Molecular interactions of the human PEX1/PEX6 AAA+ ATPase complex and in vivo mRNA editing of the PEX1-G843D mutation

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Zitierfähiger Link (URI): http://hdl.handle.net/10900/153614
http://nbn-resolving.de/urn:nbn:de:bsz:21-dspace-1536149
http://dx.doi.org/10.15496/publikation-94953
Dokumentart: Dissertation
Erscheinungsdatum: 2024-05-23
Sprache: Englisch
Fakultät: 7 Mathematisch-Naturwissenschaftliche Fakultät
Fachbereich: Biochemie
Gutachter: Dodt, Gabriele (Prof. Dr.)
Tag der mündl. Prüfung: 2024-05-16
DDC-Klassifikation: 500 - Naturwissenschaften
610 - Medizin, Gesundheit
Freie Schlagwörter: Biochemie
Peroxisomen
Proteine
PEX1
PEX6
RNA editing
SNAP-ADAR
RESTORE
peroxisomes
proteins
PEX1
PEX6
RNA editing
SNAP-ADAR
RESTORE
Biochemistry
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Abstract:

Peroxisomes, ubiquitous and highly dynamic organelles in eukaryotic cells, are crucial for human health and development. They are needed for oxidative metabolic processes, including the breakdown of fatty acids and the regulation of the cellular redox balance. These functions are conserved across species and rely on proteins encoded by nuclear DNA, which are subsequently targeted into the peroxisomal matrix after translation. Specialized proteins, known as peroxins or PEX proteins are involved in peroxisome biogenesis, ensuring a continuous influx of peroxisomal proteins. Among these, PEX1 and PEX6, members of the type II AAA+ ATPase family, are core components of the receptor export module (REM) and essential for peroxisomal matrix protein import. Together, PEX1 and PEX6 form a heterohexameric ATPase complex whose activity facilitates the release of the receptor PEX5, ensuring a steady supply of receptors for subsequent protein import. Mutations in either of the mammalian PEX1 and PEX6 proteins disrupt peroxisome biogenesis, leading to severe peroxisomal disorders and early death. A missense mutation PEX1-G843D is one of the most common causes of a milder variant of peroxisomal disorders. Some chemical and pharmacological chaperones such as betaine and diosmetin have been found to improve peroxisomal matrix protein import by restoring the PEX1-G843D function. The task of this thesis was to study the interactions of these drugs with the human PEX1/PEX6 complex and the correction of the PEX1-G843D mutation at the mRNA level. Recombinant human PEX1 and PEX6 were expressed in HEK293TT cells for protein production and the complex was purified in sequential steps. The purification is based on the knowledge that a heterohexameric AAA+ ATPase complex is formed by three subunits of PEX1 and three subunits of PEX6 proteins. A two-step purification of the complex using a C-terminal His6 tag in PEX1 and an N-terminal 2xFLAG or Strep-tag II in PEX6 was performed to get a pure complex. The purified protein complex was analyzed by microscale thermophoresis (MST) to examine binding affinity of PEX1 with the pharmacological compounds, betaine and diosmetin. The KD values for betaine were in the millimolar range while that for diosmetin fall in the micromolar range indicating stronger interaction of diosmetin with the PEX1/PEX6 complex. In collaboration with the lab of Prof. Dr. Thorsten Stafforst, the in-vivo correction of the PEX1-G843D (c.2528G>A) mRNA was studied. One approach for this was the use of ectopically expressed SNAP-ADAR proteins and transfected guide RNAs. In another approach, endogenous ADAR proteins were utilized in combination with transfected antisense nucleotides (ASOs). In both methods, the guide RNAs/ASOs specific to the point mutation site were able to partially correct the mutation, thus forming the wildtype PEX1 protein. The level of mRNA correction was calculated after Sanger sequencing and the restoration of matrix protein import was confirmed by fluorescence microscopy.

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