The mitochondrial stress-induced protein carboxyl-terminal alanine and threonine tailing (msiCAT-tailing) promotes glioblastoma tumorigenesis by modulating mitochondrial functions

Reviewer #1 (Public Review):

Summary:

Cai et al have investigated the role of msiCAT-tailed mitochondrial proteins that frequently exist in glioblastoma stem cells. Overexpression of msiCAT-tailed mitochondrial ATP synthase F1 subunit alpha (ATP5) protein increases the mitochondrial membrane potential and blocks mitochondrial permeability transition pore formation/opening. These changes in mitochondrial properties provide resistance to staurosporine (STS)-induced apoptosis in GBM cells. Therefore, msiCAT-tailing can promote cell survival and migration, while genetic and pharmacological inhibition of msiCAT-tailing can prevent the overgrowth of GBM cells.

Strengths:

The CAT-tailing concept has not been explored in cancer settings. Therefore, the present provides new insights for widening the therapeutic avenue.

Weaknesses:

Although the paper does have strengths in principle, the weaknesses of the paper are that these strengths are not directly demonstrated. The conclusions of this paper are mostly well-supported by data, but some aspects of image acquisition and data analysis need to be clarified and extended.

Reviewer #2 (Public Review):

This work explores the connection between glioblastoma, mito-RQC, and msiCAT-tailing. They build upon previous work concluding that ATP5alpha is CAT-tailed and explore how CAT-tailing may affect cell physiology and sensitivity to chemotherapy. The authors conclude that when ATP5alpha is CAT-tailed, it either incorporates into the proton pump or aggregates and that these events dysregulate MPTP opening and mitochondrial membrane potential and that this regulates drug sensitivity. This work includes several intriguing and novel observations connecting cell physiology, RQC, and drug sensitivity. This is also the first time this reviewer has seen an investigation of how a CAT tail may specifically affect the function of a protein. However, some of the conclusions in this work are not well supported. This significantly weakens the work but can be addressed through further experiments or by weakening the text.

Author response:

We are grateful for the reviewers' acknowledgment of the originality of our manuscript and its potential importance in cancer treatment. We appreciate the reviewers' critiques on certain conclusions and thank them for their thorough feedback on the manuscript. In the revised version, we will provide a more detailed clarification of the previous data and methods, bolster the existing data, and present additional evidence in support of our hypothesis. Please find below our replies to particular concerns.

In brief, to address the comments from Reviewer 1, we will make the following revisions in the manuscript:

(1) To discuss the issues regarding the specificity of ATP5⍺ CAT-tailing, we will provide new patient-derived cell lines and tumor samples and investigate the CAT-tail modifications of nuclear genome-encoded mitochondrial proteins and changes in RQC proteins within them. We will endeavor to explore the nature of NEMF modifications in GSC cells (Fig. S1A).

(2) To enhance the quality of image data, we will substitute some images (such as Fig. 1E and 3A) with higher quality images.

(3) To further understand the influence of NEMF on cancer, the effects of NEMF overexpression in GSC cells will be evaluated through testing (e.g., Fig. 3D).

(4) To further explore changes in apoptosis, we will employ additional methods to detect apoptosis, including Annexin-PI FACS assays, caspase cleavage analysis, assessing BAX-BCL2 ratios, and monitoring cytochrome c release.

(5) To further confirm the effectiveness of the CAT-tailing-mitochondria mechanism in in vivo tumor models, we will utilize a Drosophila model to study the impact of the RQC pathway and CAT-tailing mechanism on tumor proliferation in vivo. The overactivation of the Notch signaling pathway in Drosophila can stimulate malignant proliferation of neural stem cells (NSCs) through both canonical (c-Myc mediated pathway) and non-canonical (PINK1-mitochondrial-mTORC2 pathway) pathways, leading to the development of a tumor-like phenotype in the larval brain. A recent publication in PNAS Nexus (Khaket et al., PNAS Nexus, 2024) discusses the impact of the RQC pathway on c-Myc. It is possible for us to analyze the alterations in CAT-tailing on mitochondrial proteins and mitochondrial membrane potential in this Notch model and study how the RQC pathway regulates them. Moreover, tumor implantation experiments will be carried out using immunodeficient mice. Our goal is to conduct a comparative analysis of the growth of control and NEMF KD glioblastoma cell lines in animal models, alongside performing essential biochemical analyses.

Reference:

Khaket, T. P., et al. (2024). Ribosome stalling during c-myc translation presents actionable cancer cell vulnerability. PNAS nexus, 3(8), pgae321.

To address the comments from Reviewer 2, we will make the following revisions in the manuscript:

(1) The concerns raised by the reviewer regarding the authenticity of the ATP5a CAT-tail modification are duly noted. Critical control experiments will be incorporated into our study, including NEMF knockout (or NFACT domain mutants) and cycloheximide treatment, alongside other methodologies. The results of these experiments will include placements such as Fig. 1B, 1C, S3A, and S3B to improve comprehension of the CAT-tail modification on ATP5⍺.

(2) We thank the reviewer for reminding us to consider the differences between the artificial tail and the endogenous CAT-tail. A recently published study (Khan et al., 2024) provides a thorough analysis of the components of the CAT-tail. Our approach to addressing this issue involves emphasizing the use of the artificial CAT-tail sequence and adopting a more measured tone in the revised version. Additionally, we will induce the endogenous ATP5⍺-CAT-tail by express ATP5⍺-K20-non-stop in cells to validate their function in glioblastoma cells.

(3) Moreover, we aim to examine the impact of different amino acid compositions in the ATP5⍺ c-terminus extension, such as the poly (Gly-Ser) repeats noted by the reviewer, on both mitochondrial function and glioblastoma biology in our revision. By comparing the results obtained from ATP5⍺-CAT-tails with different compositions, it is anticipated that more definitive conclusions can be drawn.

(4) Additional minor revisions will be implemented to the text in accordance with the feedback given by the reviewer.

Reference:

Khan, D., Vinayak, A. A., Sitron, C. S., & Brandman, O. (2024). Mechanochemical forces regulate the composition and function of CAT tails. bioRxiv, 2024-08.