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Paolo Grumati

Assistant Investigator, TIGEM
Head of Mass Spectrometry Unit, TIGEM

Molecular Mechanisms of Autophagy and Membranes Dynamics

Autophagy is a vital catabolic mechanism employed by cells to maintain nutrient homeostasis and organellar quality control. This degradative system is conserved in eukaryotic organisms, occurs at basal levels in nearly all cell types and is increased by multiple intracellular and extracellular stimuli, including nutrient deprivation, growth factors depletion and infections. Autophagy is a dynamic process that starts with the formation of a double membrane structure (autophagosome), which engulfs a portion of the cytosol, and terminates with the fusion of the autophagosome with a lysosome. Autophagy was initially considered bereaved of substrate specificity; however, we now know that there is a fine specificity in cargo selection and an intricate molecular mechanism behind it. Usually, the cargo either contains a LC3-Interacting Region (LIR) that directly binds to the LC3s/GABARAPs or it is labeled with a tag, like ubiquitin, which binds adaptor proteins that contains a ubiquitin binding motif and a LIR domain. Selective autophagy could be crucial in protection against mammalian diseases, even more than the bulk macro-autophagy, which is primarily a homeostatic mechanism. The vast implications of the autophagy system in cellular physiology makes this process crucial for the protection against a plethora of diseases including infections, cancer, neurodegeneration and aging.

The main interest of the lab is to characterize the molecular mechanisms responsible for endoplasmic reticulum (ER) dynamics during autophagy induction. The ER is a continuous system of membrane sheets, tubules and matrices, which constantly reshape to fulfil physiological functions like: calcium homeostasis, protein quality control and secretion. An important aspect is the restoration of the original pre-stress ER network, which requires the disassembly and elimination of excess or damaged ER parts. ER membranes are rapidly remodeled by selective autophagy, ER-phagy, which is regulated by a subset of specific receptors. The molecular mechanisms responsible for receptor activation and the factors that trigger ER membrane remodeling during autophagy remain largely unexplored. 

Studying the mechanisms of ER remodeling during autophagy is not only important for acquiring new information on a fundamental cellular process but also bares major medical implications and therapeutic potential. ER-phagy plays a prominent role in the fight against viral and bacterial infections. Indeed, ER-phagy-related receptors CCPG1, FAM134B and SEC62 have been associated with several types of cancers. Moreover, mutations in ER resident proteins ARL6IP1, REEPs, SPASTIN, ATLASTINs, FAM134s and RTNs are responsible for hereditary sensory and autonomic neuropathies (HSAN).  These disorders are primarily characterized by degeneration of long projection fibers, and are often referred to as axonopathies.

The aim of our research is to investigate the involvement of the known autophagy pathways in ER remodeling; to characterize new ER-phagy receptors and their role in membrane dynamics, and to unravel the molecular mechanisms of ER-related axonal disorders. We will approach our scientific questions using state of the art technologies in gene editing (CRISPR-Cas9), proteomics (Mass Spectrometry) and Ultra Resolution Microscopy. 

Mass Spectrometry Unit

Mass Spectrometry (MS) based proteomic analysis unravels what proteins do. This high powered unbiased interrogative approach can answer important biological questions about proteins of interest such as: the signaling pathways in which they are involved, the proteins with which they interact and the post-translational modifications they are subjected to. In general, a cell phenotype reflects the proteomic status, so the mass spectrometry studies can provide fundamental biological insights on molecular mechanisms.

At the recently established state of the art Mass Spectrometry Unit in TIGEM, we offer an unbiased and quantitative description of signaling networks and determine how components of signaling cascades are affected in response to pathway perturbations. We perform complementary qualitative and quantitative MS approaches for identification and quantitation of proteins and their post-translational modifications (PTMs), both at small (e.g. interactomes) and large (whole cell- or subcellular proteome) scale.
In general, we follow a standard approach: protein samples are first treated with proteases (e.g. Trypsin and/or LysC) and then analyzed by LC-MS/MS for identification and quantification of the resulting peptides and thus the underlying proteins. Besides label-free protein quantitation based on summed peak intensities of identified peptides, we take advantage of various labelling techniques, like Stable Isotope Labelling with Amino acids in Cell culture (SILAC) and Tandem Mass Tag (TMT) reagents. The generated spectral data is analyzed using Max Quant (MPI Martinsried) and Proteome Discoverer (Thermo Scientific) in order to obtain the list of identified proteins and their abundance in samples.
Our facility is equipped with a Q Exactive HF Hybrid Quadrupole-Orbitrap with an ultra-high-field analyzer. It provides high sensitivity and acquisition speed for highly complex samples, especially in conjunction with the hyphenated UHPLC which allows the use of smaller column particles and longer columns due to higher pressure resistance of the fluidics.

Staff Scientist:

Forrester A*, De Leonibus C*, Grumati P*, Fasana E*, Staiano L, Fregno I, Raimondi A, Marazza A, Bruno G, Iavazzo M, Intartaglia D, Piemontese M, Seczynska M, van Anken E,Conte I, De Matteis MA, Dikic I, Molinari M, Settembre C. (2019) A selective ER-phagy exerts procollagen quality control via a CALNEXIN-FAM134B complex. EMBO J. 38(2) pii: e99847. doi: 10.15252/embj.201899847.

Grumati P, Morozzi G, Hölper S, Mari M, Harwardt ML, Yan R, Müller S, Reggiori F, Heilemann M, Dikic I. (2017) Full length RTN3 regulates turnover of tubular endoplasmic reticulum via selective autophagy. eLife 6; doi: 10.7554/eLife.25555.

Khaminets A*, Heinrich T*, Mari M, Grumati P, Huebner AK, Akutsu M, Liebmann L, Stolz A, Nietzsche S, Koch N, Mauthe M, Katona I, Qualmann B, Weis J, Reggiori F, Kurth I, Hübner CA, Dikic I. (2015) Regulation of endoplasmic reticulum turnover by selective autophagy. Nature 522: 354-8.

Grumati P*, Coletto L*, Schiavinato A, Castagnaro S, Bertaggia E, Sandri M, Bonaldo P. (2011) Physical exercise stimulates autophagy in normal skeletal muscles but is detrimental for collagen VI deficient muscles. Autophagy 7: 1415-23.

Grumati P*, Coletto L*, Sabatelli P, Cescon M, Angelin A, Bertaggia E, Blaauw B, Urciuolo A, Tiepolo T, Merlini L, Maraldi NM, Bernardi P, Sandri M, Bonaldo P. (2010) Autophagy is defective in collagen VI muscular dystrophies, and its reactivation rescues myofiber degeneration. Nat Med 16: 1313-20.

Paolo Grumati PhD

Cell Biology and Disease Mechanisms

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