Professional Education


  • Master of Science, Universita Degli Studi Di Napoli (2011)
  • Doctor of Philosophy, Universita Degli Studi Di Napoli (2015)

All Publications


  • The ATP-powered gymnastics of TRiC/CCT: an asymmetric protein folding machine with a symmetric origin story. Current opinion in structural biology Gestaut, D., Limatola, A., Joachimiak, L., Frydman, J. 2019; 55: 50–58

    Abstract

    The eukaryotic chaperonin TRiC/CCT is a large hetero-oligomeric complex that plays an essential role assisting cellular protein folding and suppressing protein aggregation. It consists of two rings, and each composed of eight different subunits; non-native polypeptides bind and fold in an ATP-dependent manner within their central chamber. Here, we review recent advances in our understanding of TRiC structure and mechanism enabled by application of hybrid structural methods including the integration of cryo-electron microscopy with distance constraints from crosslinking mass spectrometry. These new insights are revealing how the different TRiC/CCT subunits create asymmetry in its ATP-driven conformational cycle and its interaction with non-native polypeptides, which ultimately underlie its unique ability to fold proteins that cannot be folded by other chaperones.

    View details for PubMedID 30978594

  • Time-Resolved NMR Analysis of Proteolytic alpha-Synuclein Processing in vitro and in cellulo PROTEOMICS Limatola, A., Eichmann, C., Jacob, R., Ben-Nissan, G., Sharon, M., Binolfi, A., Selenko, P. 2018; 18 (21-22): e1800056

    Abstract

    Targeted proteolysis of the disordered Parkinson's disease protein alpha-synuclein (αSyn) constitutes an important event under physiological and pathological cell conditions. In this work, site-specific αSyn cleavage by different endopeptidases in vitro and by endogenous proteases in extracts of challenged and unchallenged cells was studied by time-resolved NMR spectroscopy. Specifically, proteolytic processing was monitored under neutral and low pH conditions and in response to Rotenone-induced oxidative stress. Further, time-dependent degradation of electroporation-delivered αSyn in intact SH-SY5Y and A2780 cells was analyzed. Results presented here delineate a general framework for NMR-based proteolysis studies in vitro and in cellulo, and confirm earlier reports pertaining to the exceptional proteolytic stability of αSyn under physiological cell conditions. However, experimental findings also reveal altered protease susceptibilities in selected mammalian cell lines and upon induced cell stress.

    View details for PubMedID 30260559

  • Intracellular repair of oxidation-damaged alpha-synuclein fails to target C-terminal modification sites NATURE COMMUNICATIONS Binolfi, A., Limatola, A., Verzini, S., Kosten, J., Theillet, F., Rose, H., Bekei, B., Stuiver, M., van Rossum, M., Selenko, P. 2016; 7: 10251

    Abstract

    Cellular oxidative stress serves as a common denominator in many neurodegenerative disorders, including Parkinson's disease. Here we use in-cell NMR spectroscopy to study the fate of the oxidation-damaged Parkinson's disease protein alpha-synuclein (α-Syn) in non-neuronal and neuronal mammalian cells. Specifically, we deliver methionine-oxidized, isotope-enriched α-Syn into cultured cells and follow intracellular protein repair by endogenous enzymes at atomic resolution. We show that N-terminal α-Syn methionines Met1 and Met5 are processed in a stepwise manner, with Met5 being exclusively repaired before Met1. By contrast, C-terminal methionines Met116 and Met127 remain oxidized and are not targeted by cellular enzymes. In turn, persisting oxidative damage in the C-terminus of α-Syn diminishes phosphorylation of Tyr125 by Fyn kinase, which ablates the necessary priming event for Ser129 modification by CK1. These results establish that oxidative stress can lead to the accumulation of chemically and functionally altered α-Syn in cells.

    View details for DOI 10.1038/ncomms10251

    View details for Web of Science ID 000369020300001

    View details for PubMedID 26807843

    View details for PubMedCentralID PMC4737712