m‐AAA protease‐driven membrane dislocation allows intramembrane cleavage by rhomboid in mitochondria
TL;DR: Findings reveal for the first time a non‐proteolytic function of the m‐AAA protease during mitochondrial biogenesis and rationalise the requirement of a preceding step for intramembrane cleavage by rhomboid.
Abstract: Maturation of cytochrome c peroxidase (Ccp1) in mitochondria occurs by the subsequent action of two conserved proteases in the inner membrane: the m-AAA protease, an ATP-dependent protease degrading misfolded proteins and mediating protein processing, and the rhomboid protease Pcp1, an intramembrane cleaving peptidase. Neither the determinants preventing complete proteolysis of certain substrates by the m-AAA protease, nor the obligatory requirement of the m-AAA protease for rhomboid cleavage is currently understood. Here, we describe an intimate and unexpected functional interplay of both proteases. The m-AAA protease mediates the ATP-dependent membrane dislocation of Ccp1 independent of its proteolytic activity. It thereby ensures the correct positioning of Ccp1 within the membrane bilayer allowing intramembrane cleavage by rhomboid. Decreasing the hydrophobicity of the Ccp1 transmembrane segment facilitates its dislocation from the membrane and renders rhomboid cleavage m-AAA protease-independent. These findings reveal for the first time a non-proteolytic function of the m-AAA protease during mitochondrial biogenesis and rationalise the requirement of a preceding step for intramembrane cleavage by rhomboid.
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Dissertation•
01 Jan 2010
TL;DR: It is shown here that the division protein DivIC can protect FtsL against RasP cleavage, and it could be shown that a recognition motif within the cytosolic N-terminal domain of FTSL is essential for degradation by RasP.
Abstract: Cell division in Bacillus subtilis is a highly regulated process. Division takes place precisely at midcell resulting in two equally sized daughter cells. It is important that the divisome is disassembled after division is completed and does not directly re-assemble. Otherwise a new cycle of division is initiated close to the new formed cell pole, resulting in non viable, DNA-less mini cells. This study analyses the role of the intramembrane protease RasP in preventing divisome re-assembly. RasP degrades the late cell division protein FtsL in vivo. We tried to establish an in vitro assay to investigate this proteolysis. Both proteins were purified, but RasP seems to be partly unfolded after solubilisation. Therefore a heterolous co-expression system in E. coli was established instead. It is shown here that the division protein DivIC can protect FtsL against RasP cleavage. This stabilisation is achieved by inhibiting substrate recognition. It could be shown that a recognition motif within the cytosolic N-terminal domain of FtsL is essential for degradation by RasP. FtsL and DivIC tightly interact with each other. Direct interaction of the N-terminal domains blocks accessibility of the FtsL substrate recognition motif. Hence, as long as FtsL is incorporated in the divisome, RasP cleavage is impaired. After the division complex disassembles RasP is able to degrade FtsL. This cleavage removes FtsL from the membrane. Using fluorescence microscopy it was shown that the cytosolic cleavage product is then rapidly degraded by general proteolysis. A complex network of the late division proteins FtsL, DivIC and DivIB most likely provides a scaffold for cytokinesis. Since these proteins are strongly interdependent on each other for correct assembly, complete degradation of FtsL should efficiently prevent re-assembly of the divisome close to the new cell pole.
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Cites background from "m‐AAA protease‐driven membrane disl..."
..., 2001] and the full-length yeast protein cytochrome c peroxidase (Ccp1) is cleaved by the the rhomboid protease Pcp1 [Tatsuta et al., 2007]....
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Dissertation•
20 Mar 2009
01 Jan 2012
TL;DR: This work shows that PfSPP is a druggable enzyme and that parasites are extremely vulnerable to its inhibition, and evidence is presented that suggests this enzyme may play an important role in the parasite's endoplasmic reticulum stress-response.
