However, the impact of chemotherapy on ClpXP expression and if expression of ClpXP changes at relapse need to be addressed. In addition, it will be important to understand the mechanism of resistance to ClpP inhibitors and activators and identify the strategies to overcome them. novel therapeutic approach for malignancy. inner membrane, intermembrane space, mitochondrial Ca2+ uniporter, phosphatidylethanolamine. For example, OMA1 (Metalloendopeptidase OMA1) is usually a processing peptidase located in the mitochondrial inner membrane and intermembrane space. OMA1 cleaves the inner mitochondrial protein OPA1(Dynamin-like 120?kDa protein) to regulate mitochondrial dynamics. Upon loss of mitochondrial membrane potential, OMA1 cleaves OPA1, resulting in OPA1 inactivation and decreased mitochondrial fusion17. High temperature requirement peptidase 2 (HTRA2) (also called OMI) is usually another protease in the mitochondrial intermembrane space, which plays a critical role in maintaining mitochondrial cristae structure by interacting and degrading its substrate in the mitochondrial intermembrane space bridging (MIB) complex, inner membrane mitochondrial protein (IMMT)18. HTRA2 is also released into the cytoplasm during apoptosis where it binds and inhibits Baculoviral IAP Repeat Containing (BIRC) proteins (also called inhibitor of apoptosis proteins, IAPs), leading to an increase in caspase activity19,20. Among these proteases, the ATP-dependent proteases are active in all mitochondrial compartments and represent core components of the mitochondrial proteolytic system performing both quality control and regulatory functions13,21. The users of this family are the Lon protease localized to the mitochondrial matrix, the Efonidipine hydrochloride homologous i-AAA, and m-AAA proteases localized to the inner mitochondrial membrane, and the ClpXP complex localized to the mitochondrial matrix (the serine protease ClpP and the AAA+ATPase ClpX) (Fig. ?(Fig.11)10,22C24. These proteases degrade inner membrane proteins including subunits of respiratory complexes and translocases, as well as proteins within the matrix, intermembrane space, and outer membrane. Open in a separate window Fig. 1 Schematic representation of ATP-dependent proteases.Mammalian mitochondria contains four proteases of the AAA+ superfamily to modulate protein quality control. The Lon protease 1, and ClpXP complex in the matrix and the i-AAA, m-AAA proteases in IM. OMM outer mitochondrial membrane, IMS intermembrane space, IMM inner mitochondrial membrane. This review focuses on the mitochondrial ClpP protease and its regulatory subunit ClpX (referred to as the ClpXP complex) that reside in the mitochondrial matrix. The reader is referred to other excellent reviews discussing other mitochondrial proteases13,15,25,26. We will discuss the molecular characteristics and biological roles of mitochondrial Efonidipine hydrochloride ClpXP and potential therapeutic strategies to target this protease for cancer therapy. Mitochondrial ClpP ClpP is located in the mitochondrial matrix of a diverse range of eukaryotes from C. elegans to human, although homologs are not found in yeast. In humans, ClpP is encoded on chromosome 1927. Once translated in the cytosol, it is directed to the mitochondrial matrix by a 56-residue N-terminal targeting sequence. This sequence is cleaved upon protein maturation in the mitochondrial matrix1. Mature human ClpP (hClpP) has 277 amino acids and shares high sequence similarity (71%) and identity (56%) with ClpP. However, mammalian ClpP, including the human homolog, has an extended 28 residues at its C-terminus (Fig. ?(Fig.22)28,29. This C-terminal extension forms an unstructured flexible loop which extends out of the surface of the oligomer. The role of this sequence is not well understood, but seems necessary for the stability of the protease, the assembly of the functional ClpP heptamer, and its affinity for its chaperone ClpX28. Open in a separate window Fig. 2 Structure and interaction of ClpP and ClpX.a Domain organization of ClpX (top) and ClpP (bottom) with catalytic residues of Ser153, His178, and Asp227. MTS mitochondrial targeting sequence, ZBD zinc-binding domain; AAA+, ATPases associated with diverse cellular activities. b Schematic representation of the ClpX and ClpP interaction and proteolytic cycle. c Top view of hexameric ClpX (top) and heptameric ClpP (bottom). Much of our understanding of the structure and function of human ClpP has been derived from studies of the bacterial homolog and the crystal structure of human mitochondrial at 