RagD auto-activating mutations impair MiT/TFE activity in kidney tubulopathy and cardiomyopathy syndrome

Heterozygous mutations in the gene encoding RagD GTPase were shown to cause a novel autosomal dominant condition characterized by kidney tubulopathy and cardiomyopathy. We previously demonstrated that RagD, and its paralogue RagC, mediate a non-canonical mTORC1 signaling pathway that inhibits the activity of TFEB and TFE3, transcription factors of the MiT/TFE family and master regulators of lysosomal biogenesis and autophagy. Here we show that RagD mutations causing kidney tubulopathy and cardiomyopathy are “auto- activating”, even in the absence of Folliculin, the GAP responsible for RagC/D activation, and cause constitutive phosphorylation of TFEB and TFE3 by mTORC1, without affecting the phosphorylation of “canonical” mTORC1 substrates, such as S6K. By using HeLa and HK-2 cell lines, human induced pluripotent stem cell-derived cardiomyocytes and patient-derived primary fibroblasts, we show that RRAGD auto-activating mutations lead to inhibition of TFEB and TFE3 nuclear translocation and transcriptional activity, which impairs the response to lysosomal and mitochondrial injury. These data suggest that inhibition of MiT/TFE factors plays a key role in kidney tubulopathy and cardiomyopathy syndrome.


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For graphs the exact p value for all the experiments is present in the Source data file. The Whole-Exome Sequencing (WES) data are deposited in Sequence Read Archive (SRA) of NCBI repository (BioProject ID:PRJNA960632, available at the following link https://www.ncbi.nlm.nih.gov/bioproject/960632 ). All other data are available from the corresponding author on request.
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The characteristics of the population involved in the study is exclusively based on the genotype (RRAGD gene variant) and on the presence of signs of the disease. Age range from 10 to 63 years old.
The human participants involved in the study belong to the same family and have been selected accordingly to their RRAGD genotype. No additional families have been found with the same mutation so no additional recruitment from other families has been possible.
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The sample size was chosen based on previous experience with respect to how many independent number of cells per treatment/group are required to reliably detect biologically meaningful differences among groups. Furthermore, most of the data have been carried out by High Content Imaging in which thousands of cells/group/treatement were analyzed. All the relevant information about sample size are reported in the figure legends.
We did not apply any exclusion criteria All experiments were carried out under standard and clearly defined conditions (based on our experience and on the literature), and were replicated successfully by at least one researcher. All attempts at replication were successful and the relative data are available in the Source Data File.
The cells used in the manuscript were randomly assigned to the experimental group/treatment. In case of experiments involving treatments such as nutrients manipulation/ drug administration, untreated and treated cells came from the very same culture and an homogeneous cell suspension was divided into many wells and either left untreated or subjected to the treatment.
The antibodies used in this manuscript were chosen based on previous literature. Information about the validation of the primary antibodies used are accessible on the manufacturer's websites (reported above) along with references in which the antibodies were used/tested. The antibodies used are the most diffused and trusted in the field of the lysosomes, autophagy, and mTOR pathway analysis (Napolitano et al., Nature 2020. PMID: 32612235) Hela and HK-2 cells were purchased from ATCC (Hela CCL/2; HK-2 CRL-2190). FLCN-KO Hela and FLCN-KO HK-2 cell lines were generated in this study.