Substrate recognition and processing by a Walker B mutant of the human mitochondrial AAA+ protein CLPX

Lowth, Bradley R.; Kirstein, Janine; Saiyed, Tamanna; Brötz‐Oesterhelt, Heike; Morimoto, Richard I.; Truscott, Kaye N.; Dougan, David A.

doi:10.1016/j.jsb.2012.06.001

Cited by 41 publications

(60 citation statements)

References 57 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, it is also important to note that recombinant ClpXP, which consists of mouse CLPX and human CLPP, is able to digest the general ClpXP substrate "casein," confirming that mammalian ClpXP is an active protease. Indeed, other groups have previously reported on the proteolytic ability of human ClpXP using casein as a substrate (15,25), although the specific substrate used by human ClpXP remains unclear. Thus, based on our results, we hypothesize that ClpXP can recognize and modify ALAS1 for degradation under special conditions, such as in the presence of heme, as in our assay conditions.…”

Section: Discussionmentioning

confidence: 99%

“…Such experiments are ongoing projects in our laboratory, and we are actively attempting to express and purify these recombinant proteins to create an in vitro assay system. Although a procedure for purifying recombinant active human ClpXP protein has already been reported (25), a method for purifying recombinant human ALAS1 protein has not yet been established. Indeed, human ALAS1 could not be purified using the same methods that we used to purify recombinant ALAS2 (26,27) because the recombinant ALAS1 protein degraded during purification (data not shown).…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Novel Mechanisms for Heme-dependent Degradation of ALAS1 Protein as a Component of Negative Feedback Regulation of Heme Biosynthesis

Kubota

Nomura

Katoh

et al. 2016

Journal of Biological Chemistry

View full text Add to dashboard Cite

In eukaryotic cells, heme production is tightly controlled by heme itself through negative feedback-mediated regulation of nonspecific 5-aminolevulinate synthase (ALAS1), which is a rate-limiting enzyme for heme biosynthesis. However, the mechanism driving the heme-dependent degradation of the ALAS1 protein in mitochondria is largely unknown. In the current study, we provide evidence that the mitochondrial ATP-dependent protease ClpXP, which is a heteromultimer of CLPX and CLPP, is involved in the heme-dependent degradation of ALAS1 in mitochondria. We found that ALAS1 forms a complex with ClpXP in a heme-dependent manner and that siRNA-mediated suppression of either CLPX or CLPP expression induced ALAS1 accumulation in the HepG2 human hepatic cell line. We also found that a specific heme-binding motif on ALAS1, located at the N-terminal end of the mature protein, is required for the heme-dependent formation of this protein complex. Moreover, hemin-mediated oxidative modification of ALAS1 resulted in the recruitment of LONP1, another ATP-dependent protease in the mitochondrial matrix, into the ALAS1 protein complex. Notably, the heme-binding site in the N-terminal region of the mature ALAS1 protein is also necessary for the heme-dependent oxidation of ALAS1. These results suggest that ALAS1 undergoes a conformational change following the association of heme to the heme-binding motif on this protein. This change in the structure of ALAS1 may enhance the formation of complexes between ALAS1 and ATP-dependent proteases in the mitochondria, thereby accelerating the degradation of ALAS1 protein to maintain appropriate intracellular heme levels.Heme is an essential molecule to almost all organisms. Heme functions as a prosthetic group on several types of proteins, including cytochromes, catalases, hemoglobin, and myoglobin. Moreover, it has been reported that heme is also involved in numerous regulatory systems in mammals (1), including those that govern transcription (2), translation (3), microRNA processing (4), and the circadian rhythm (5). Excess quantities of heme or heme precursors result in the generation of reactive oxygen species, causing oxidative stress in cells (6). Thus, the production of heme and heme precursors must be tightly regulated. This regulation occurs through the precise control of the intracellular expression of nonspecific 5-aminolevulinatye synthases (ALAS-N or ALAS1). ALAS1 is the first and the ratelimiting enzyme of the heme biosynthetic pathway in mammalian cells, except for in erythroid cells, in which erythroid-specific 5-aminolevulinate synthase (ALAS-E or ALAS2) regulates the first step of heme biosynthesis (7). Although ALAS2 expression increases during erythroid differentiation, ALAS1 expression is suppressed by heme at the transcriptional, translational, and post-translational levels (8). ALAS1 and ALAS2 are encoded by independent genes (9); however, they both contain a conserved amino acid sequence called the heme regulatory motif (HRM), 2 which is involved in heme-dependent inhib...