2.1??.ONC201 is the first-in-class imipridones that is in clinical trials for multiple advanced cancer. OMA1 (Metalloendopeptidase OMA1) is a processing peptidase located in the mitochondrial inner membrane and intermembrane space. OMA1 cleaves the inner mitochondrial protein OPA1(Dynamin-like FZD3 120?kDa protein) to regulate mitochondrial dynamics. Upon loss of mitochondrial membrane potential, OMA1 cleaves OPA1, resulting in OPA1 inactivation and decreased mitochondrial fusion17. High temperature requirement peptidase 2 (HTRA2) (also called OMI) is another protease in the mitochondrial intermembrane space, which plays a critical role in maintaining mitochondrial cristae structure by interacting and degrading its substrate in the mitochondrial intermembrane space bridging (MIB) complex, inner membrane mitochondrial protein (IMMT)18. HTRA2 is also released into the cytoplasm during apoptosis where it binds and inhibits Baculoviral IAP Repeat Containing (BIRC) proteins (also called inhibitor of apoptosis proteins, IAPs), leading to an increase in caspase activity19,20. Among these proteases, the ATP-dependent proteases are active in all mitochondrial compartments and represent core components of the mitochondrial proteolytic system performing both quality control and regulatory functions13,21. The members of this family are the Lon protease localized to the mitochondrial matrix, the homologous i-AAA, and m-AAA proteases localized to the inner mitochondrial membrane, and the ClpXP complex localized to the mitochondrial matrix (the serine protease ClpP and the AAA+ATPase ClpX) (Fig. ?(Fig.11)10,22C24. These proteases degrade inner membrane proteins including subunits of respiratory complexes and translocases, as well as proteins within the matrix, intermembrane space, and outer membrane. Open in a separate window Fig. 1 Schematic Efonidipine hydrochloride representation of ATP-dependent proteases.Mammalian mitochondria contains four proteases of the AAA+ superfamily to modulate protein quality control. The Lon protease 1, and ClpXP complex in the matrix and the i-AAA, m-AAA proteases in IM. OMM outer mitochondrial membrane, IMS intermembrane space, IMM inner mitochondrial membrane. This review focuses on the mitochondrial ClpP protease and its regulatory subunit ClpX (referred to as the ClpXP complex) that reside in the mitochondrial matrix. The reader is referred to other excellent reviews discussing other mitochondrial proteases13,15,25,26. We will discuss the molecular characteristics and biological roles of mitochondrial ClpXP and potential therapeutic strategies to target this protease for cancer therapy. Mitochondrial ClpP ClpP is located in the mitochondrial matrix of a diverse range of eukaryotes from C. elegans to human, although homologs are not found in yeast. In humans, ClpP is encoded on chromosome 1927. Once translated in the cytosol, it is directed to the mitochondrial matrix by a 56-residue N-terminal targeting sequence. This sequence is cleaved upon protein maturation in the mitochondrial matrix1. Mature human ClpP (hClpP) has 277 Efonidipine hydrochloride amino acids and shares high sequence similarity (71%) and identity (56%) with ClpP. However, mammalian ClpP, including the human homolog, has an extended 28 residues at its C-terminus (Fig. ?(Fig.22)28,29. This C-terminal extension forms an unstructured flexible loop which extends out of the surface of the oligomer. The role of this sequence is not well understood, but seems necessary for the stability of the protease, the assembly of the functional ClpP heptamer, and its affinity for its chaperone ClpX28. Open in a separate window Fig. 2 Structure and interaction of ClpP and ClpX.a Domain organization of ClpX (top) and ClpP (bottom) with catalytic residues of Ser153, His178, and Asp227. MTS mitochondrial targeting sequence, ZBD zinc-binding domain; AAA+, ATPases associated with diverse cellular activities. b Schematic representation of the ClpX and ClpP interaction and proteolytic cycle. c Top view of hexameric ClpX (top) and heptameric ClpP (bottom). Much of our understanding of the structure and function of human ClpP has been derived from studies of the bacterial homolog and the crystal structure of human mitochondrial at 2.1?? (PDB: 1TG6)28,30. Similar to the bacterial enzyme, functional mitochondrial ClpP is a large cylindrical tetradecamer of two identical stable heptameric rings enclosing a large aqueous chamber. Each ClpP monomer has a compact body, called the head region, and a unique expanded / unit called the handle region. Heads of seven monomers.