Abstract: Plasmodium falciparum is a protozoan parasite and the causative agent of malaria, which kills upwards of 1 million people annually. With the increasing prevalence of drug-resistant parasites, considerable interest now exists in the identification of new biological targets for the development of new malaria chemotherapeutics. However, given the genetic intractability inherent in studying P. falciparum, it is imperative that novel approaches be developed if we are to understand the role of essential enzymes. My work presented here focuses on the development and use of chemical tools to study malarial proteases, a class of enzymes that have been shown to play essential roles throughout the parasite lifecycle, but the majority of which though are still uncharacterized. In Chapter 2 I develop a novel set of activity-based probes (ABPs) based on the natural product metallo-aminopeptidase (MAP) inhibitor bestatin. I show the bestatin-based ABP allows the functional characterization of MAP activity within a complex proteome. In Chapter 3, I utilize an extended library of bestatin-based ABPs to define the function of two essential malarial MAPs, PfA-M1 and Pf-LAP. I find that PfA-M1 is necessary in the proteolysis of hemoglobin and that lethal inhibition starves parasites of amino acids. I also show that Pf-LAP has a role other than hemoglobin digestion, as parasites are susceptible to its inhibition prior to the onset of this process. In Chapter 4, I use a suite of specific small molecules to validate the P. falciparum signal peptide peptidase (PfSPP) as a drug target. This work shows that PfSPP is a druggable enzyme and that parasites are extremely vulnerable to its inhibition. Evidence is also presented that suggests this enzyme may play an important role in the parasite's endoplasmic reticulum stress-response. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Pharmacology First Advisor Doron C. Greenbaum
TL;DR: In this article , the degradative pathways for mutant forms of three mitochondrial matrix proteins (mas1-1HA, mas2-11HA, and tim44-8HA) in Saccharomyces cerevisiae were assessed.
References
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TL;DR: The structural organization of AAA+ proteins, the conformational changes they undergo, the range of different reactions they catalyse, and the diseases associated with their dysfunction are reviewed.
Abstract: The AAA+ (ATPases associated with various cellular activities) family is a large and functionally diverse group of enzymes that are able to induce conformational changes in a wide range of substrate proteins. The family's defining feature is a structurally conserved ATPase domain that assembles into oligomeric rings and undergoes conformational changes during cycles of nucleotide binding and hydrolysis. Here, we review the structural organization of AAA+ proteins, the conformational changes they undergo, the range of different reactions they catalyse, and the diseases associated with their dysfunction.
1,137 citations
"m‐AAA protease‐driven membrane disl..." refers background in this paper
...This loop contains an aromatichydrophobic-glycine motif (FVG in Yta10 and Yta12), which is conserved within AAAþ proteins (Figure 7A) and has been linked to substrate translocation in other AAA proteins (Sauer et al, 2004; Hanson and Whiteheart, 2005)....
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...At the same time, they conduct the quality surveillance of cellular proteins and degrade misfolded proteins to peptides (Sauer et al, 2004; Ciechanover, 2005; Hanson and Whiteheart, 2005)....
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...Conserved residues in the pore loop are essential for Ccp1 processing Most AAAþ proteins form hexameric ring structures that allow substrates to enter the central channel (Sauer et al, 2004; Hanson and Whiteheart, 2005)....
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...This loop contains an aromatichydrophobic-glycine motif (FVG in Yta10 and Yta12), which is conserved within AAA proteins (Figure 7A) and has been linked to substrate translocation in other AAA proteins (Sauer et al, 2004; Hanson and Whiteheart, 2005)....
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...Ccp1 processing Most AAA proteins form hexameric ring structures that allow substrates to enter the central channel (Sauer et al, 2004; Hanson and Whiteheart, 2005)....
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TL;DR: In this paper, the ubiquitin-proteasome system resolved the enigma of how cellular proteins are degraded in the lysosome and showed that non-lysosomal pathways have an important role in intracellular proteolysis, although their identity and mechanisms of action remained obscure.
Abstract: How the genetic code is translated into proteins was a key focus of biological research before the 1980s, but how these proteins are degraded remained a neglected area With the discovery of the lysosome, it was suggested that cellular proteins are degraded in this organelle However, several independent lines of experimental evidence strongly indicated that non-lysosomal pathways have an important role in intracellular proteolysis, although their identity and mechanisms of action remained obscure The discovery of the ubiquitin–proteasome system resolved this enigma
1,009 citations
TL;DR: M mammalian mitochondrial function and morphology is regulated through processing of OPA1 in a ΔΨ‐dependent manner through proteolytic cleavage of Mgm1, the yeast homolog of O PA1.
