Mutational analysis of the antizyme-binding element reveals critical residues for the function of ornithine decarboxylase

Bruno Ramos-Molina; Ana Lambertos; Andrés J López-Contreras; Rafael Peñafiel

doi:10.1016/j.bbagen.2013.07.003

Mutational analysis of the antizyme-binding element reveals critical residues for the function of ornithine decarboxylase

Bruno Ramos-Molina, Ana Lambertos, Andrés J López-Contreras, Rafael Peñafiel

4 Citations (Scopus)

Abstract

BACKGROUND: Ornithine decarboxylase (ODC), the key enzyme in the polyamine biosynthetic pathway, is highly regulated by antizymes (AZs), small proteins that bind and inhibit ODC and increase its proteasomal degradation. Early studies delimited the putative AZ-binding element (AZBE) to the region 117-140 of ODC. The aim of the present work was to study the importance of certain residues of the region 110-142 that includes the AZBE region for the interaction between ODC and AZ1 and the ODC functionality.

METHODS: Computational analysis of the protein sequences of the extended AZBE site of ODC and ODC paralogues from different eukaryotes was used to search for conserved residues. The influence of these residues on ODC functionality was studied by site directed mutagenesis, followed by different biochemical techniques.

RESULTS: The results revealed that: a) there are five conserved residues in ODC and its paralogues: K115, A123, E138, L139 and K141; b) among these, L139 is the most critical one for the interaction with AZs, since its substitution decreases the affinity of the mutant protein towards AZs; c) all these conserved residues, with the exception of A123, are critical for ODC activity; d) substitutions of K115, E138 or L139 diminish the formation of ODC homodimers.

CONCLUSIONS: These results reveal that four of the invariant residues of the AZBE region are strongly related to ODC functionality.

GENERAL SIGNIFICANCE: This work helps to understand the interaction between ODC and AZ1, and describes various new residues involved in ODC activity, a key enzyme for cell growth and proliferation.

Original language	English
Journal	B B A - Reviews on Cancer
Volume	1830
Issue number	11
Pages (from-to)	5157-65
Number of pages	9
ISSN	0006-3002
DOIs	https://doi.org/10.1016/j.bbagen.2013.07.003
Publication status	Published - Nov 2013
Externally published	Yes

Keywords

Amino Acid Sequence
Binding Sites
Cell Line
DNA Mutational Analysis
HEK293 Cells
Humans
Models, Molecular
Molecular Sequence Data
Mutagenesis, Site-Directed
Mutant Proteins
Ornithine Decarboxylase
Protein Binding
Protein Conformation
Proteins

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10.1016/j.bbagen.2013.07.003

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@article{6628d97f947f4051bcd2956a074fd83d,

title = "Mutational analysis of the antizyme-binding element reveals critical residues for the function of ornithine decarboxylase",

abstract = "BACKGROUND: Ornithine decarboxylase (ODC), the key enzyme in the polyamine biosynthetic pathway, is highly regulated by antizymes (AZs), small proteins that bind and inhibit ODC and increase its proteasomal degradation. Early studies delimited the putative AZ-binding element (AZBE) to the region 117-140 of ODC. The aim of the present work was to study the importance of certain residues of the region 110-142 that includes the AZBE region for the interaction between ODC and AZ1 and the ODC functionality.METHODS: Computational analysis of the protein sequences of the extended AZBE site of ODC and ODC paralogues from different eukaryotes was used to search for conserved residues. The influence of these residues on ODC functionality was studied by site directed mutagenesis, followed by different biochemical techniques.RESULTS: The results revealed that: a) there are five conserved residues in ODC and its paralogues: K115, A123, E138, L139 and K141; b) among these, L139 is the most critical one for the interaction with AZs, since its substitution decreases the affinity of the mutant protein towards AZs; c) all these conserved residues, with the exception of A123, are critical for ODC activity; d) substitutions of K115, E138 or L139 diminish the formation of ODC homodimers.CONCLUSIONS: These results reveal that four of the invariant residues of the AZBE region are strongly related to ODC functionality.GENERAL SIGNIFICANCE: This work helps to understand the interaction between ODC and AZ1, and describes various new residues involved in ODC activity, a key enzyme for cell growth and proliferation.",

keywords = "Amino Acid Sequence, Binding Sites, Cell Line, DNA Mutational Analysis, HEK293 Cells, Humans, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, Mutant Proteins, Ornithine Decarboxylase, Protein Binding, Protein Conformation, Proteins",

author = "Bruno Ramos-Molina and Ana Lambertos and L{\'o}pez-Contreras, {Andr{\'e}s J} and Rafael Pe{\~n}afiel",

note = "{\textcopyright} 2013.",

year = "2013",

month = nov,

doi = "10.1016/j.bbagen.2013.07.003",

language = "English",

volume = "1830",

pages = "5157--65",

journal = "Biochimica et Biophysica Acta - Reviews on Cancer",

issn = "0304-419X",

publisher = "Elsevier",

number = "11",

}

TY - JOUR

T1 - Mutational analysis of the antizyme-binding element reveals critical residues for the function of ornithine decarboxylase

