טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentLevy Shiri
SubjectLACTB2,a Metallo-Beta-Lactamase Protein,is a Novel
Endoribonuclease Located in the Mitochondria
DepartmentDepartment of Biology
Supervisor Professor Gadi Schuster
Full Thesis textFull thesis text - English Version


Abstract

Mitochondria are critical to numerous cellular processes, including energy metabolism, cell apoptosis, signaling, and metabolic pathways. Therefore, unraveling the mechanism of RNA degradation pathway in human mitochondria is of great importance. A candidate family of proteins responsible for nucleic acid degradation found in all three domains of life is the Metallo-β-lactamase (MBL) superfamily. Among the MBL proteins known to degrade RNA are: CPSF-73, RNase J, and tRNase Z. Based on these findings we hypothesized MBL proteins may be responsible for RNA turnover in mammalian mitochondria.

In this study, we discovered a new role for a MBL protein found in mitochondrial mouse proteomics and containing all five motifs of MBL. Our stated hypothesis was that the annotation free, Metallo beta Lactamase like protein 2 (LACTB2) was present in the mitochondria and could be the “missing” ribonuclease. The gene of LACTB2 is encoded in the nucleus and contains a transit peptide signal shuttling to the mitochondria. Based on in silico structural modeling, LACTB2 revealed a substantial similarity of the superimposed active site to the MBL ribonuclease CPSF-73. The results disclosed the evidential localization of endogenous LACTB2 to human mitochondria as a soluble monomeric protein. Overexpression and purification of recombinant human LACTB2 revealed, for the first time, the robust endoribonuclease activity of LACTB2 on ssRNA with preference to A/U rich sequences. We also constructed defective mutants that damaged the wild type optimal activity and located a new RNA binding groove on the protein. A knocked-down of LACTB2 in cultured cells caused a moderate but significant transcript accumulation of almost all mitochondrial transcripts and its overexpression led to the opposite observation. Furthermore, manipulation of LACTB2 expressions resulted in cellular morphological deformation and cell death. Together, the study discovered the involvement of LACTB2, a MBL protein, as an endoribonuclease essential for mitochondria functioning in human cells.