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Novel Mechanisms for Heme-dependent Degradation of ALAS1 Protein as a Component of Negative Feedback Regulation of Heme Biosynthesis

Kubota

Nomura

Katoh

et al. 2016

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Protein concentrations refer to the protomer and were determined using Protein Assay (Bio‐Rad, Hercules, CA, USA) with bovine serum albumin (Thermo Scientific, Rockford, IL, USA) as a standard. Human CLPX was expressed and purified as described previously (24), except that affinity chromatography was performed under gravity flow using Ni‐NTA agarose. In addition, an extra 10 column volumes of binding buffer containing 80 mM imidazole was used prior to the elution step.…”

Section: Methodsmentioning

confidence: 99%

“…Cross‐linking of SDH5 (10 μM) was performed using 0.05% (v/v) glutaraldehyde, in 50 mM HEPES–KOH (pH 8), 150 mM NaCl, 0.025% (v/v) Triton X‐100, and 20 mM MgCl 2 (with or without 1 mM DTT, as indicated), as described previously (24), and separated by Tris‐Tricine sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE).…”

Section: Methodsmentioning

confidence: 99%

Mitochondrial matrix proteostasis is linked to hereditary paraganglioma: LON‐mediated turnover of the human flavinylation factor SDH5 is regulated by its interaction with SDHA

et al. 2014

Self Cite

View full text Add to dashboard Cite

Mutations in succinate dehydrogenase (SDH) subunits and assembly factors cause a range of clinical conditions. One such condition, hereditary paraganglioma 2 (PGL2), is caused by a G78R mutation in the assembly factor SDH5. Although SDH5(G78R) is deficient in its ability to promote SDHA flavinylation, it has remained unclear whether impairment to its import, structure, or stability contributes to its loss of function. Using import-chase analysis in human mitochondria isolated from HeLa cells, we found that the import and maturation of human SDH5(G78R) was normal, while its stability was reduced significantly, with ~25% of the protein remaining after 180 min compared to ~85% for the wild-type protein. Notably, the metabolic stability of SDH5(G78R) was restored to wild-type levels by depleting mitochondrial LON (LONM). Degradation of SDH5(G78R) by LONM was confirmed in vitro; however, in contrast to the in organello analysis, wild-type SDH5 was also rapidly degraded by LONM. SDH5 instability was confirmed in SDHA-depleted mitochondria. Blue native PAGE showed that imported SDH5(G78R) formed a transient complex with SDHA; however, this complex was stabilized in LONM depleted mitochondria. These data demonstrate that SDH5 is protected from LONM-mediated degradation in mitochondria by its stable interaction with SDHA, a state that is dysregulated in PGL2.

show abstract

“…Interestingly, in the case of B. subtilis ClpP, ADEP not only triggers opening of the pore, but also triggers oligomerization of ClpP from free 'inactive' monomers to 'active' tetradecamers [Kirstein et al, 2009a], a step that is normally controlled by the cognate unfoldase, ClpC [Kirstein et al, 2006]. Similarly, ADEP activation of human ClpP for unregulated degradation [Lowth et al, 2012] is also likely to result from assembly of the ClpP tetradecamer, a process that normally requires the assistance of human ClpX [Kang et al, 2005]. Indeed, this activation also appears to be a competitive inhibitor of unfoldase binding to ClpP, preventing the regulated degradation of substrates that would normally be delivered to ClpP by the unfoldase component [Kirstein et al, 2006].…”

Section: Clpp As a Novel Antibiotic Targetmentioning

confidence: 99%

Control of Protein Function through Regulated Protein Degradation: Biotechnological and Biomedical Applications

Nagpal¹,

Tan²,

Truscott³

et al. 2013

Microb Physiol

Self Cite

View full text Add to dashboard Cite

Targeted protein degradation is crucial for the correct function and maintenance of a cell. In bacteria, this process is largely performed by a handful of ATP-dependent machines, which generally consist of two components - an unfoldase and a peptidase. In some cases, however, substrate recognition by the protease may be regulated by specialized delivery factors (known as adaptor proteins). Our detailed understanding of how these machines are regulated to prevent uncontrolled degradation within a cell has permitted the identification of novel antimicrobials that dysregulate these machines, as well as the development of tunable degradation systems that have applications in biotechnology. Here, we focus on the physiological role of the ClpP peptidase in bacteria, its role as a novel antibiotic target and the use of protein degradation as a biotechnological approach to artificially control the expression levels of a protein of interest.

show abstract

Substrate recognition and processing by a Walker B mutant of the human mitochondrial AAA+ protein CLPX

Cited by 41 publications

References 57 publications

Novel Mechanisms for Heme-dependent Degradation of ALAS1 Protein as a Component of Negative Feedback Regulation of Heme Biosynthesis

Novel Mechanisms for Heme-dependent Degradation of ALAS1 Protein as a Component of Negative Feedback Regulation of Heme Biosynthesis

Mitochondrial matrix proteostasis is linked to hereditary paraganglioma: LON‐mediated turnover of the human flavinylation factor SDH5 is regulated by its interaction with SDHA

Control of Protein Function through Regulated Protein Degradation: Biotechnological and Biomedical Applications

Contact Info

Product

Resources

About