Archives

  • 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • br Materials and method br Result and discussion br

    2022-11-04


    Materials and method
    Result and discussion
    Conclusion
    Acknowledgments This work is partly supported by the University Research Committee (URC) of the Senate of The Federal University of Technology, Akure. The authors thanked IFS for the funds (F/4449 1F and F/4449-2F) used to purchase the equipments.
    Specifications table [please fill in right-hand column of the table below] Value of the data [describe in 3–5 bulleted points why this data is of value to the scientific community]
    Data, experimental design, materials and methods
    Acknowledgments Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award numbers R01GM065546 and R01GM093123. Part of the research was performed at the Advanced Light Source, which is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. The Advanced Light Source is a national user facility operated by Lawrence Berkeley National Laboratory on behalf of the Department of Energy, Office of Basic Energy Sciences, through the Integrated Diffraction Analysis Technologies (IDAT) program, supported by DOE Office of Biological and Environmental Research. Additional support comes from the National Institute of Health Project MINOS (R01GM105404).
    Introduction The lysine catabolic enzyme aldehyde dehydrogenase 7A1 (ALDH7A1) catalyzes the NAD+-dependent oxidation of α-aminoadipate semialdehyde (AASAL) to α-aminoadipate (Fig. 1A). Misregulation of ALDH7A1 and other members of the ALDH superfamily has been observed in cancer stem GSK1904529A and linked to cancer stem cell metastasis [1], [2]. Defects in ALDH function are also associated with numerous inherited metabolic disorders. Notably, certain mutations in the ALDH7A1 gene cause pyridoxine-dependent epilepsy (PDE), a recessive, inherited disease that results in seizures [3], [4]. According to the Human Gene Mutation Database [5], 47 disease-related mutations, both missense and nonsense, have been mapped to the ALDH7A1 gene. 39 of the 47 are linked to PDE. A decade ago, Mills et al. proposed that the pathology of PDE involves inactivation of the enzyme cofactor pyridoxal 5'-phosphate (PLP) [3]. Specifically, diminished ALDH7A1 activity leads to increased levels of AASAL and its cyclized form, Δ1-piperideine-6-carboxylic acid (P6C, Fig. 1). The elevated P6C concentration promotes covalent inactivation of PLP via the Knoevenagel condensation (Fig. 1B) [3]. Given that roughly 4% of all enzymes employ PLP as a cofactor [6], reduced PLP levels could have a variety of adverse physiological consequences. Increased incidence of seizures is believed to be one of these [3]. Elucidating the functional impact of ALDH7A1 mutations is key GSK1904529A to understanding the molecular basis of PDE. High-resolution structures of ALDH7A1 complexed with NAD+ and α-aminoadipate have revealed the protein fold, the apparent quaternary structure, and the residues involved in substrate- and cofactor binding [7], [8], [9]. Despite the availability of structural information for the wild-type enzyme, it is difficult to predict the impact of ALDH7A1 point-mutations on catalytic activity and three-dimensional structure, particularly those remote from the active site. In this context, it is notable that approximately 12% of all disease-related mutations are located in protein-protein interfaces of oligomeric proteins [10]. Indeed, nearly 20% of the known PDE-associated missense mutations reside in, or near, the interfaces of the ALDH7A1 tetramer: P78L [11], G83E [12], A129P [11], G137V [12], G138V [13], A149E [11], G255D [14], and G263E [15]. To better understand the impact of these mutations, we expressed recombinant versions of the eight aforementioned PDE-related mutant enzymes in Escherichia coli and compared their self-association behavior to that of wild-type ALDH7A1. The wild-type protein forms a catalytically active, high-affinity dimer that can further associate, at higher concentrations, to form a tetramer. Two of the variant proteins did not express appreciable amounts of soluble protein (P78L, G83E), precluding detailed analysis. The other six, although soluble, are catalytically inactive and display aberrant self-association behavior. Specifically, both monomeric and dimeric states are populated at low concentration, and the dimers are apparently incapable of forming the tetrameric dimer-of-dimers observed for wild-type ALDH7A1. These results suggest that the disease-related mutations result in the production of misfolded monomers incapable of associating into active oligomers.