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. 2024 Jul 10:12:1423208.
doi: 10.3389/fcell.2024.1423208. eCollection 2024.

Mitochondrial inorganic polyphosphate is required to maintain proteostasis within the organelle

Renata T Da Costa  1 , Pedro Urquiza #  1 , Matheus M Perez #  1 , YunGuang Du  2 , Mei Li Khong  3   4 , Haiyan Zheng  5 , Mariona Guitart-Mampel  1 , Pia A Elustondo  6 , Ernest R Scoma  1 , Vedangi Hambardikar  1 , Beatrix Ueberheide  7 , Julian A Tanner  4   8   9 , Alejandro Cohen  6 , Evgeny V Pavlov  10 , Cole M Haynes  2 , Maria E Solesio  1
Affiliations

Mitochondrial inorganic polyphosphate is required to maintain proteostasis within the organelle

Renata T Da Costa et al. Front Cell Dev Biol. .

Abstract

The existing literature points towards the presence of robust mitochondrial mechanisms aimed at mitigating protein dyshomeostasis within the organelle. However, the precise molecular composition of these mechanisms remains unclear. Our data show that inorganic polyphosphate (polyP), a polymer well-conserved throughout evolution, is a component of these mechanisms. In mammals, mitochondria exhibit a significant abundance of polyP, and both our research and that of others have already highlighted its potent regulatory effect on bioenergetics. Given the intimate connection between energy metabolism and protein homeostasis, the involvement of polyP in proteostasis has also been demonstrated in several organisms. For example, polyP is a bacterial primordial chaperone, and its role in amyloidogenesis has already been established. Here, using mammalian models, our study reveals that the depletion of mitochondrial polyP leads to increased protein aggregation within the organelle, following stress exposure. Furthermore, mitochondrial polyP is able to bind to proteins, and these proteins differ under control and stress conditions. The depletion of mitochondrial polyP significantly affects the proteome under both control and stress conditions, while also exerting regulatory control over gene expression. Our findings suggest that mitochondrial polyP is a previously unrecognized, and potent component of mitochondrial proteostasis.

Keywords: mitochondria; mitochondrial inorganic polyphosphate; polyP; protein homeostasis; proteostasis.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Lack of mitochondrial polyP deleteriously affects protein homeostasis within the organelle. (A). Significant immunoblots showing the aggregation behavior of TUFM under control conditions (25°C) and after HS (30 min, 45°C). Protein content was normalized before separation of the soluble and insoluble mitochondrial fractions by centrifugation. Original uncropped membranes are presented in the Supplementary Material. At least three independent experiments were conducted. (B). IPA analysis of the proteomics data obtained from the soluble and insoluble mitochondrial protein fractions of the Wt and MitoPPX HEK293, both under control conditions and after HS. The main canonical pathways; and diseases and functions which are predicted to be affected based on the LC-MS/MS are represented in the graphs. Data is represented as the values obtained in MitoPPX vs. those obtained in Wt cells. Selection of the canonical pathways; and disease and functions were conducted among those with p-values ≤0.05, considering z-scores and relationship with mitochondrial physiology and protein homeostasis. Three independent sets of cells were used to obtained the proteomics data. Data sets obtained in the mass spectrometry experiments are included in Supplementary Table S1.
FIGURE 2
FIGURE 2
Mitochondrial polyP is able to bind proteins. This binding is affected by the presence of different stressors. (A) Coomassie-stained SDS-PAGE showing the crude extracts pulled-down with polyP (lanes 2, 3, and 4). The treatments (HS or H2O2) and the pull-down were conducted on isolated mitochondria from mice liver. HS was conducted for 30 min at 45°C, and the treatment with H2O2 was conducted for 30 min, using 200 μM. (B) Venn Diagrams showing the number of proteins that were present in each of the studied conditions and the overlapping areas. Proteins were detected by LC-MS/MS (C). Scatter plots showing unique proteins that were present in the comparison between the different conditions. To show this comparison, the results were plotted as PSMs vs. % of sequence coverage. The outliers are the proteins that are more abundant in each of the comparisons, and each dot represents a different protein. Following the standards in the pull-down field, this experiment was conducted only once. The name of these proteins, the exact number of PSMs, and the complete LC-MS/MS results are included in Supplementary Table S2.
FIGURE 3
FIGURE 3
The enzymatic depletion of mitochondrial polyP is a potent regulator of mammalian gene expression. qRT-PCR assays were conducted on samples obtained from at least three independent experiments. Specifically, we assayed the expression levels of some key genes involved on the maintenance of (A) Mitochondrial proteostasis (DDIT3, ATF5, SIRT3, SOD2, HSPD1, and HSPE1), and (B). Mitochondrial physiology, further than proteostasis (PRKN, DNML1, and TOMM20) in Wt and MitoPPX HEK293 cells. Experiments were conducted under control conditions and after treatment with rotenone (4 and 6 μM for 3 h). Values were standardized with those found in Wt control. The statistical analysis was carried out by one-way ANOVA with Tukey’s post-test for multiple comparisons; with the exemption of HSPE1, whose statistical analysis was carried out with Brown-Forsythe and Welch ANOVA due to the unequal variances between groups compared. Data is expressed as mean ± SEM from at least three independent experiments. Unless otherwise indicated, the differences between the groups are not significant. *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.
FIGURE 4
FIGURE 4
Proteins involved in the maintenance of mitochondrial proteostasis are affected by the depletion of mitochondrial polyP. Representative immunoblots analysis and quantification of protein levels obtained from cell lysates. In this case, we followed the same rationale as for the studies of gene expression, and we assayed some key proteins involved in the maintenance of (A). Mitochondrial proteostasis (CHOP, Sirt3, SOD2, Hsp60, Hsp10, and FoxO3a), and (B). Mitochondrial physiology, further than proteostasis (TOM20, Parkin, and LC3 I/II). To conduct these experiments, Wt and MitoPPX cells were used under control conditions and after treatment with rotenone 4 μM for 3 h to increase protein dyshomeostasis via a rise in the generation of mitochondrial ROS. Original uncropped membranes are included in the Supplementary Material. Data is expressed as mean ± SEM from at least three independent experiments. One-way ANOVA followed by Tukey’s multiple comparisons test was performed to determine statistical significance. Otherwise indicated, the differences between the groups are not significant. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.
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Grants and funding

The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This study was supported by the National Institutes of Health (1K99AG055701-01A1 and 4R00AG055701-03 to MS) and by the Start Up funds from Rutgers University to MS. At her present institution, currently, MGM is funded by CD21/00019 (ISCIII – FSE+). This project was also partially funded thanks to the 5R35GM139615 from the National institutes of Health to EP.

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