Investigation into the Function of the Mitochondrial Intermembrane Space Protein ClpB
Creators: name: LaVallee, Jacquelyn Marie
Publisher: name: Brandeis University
Year: 2024
Subjects: Biochemistry, Proteins

description: Human caseinolytic peptidase B (CLPB) protein is an enzyme found in the intermembrane space (IMS) of the mitochondria but with a largely unknown function. It belongs to the AAA+ family of proteins that couples ATP hydrolysis to perform mechanical work. CLPB is thought to be an analog of the prokaryotic ClpB (caseinolytic peptidase B) protein and the lower eukaryotic yeast heat shock proteins (Hsp) 78 and Hsp104, each of which works to resolubilize proteins following stress events1–3. Although CLPB maintains an AAA+ domain capable of ATP hydrolysis and does demonstrate disaggregation activity2,3, a gained ankyrin repeat domain suggests a more specific role in client binding, potentially modulating function within the intermembrane space with greater specificity than other ClpB homologs. While CLPB may possess some disaggregase activity because aggregation phenotypes in the mitochondrial intermembrane space are unknown, the ankyrin domain suggests targeted protein disaggregation instead of the more generalized ClpB/Hsp disaggregation seen in bacteria and yeast. In addition, recent candidates for native CLPB clients have begun to emerge through several proteomic studies2,4,5; Optic atrophy 1 (OPA1) is a GTP-dependent dynamin-like protein that has become a prominent choice. Initial pull-down studies in whole-cell lysates implicate CLPB association with OPA14, but a more finely tuned assay is necessary to confirm this interaction. OPA1 plays a role in fission and fusion events in the inner mitochondrial membrane. Two isoforms of OPA1 exist, the membrane-bound, long-form, and the soluble short form which work in concert to mediate membrane fusion events6. There is overlap in the characteristic phenotypes seen in vivo in a CLPB knockout in human acute myeloid leukemia cells, where ablation of CLPB causes an imbalance in the long to short forms of OPA12,4. Each separately causes similar mitochondrial fragmentation and changes to cristae morphology4. This taken together suggests that CLPB may interact closely with OPA1, potentially modulating its membrane fusion responsibilities. OPA1 is a good candidate to test as a CLPB substrate initially because of the breadth of published literature describing its function. If OPA1 is a substrate of CLPB, previously conducted work will act as a baseline to describe how OPA1 levels and function change following modification of CLPB.
description: With little understanding of the function of CLPB and no known substrates, this presents a significant gap in our understanding of how CLPB modulates the IMS. By determining the mechanism of how CLPB interacts with substrate and determining specific substrates we can make impactful strides in understanding the phenotypic clinical outcomes that arise because of genotypic modifications to CLPB.
description: One aim of this research was to determine the functional role of the ankyrin repeat domain of CLPB and determine how CLPB modulates substrate in the mitochondrial IMS. Since specific substrate interacting partners have not been defined, a second aim was to determine if OPA1 is an interacting partner with CLPB and further define the biochemical interactions between CLPB and OPA1. Modeling the structure of CLPB will be useful towards understanding the domain structure and useful in determining possible biochemical interactions. Characterizing the physiological functions of CLPB will be the gateway to further defining interacting partners.