Abstract: The dynamin-like GTPase OPA1, a causal gene product of human dominant optic atrophy, functions in mitochondrial fusion and inner membrane remodeling. It has several splice variants and even a single variant is found as several processed forms, although their functional significance is unknown. In yeast, mitochondrial rhomboid protease regulates mitochondrial function and morphology through proteolytic cleavage of Mgm1, the yeast homolog of OPA1. We demonstrate that OPA1 variants are synthesized with a bipartite-type mitochondrial targeting sequence. During import, the matrix-targeting signal is removed and processed forms (L-isoforms) are anchored to the inner membrane in type I topology. L-isoforms undergo further processing in the matrix to produce S-isoforms. Knockdown of OPA1 induced mitochondrial fragmentation, whose network morphology was recovered by expression of L-isoform but not S-isoform, indicating that only L-isoform is fusion-competent. Dissipation of membrane potential, expression of m-AAA protease paraplegin, or induction of apoptosis stimulated this processing along with the mitochondrial fragmentation. Thus, mammalian mitochondrial function and morphology is regulated through processing of OPA1 in a ΔΨ-dependent manner.
810 citations
"m‐AAA protease‐driven membrane disl..." refers background in this paper
...Of note, proteolytic processing of the Mgm1 homologue OPA1 in mammalian cells has recently been linked to an m-AAA protease (Ishihara et al, 2006)....
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TL;DR: The very large peptide bond ASP, -96 +/- 6 cal/mol/A2, profoundly affects the results of computational comparisons of protein stability which use ASPs derived from octanol-water partitioning data.
Abstract: Octanol-to-water solvation free energies of acetyl amino amides (Ac-X-amides) [Fauchere, J.L., & Pliska, V. (1983) Eur. J. Med. Chem. --Chim. Ther. 18,369] form the basis for computational comparisons of protein stabilities by means of the atomic solvation parameter formalism of Eisenberg and McLachlan [(1986) Nature 319, 199]. In order to explore this approach for more complex systems, we have determined by octanol-to-water partitioning the solvation energies of (1) the guest (X) side chains in the host-guest pentapeptides AcWL-X-LL, (2) the carboxy terminus of the pentapeptides, and (3) the peptide bonds of the homologous series of peptides AcWLm (m = 1-6). Solvation parameters were derived from the solvation energies using estimates of the solvent-accessible surface areas (ASA) obtained from hard-sphere Monte Carlo simulations. The measurements lead to a side chain solvation-energy scale for the pentapeptides and suggest the need for modifying the Asp, Glu, and Cys values of the "Fauchere-Pliska" solvation-energy scale fro the Ac-X-amides. We find that the unfavorable solvation energy of nonpolar residues can be calculated accurately by a solvation parameter of 22.8 +/- 0.8 cal/mol/A2, which agrees satisfactorily with the AC-X-amide data and thereby validates the Monte Carlo ASA results. Unlike the Ac-X-amide data, the apparent solvation energies of the uncharged polar residues are also largely unfavorable. This unexpected finding probably results, primarily, from differences in conformation and hydrogen bonding in octanol and buffer but may also be due to the additional flaking peptide bonds of the pentapeptides. The atomic solvation parameter (ASP) for the peptide bond is comparable to the ASP of the charged carboxy terminus which is an order of magnitude larger than the ASP of the uncharged polar side chains of the Ac-X-amides. The very large peptide bond ASP, -96 +/- 6 cal/mol/A2, profoundly affects the results of computational comparisons of protein stability which use ASPs derived from octanol-water partitioning data.
538 citations
"m‐AAA protease‐driven membrane disl..." refers methods in this paper
...We determined the hydrophobicity of this region using the membrane protein explorer (MPEx) programme, which is based on experimentally derived Wimley–White hydropathy scale (Wimley et al, 1996)....
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TL;DR: Exciting progress has been made in understanding how AAA(+) machines recognize specific proteins as targets and then carry out ATP-dependent dismantling of the tertiary and/or quaternary structure of these molecules during the processes of protein degradation and the disassembly of macromolecular complexes.
460 citations
"m‐AAA protease‐driven membrane disl..." refers background in this paper
...Conserved residues in the pore loop are essential for Ccp1 processing Most AAAþ proteins form hexameric ring structures that allow substrates to enter the central channel (Sauer et al, 2004; Hanson and Whiteheart, 2005)....
[...]
...At the same time, they conduct the quality surveillance of cellular proteins and degrade misfolded proteins to peptides (Sauer et al, 2004; Ciechanover, 2005; Hanson and Whiteheart, 2005)....
[...]
...ATP-dependent unfolding of substrates allows substrate entry into barrel-like proteolytic chambers and results in complete degradation (Sauer et al, 2004)....
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...This loop contains an aromatichydrophobic-glycine motif (FVG in Yta10 and Yta12), which is conserved within AAAþ proteins (Figure 7A) and has been linked to substrate translocation in other AAA proteins (Sauer et al, 2004; Hanson and Whiteheart, 2005)....
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