (fkh) protein. We have previously described the isolation, from rodent tissue, of an extensive family of tissue-specific HNF-3/fkh homolog (HFH) genes sharing homology in their winged helix motifs. In this report, we have determined the preferred DNA-binding consensus sequence for the HNF-3 beta protein as well as for two divergent family members, HFH-1 and HFH-2. We show that these HNF-3/fkh proteins bind to distinct DNA sites and that the specificity of protein recognition is dependent on subtle nucleotide alterations in the site. The HNF-3, HFH-1, and HFH-2 consensus binding sequences were also used to search DNA regulatory regions to identify potential target genes. Furthermore, an analysis of the DNA-binding properties of a series of HFH-1/HNF-3 beta protein chimeras has allowed us to identify a 20-amino-acid region, located adjacent to the DNA recognition helix, which contributes to DNA-binding specificity. These sequences are not involved in base-specific contacts and include residues which diverge within the HNF-3/fkh family. Replacement of this 20-amino-acid region in HNF-3 beta with corresponding residues from HFH-1 enabled the HNF-3 beta recognition helix to bind only HFH-1-specific DNA-binding sites. We propose a model in which this 20-amino-acid flanking region influences the DNA-binding properties of the recognition helix.

scholarly journals Rho1p-Bni1p-Spa2p Interactions: Implication in Localization of Bni1p at the Bud Site and Regulation of the Actin Cytoskeleton inSaccharomyces cerevisiae

1998 ◽  
Vol 9 (5) ◽  
pp. 1221-1233 ◽  
Author(s):  
Takeshi Fujiwara ◽  
Kazuma Tanaka ◽  
Akihisa Mino ◽  
Mitsuhiro Kikyo ◽  
Kazuo Takahashi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Actin Cytoskeleton ◽  
Binding Protein ◽  
Microscopic Analysis ◽  
Mitogen Activated Protein Kinase ◽  
Gene Encoding ◽  
Amino Acid Region ◽  
Genes Encoding ◽  
Mitogen Activated Protein ◽  
Acid Region

Rho1p is a yeast homolog of mammalian RhoA small GTP-binding protein. Rho1p is localized at the growth sites and required for bud formation. We have recently shown that Bni1p is a potential target of Rho1p and that Bni1p regulates reorganization of the actin cytoskeleton through interactions with profilin, an actin monomer-binding protein. Using the yeast two-hybrid screening system, we cloned a gene encoding a protein that interacted with Bni1p. This protein, Spa2p, was known to be localized at the bud tip and to be implicated in the establishment of cell polarity. The C-terminal 254 amino acid region of Spa2p, Spa2p(1213–1466), directly bound to a 162-amino acid region of Bni1p, Bni1p(826–987). Genetic analyses revealed that both thebni1 and spa2 mutations showed synthetic lethal interactions with mutations in the genes encoding components of the Pkc1p-mitogen-activated protein kinase pathway, in which Pkc1p is another target of Rho1p. Immunofluorescence microscopic analysis showed that Bni1p was localized at the bud tip in wild-type cells. However, in the spa2 mutant, Bni1p was not localized at the bud tip and instead localized diffusely in the cytoplasm. A mutant Bni1p, which lacked the Rho1p-binding region, also failed to be localized at the bud tip. These results indicate that both Rho1p and Spa2p are involved in the localization of Bni1p at the growth sites where Rho1p regulates reorganization of the actin cytoskeleton through Bni1p.

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