Browsing by Subject "INTERACTOME"

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  • Venditti, Rossella; Rega, Laura Rita; Masone, Maria Chiara; Santoro, Michele; Polishchuk, Elena; Sarnataro, Daniela; Paladino, Simona; D'Auria, Sabato; Varriale, Antonio; Olkkonen, Vesa M.; Di Tullio, Giuseppe; Polishchuk, Roman; De Matteis, Maria Antonietta (2019)
    ER-TGN contact sites (ERTGoCS) have been visualized by electron microscopy, but their location in the crowded perinuclear area has hampered their analysis via optical microscopy as well as their mechanistic study. To overcome these limits we developed a FRET-based approach and screened several candidates to search for molecular determinants of the ERTGoCS. These included the ER membrane proteins VAPA and VAPB and lipid transfer proteins possessing dual (ER and TGN) targeting motifs that have been hypothesized to contribute to the maintenance of ERTGoCS, such as the ceramide transfer protein CERT and several members of the oxysterol binding proteins. We found that VAP proteins, OSBP1, ORP9, and ORP10 are required, with OSBP1 playing a redundant role with ORP9, which does not involve its lipid transfer activity, and ORP10 being required due to its ability to transfer phosphatidylserine to the TGN. Our results indicate that both structural tethers and a proper lipid composition are needed for ERTGoCS integrity.
  • Doccini, Stefano; Morani, Federica; Nesti, Claudia; Pezzini, Francesco; Calza, Giulio; Soliymani, Rabah; Signore, Giovanni; Rocchiccioli, Silvia; Kanninen, Katja M.; Huuskonen, Mikko T.; Baumann, Marc; Simonati, Alessandro; Lalowski, Maciej; Santorelli, Filippo M. (2020)
    CLN5 disease is a rare form of late-infantile neuronal ceroid lipofuscinosis (NCL) caused by mutations in theCLN5genethat encodes a protein whose primary function and physiological roles remains unresolved. Emerging lines of evidence point to mitochondrial dysfunction in the onset and progression of several forms of NCL, offering new insights into putative biomarkers and shared biological processes. In this work, we employed cellular and murine models of the disease, in an effort to clarify disease pathways associated with CLN5 depletion. A mitochondria-focused quantitative proteomics approach followed by functional validations using cell biology and immunofluorescence assays revealed an impairment of mitochondrial functions in different CLN5 KO cell models and inCln5−/−cerebral cortex, which well correlated with disease progression. A visible impairment of autophagy machinery coupled with alterations of key parameters of mitophagy activation process functionally linked CLN5 protein to the process of neuronal injury. The functional link between impaired cellular respiration and activation of mitophagy pathways in the human CLN5 disease condition was corroborated by translating organelle-specific proteome findings to CLN5patients’fibroblasts. Our study highlights the involvement of CLN5 in activation of mitophagy and mitochondrial homeostasis offering new insights into alternative strategies towards the CLN5 disease treatment.