AU - Ramos-Molina, Bruno

AU - Lambertos, Ana

AU - López-Contreras, Andrés J

AU - Peñafiel, Rafael

PY - 2013/11

Y1 - 2013/11

N2 - BACKGROUND: Ornithine decarboxylase (ODC), the key enzyme in the polyamine biosynthetic pathway, is highly regulated by antizymes (AZs), small proteins that bind and inhibit ODC and increase its proteasomal degradation. Early studies delimited the putative AZ-binding element (AZBE) to the region 117-140 of ODC. The aim of the present work was to study the importance of certain residues of the region 110-142 that includes the AZBE region for the interaction between ODC and AZ1 and the ODC functionality.METHODS: Computational analysis of the protein sequences of the extended AZBE site of ODC and ODC paralogues from different eukaryotes was used to search for conserved residues. The influence of these residues on ODC functionality was studied by site directed mutagenesis, followed by different biochemical techniques.RESULTS: The results revealed that: a) there are five conserved residues in ODC and its paralogues: K115, A123, E138, L139 and K141; b) among these, L139 is the most critical one for the interaction with AZs, since its substitution decreases the affinity of the mutant protein towards AZs; c) all these conserved residues, with the exception of A123, are critical for ODC activity; d) substitutions of K115, E138 or L139 diminish the formation of ODC homodimers.CONCLUSIONS: These results reveal that four of the invariant residues of the AZBE region are strongly related to ODC functionality.GENERAL SIGNIFICANCE: This work helps to understand the interaction between ODC and AZ1, and describes various new residues involved in ODC activity, a key enzyme for cell growth and proliferation.

AB - BACKGROUND: Ornithine decarboxylase (ODC), the key enzyme in the polyamine biosynthetic pathway, is highly regulated by antizymes (AZs), small proteins that bind and inhibit ODC and increase its proteasomal degradation. Early studies delimited the putative AZ-binding element (AZBE) to the region 117-140 of ODC. The aim of the present work was to study the importance of certain residues of the region 110-142 that includes the AZBE region for the interaction between ODC and AZ1 and the ODC functionality.METHODS: Computational analysis of the protein sequences of the extended AZBE site of ODC and ODC paralogues from different eukaryotes was used to search for conserved residues. The influence of these residues on ODC functionality was studied by site directed mutagenesis, followed by different biochemical techniques.RESULTS: The results revealed that: a) there are five conserved residues in ODC and its paralogues: K115, A123, E138, L139 and K141; b) among these, L139 is the most critical one for the interaction with AZs, since its substitution decreases the affinity of the mutant protein towards AZs; c) all these conserved residues, with the exception of A123, are critical for ODC activity; d) substitutions of K115, E138 or L139 diminish the formation of ODC homodimers.CONCLUSIONS: These results reveal that four of the invariant residues of the AZBE region are strongly related to ODC functionality.GENERAL SIGNIFICANCE: This work helps to understand the interaction between ODC and AZ1, and describes various new residues involved in ODC activity, a key enzyme for cell growth and proliferation.

KW - Amino Acid Sequence

KW - Binding Sites

KW - Cell Line

KW - DNA Mutational Analysis

KW - HEK293 Cells

KW - Humans

KW - Models, Molecular

KW - Molecular Sequence Data

KW - Mutagenesis, Site-Directed

KW - Mutant Proteins

KW - Ornithine Decarboxylase

KW - Protein Binding

KW - Protein Conformation

KW - Proteins

U2 - 10.1016/j.bbagen.2013.07.003

DO - 10.1016/j.bbagen.2013.07.003

M3 - Journal article

C2 - 23872168

SN - 0304-419X

VL - 1830

SP - 5157

EP - 5165

JO - Biochimica et Biophysica Acta - Reviews on Cancer

JF - Biochimica et Biophysica Acta - Reviews on Cancer

IS - 11

ER -

Mutational analysis of the antizyme-binding element reveals critical residues for the function of ornithine decarboxylase

Abstract

Keywords

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