Conserved amino acids at the C-terminus of rat phospholipase D1 are essential for enzymatic activity

The binding of cyclin A to p34cdc2 and p32cdk2 and the protein kinase activity of the complexes has been measured by cell-free translation of the corresponding mRNA in extracts of frog eggs, followed by immunoprecipitation. A variety of mutant cyclin A molecules have been constructed and tested in this assay. Small deletions and point mutations of highly conserved residues in the 100-residue "cyclin box" abolish binding and activation of both p34cdc2 and p32cdk2. By contrast, large deletions at the N-terminus have no effect on kinase binding and activation, until they remove residues beyond 161, where the first conserved amino acids are found in all known examples of cyclin A. At the C-terminus, removal of 14 or more amino acids abolishes activity. We also demonstrate that deletion of, or point mutations, in the cyclin A homologue of the 10-residue "destruction box," previously described in cyclin B (Glotzer et al., 1991) abolish cyclin proteolysis at the transition from M-phase to interphase.

Download Full-text

Cancer associated mutations in Sec61γ alter the permeability of the ER translocase

PLoS Genetics ◽

10.1371/journal.pgen.1009780 ◽

2021 ◽

Vol 17 (8) ◽

pp. e1009780

Author(s):

Christopher M. Witham ◽

Aleshanee L. Paxman ◽

Lamprini Baklous ◽

Robert F. L. Steuart ◽

Benjamin L. Schulz ◽

...

Keyword(s):

Amino Acids ◽

Protein Complex ◽

Integral Membrane Proteins ◽

Permeability Barrier ◽

Malignant Cells ◽

Conducting Channel ◽

Genome Database ◽

C Terminus ◽

Progression Of Disease ◽

Conserved Amino Acids

Translocation of secretory and integral membrane proteins across or into the ER membrane occurs via the Sec61 complex, a heterotrimeric protein complex possessing two essential sub-units, Sec61p/Sec61α and Sss1p/Sec61γ and the non-essential Sbh1p/Sec61β subunit. In addition to forming a protein conducting channel, the Sec61 complex maintains the ER permeability barrier, preventing flow of molecules and ions. Loss of Sec61 integrity is detrimental and implicated in the progression of disease. The Sss1p/Sec61γ C-terminus is juxtaposed to the key gating module of Sec61p/Sec61α and is important for gating the translocon. Inspection of the cancer genome database identifies six mutations in highly conserved amino acids of Sec61γ/Sss1p. We identify that five out of the six mutations identified affect gating of the ER translocon, albeit with varying strength. Together, we find that mutations in Sec61γ that arise in malignant cells result in altered translocon gating dynamics, this offers the potential for the translocon to represent a target in co-therapy for cancer treatment.

Download Full-text

The role of Asp-49 and other conserved amino acids in phospholipases A2 and their importance for enzymatic activity

Journal of Cellular Biochemistry ◽

10.1002/jcb.240390404 ◽

1989 ◽

Vol 39 (4) ◽

pp. 379-390 ◽

Cited By ~ 82

Author(s):

Carel J. van den Bergh ◽

Arend J. Slotboom ◽

Hubertus M. Verheij ◽

Gerard H. de Haas

Keyword(s):

Amino Acids ◽

Enzymatic Activity ◽

Phospholipases A2 ◽

Conserved Amino Acids

Download Full-text

C Terminus of DJ-1 Determines Its Homodimerization, Deglycation Activity and Suppression of Ferroptosis

10.1101/2020.03.26.992446 ◽

2020 ◽

Author(s):

Li Jiang ◽

Xiaobing Chen ◽

Qian Wu ◽

Haiying Zhu ◽

Chengyong Du ◽

...

Keyword(s):

Amino Acids ◽

Parkinson Disease ◽

Enzymatic Activity ◽

Early Onset ◽

Wild Type ◽

Functional Protein ◽

Point Mutant ◽

C Terminus ◽

Antioxidative Stress ◽

The Relationship

AbstractDJ-1 is a multi-functional protein related to cancer and autosomal early-onset Parkinson disease (PD). Besides the well-documented antioxidative stress activity, recent studies suggest that DJ-1 has the deglycation enzymatic activity and the anti-ferroptosis function. Although it has been demonstrated that DJ-1 forms the homodimerization, which dictates its antioxidative stress activity, the relationship between the dimeric structure and newly reported activities remains largely elusive. In this study, we find that the deletion mutation of the last 3 amino acids at C terminus of DJ-1 disrupts its homodimerization in both transfected and purified DJ-1 protein. Further study shows that hydrophobic L187 residue is of great importance for DJ-1 homodimerization. In addition, the ability in methylglyoxal detoxification is almost abolished in the mutation of deleting last 3 residues at C terminus (ΔC3) and point mutant L187E compared with wild type DJ-1 (DJ-1 WT). We also find that the suppression of ferroptosis is fully inhibited by ΔC3 and L187E while partially suppressed by V51C. Thus, our findings show that C terminus of DJ-1 is crucial for its homodimerization, deglycation activity and suppression of ferroptosis.

Download Full-text

Site-directed mutagenesis of coumarin-type anticoagulant-sensitive VKORCI. Evidence that highly conserved amino acids define structural requirements for enzymatic activity and inhibition by warfarin

Thrombosis and Haemostasis ◽

10.1160/th05-02-0082 ◽

2005 ◽

Cited By ~ 4

Author(s):

Simone Rost ◽

Andreas Fregin ◽

Mirja Hünerberg ◽

Carville G. Bevans ◽

Clemens R. Müller ◽

...

Keyword(s):

Amino Acids ◽

Enzymatic Activity ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Structural Requirements ◽

Conserved Amino Acids

Download Full-text

Human Platelet Antigen 3 (HPA-3): Localization of the Determinant of the Alloantibody Leka (HPA-3a) to the C-Terminus of Platelet Glycoprotein IIb Heavy Chain and Contribution of O-Linked Carbohydrates

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651638 ◽

1993 ◽

Vol 69 (05) ◽

pp. 485-489 ◽

Cited By ~ 23

Author(s):

Isabelle Djaffar ◽

Didier Vilette ◽

Dominique Pidard ◽

Jean-Luc Wautier ◽

Jean-Philippe Rosa

Keyword(s):

Amino Acids ◽

Heavy Chain ◽

Human Platelet ◽

Platelet Glycoprotein ◽

Fibrinogen Receptor ◽

C Terminus ◽

Platelet Antigen ◽

Human Platelet Antigen ◽

Determinant Structure ◽

Polymerase Chain

SummaryThe human platelet antigen (HPA) 3 system is expressed on GPIIb, one subunit of GPIIb-IIIa, the platelet fibrinogen receptor. It was recently shown that HPA-3 was associated with an Ile843/Ser polymorphism. To investigate further HPA-3 determinant structure, we localized an HPA-3a determinant, recognized by the alloantiserum Leka, within the last 29 amino acids of GPIIbα. This region encompasses the polymorphic Ile843, which, as expected, is substituted into Ser in Leka-negative individuals, as shown by DNA sequence after polymerase chain reaction on platelet RNA. In addition, contribution of glycosylation to the determinant structure was demonstrated since the Leka antigenicity was strongly decreased after specifically removing nonterminal O-linked sugars, but not terminal sialic acids. We have thus refined the localization of an HPA-3a determinant within the last 29 amino acids, including Ile843, of GPIIb heavy chain, and shown that the Leka HPA-3a determinant is dependent, in part, upon the serine-linked carbohydrates adjacent to Ile/Ser843.

Download Full-text