Periodic Reporting for period 4 - SYSMET (Systems Biology of Membrane Trafficking)
Periodo di rendicontazione: 2020-07-01 al 2022-06-30
Membrane trafficking is fundamental for the general homeostasis of the cell and for that of cell organelles as far as for the transport to and from the extracellular medium. Furthermore it has become clear that cell and organelle homeostasis not only depends on correct functioning of conventional membrane trafficking pathways, but also on the inter-organelle communication that occurs at the level of membrane contact sites, i.e. sites where two different organelles come very close each other (at a distance lower than 30nm).
Although we have gained detailed knowledge on the molecular organization of the many different membrane trafficking machineries a global view of their function and regulation is lacking. To date membrane trafficking is often regarded as a constitutive process with a high degree of functional redundancy. However, the fact that mutations of single genes encoding for membrane trafficking components with ubiquitous expression give rise to tissue-specific human diseases and that discrete sets of trafficking genes have differential effects on tissue development challenge this view.
Using a combination of state-of the-art technologies, we applied a systems biology approach to delineate a physiological and functional spatiotemporal map of membrane trafficking and membrane contact site genes and proteins (membrane trafficking modules), starting from our curated list of 1,187 genes representing ER, Golgi, Endosomes and Lysosomes.
The overall objectives we aim to reach with the above integrated approach are to advance our knowledge on the physiological and pathological relevance of conventional membrane trafficking pathways and of membrane contact sites by establishing a physiological and functional map of membrane trafficking and membrane contact sites genes and studying their role in relevant cell and organism systems in health and disease state.
Although we have gained detailed knowledge on the molecular organization of the many different membrane trafficking machineries a global view of their function and regulation is lacking. To date membrane trafficking is often regarded as a constitutive process with a high degree of functional redundancy. However, the fact that mutations of single genes encoding for membrane trafficking components with ubiquitous expression give rise to tissue-specific human diseases and that discrete sets of trafficking genes have differential effects on tissue development challenge this view.
Using a combination of state-of the-art technologies, we applied a systems biology approach to delineate a physiological and functional spatiotemporal map of membrane trafficking and membrane contact site genes and proteins (membrane trafficking modules), starting from our curated list of 1,187 genes representing ER, Golgi, Endosomes and Lysosomes.
The overall objectives we aim to reach with the above integrated approach are to advance our knowledge on the physiological and pathological relevance of conventional membrane trafficking pathways and of membrane contact sites by establishing a physiological and functional map of membrane trafficking and membrane contact sites genes and studying their role in relevant cell and organism systems in health and disease state.
Identifying regulatory networks in membrane trafficking
Based on the co-expression of membrane trafficking genes we identified 23 consensus Membrane Trafficking Modules and we experimentally validated some of them. We studied the ER and the Golgi complex contact sites (Venditti et al., PMID: 30659100), and showed that they control the Golgi levels of PI4P, a lipid governing the function of the Golgi complex (Venditti et al., PMID: 30659099). We identified the protein FAPP1 as a PI4P sensor at the ER-Golgi contact sites. FAPP1 belongs to module 14 and it shows, in this module, a high degree of co-expression with GOLPH3, a PI4P effector and an oncogene. Indeed, we have obtained results indicating that FAPP1 controls GOLPH3 activity in promoting cell migration and invasion by regulating PI4P at the Golgi complex, thus showing that the definition of the membrane trafficking modules is highly informative and instrumental in identifying biologically relevant processes.
Membrane trafficking in human disease
Inherited Diseases We obtained evidence that: 1. OCRL (mutated in OculoCerebroRenal Lowe syndrome) is involved in the lysosome cargo response, a novel signalling pathway that we have uncovered on lysosomes (De Leo et al. PMID: 27398910); 2. TRAPPC2 (mutated in spondyloepiphyseal dysplasia tarda, SEDT) plays a pivotal role the biogenesis of stress granules (Zappa et al., PMID: 31429971); and 3. VAPB (mutated in Amyotrophic Lateral Sclerosis 8) is required to maintain the PI4P balance in endo-lysosomes (Genevini et al., PMID: 30745341) and controls a bidirectional relationship between the ER and mitochondria.
We developed a Medaka fish model, which reproduces the SEDT phenotype and which is amenable to drug screening. We have developed a pipeline for high content screening, identified hits which are able to correct the endocytic phenotype in Lowe syndrome cell and models (that we contributed to characterize in Festa et al., PMID: 30590522). One compound (CpdA) was particularly effective in rescuing the function of proximal tubular cells in these mice. The CpdA producer company is in advanced clinical studies for CpdA, in which it demonstrated to have efficacy in a number of clinical indications and also showed a remarkable safety profile. Prompted by our results the company decided that a patent application and an Orphan Drug Designation request for CpdA in Lowe syndrome should be submitted in the next months. Importantly a Phase II exploratory study will be launched in 2023 in collaboration with the company.
COVID19 The COVID-19 pandemic exposed our ignorance about many of the basic mechanisms, including those driving membrane remodeling, at the coronavirus-host interface. Thus, also prompted by the availability of the ERC, in reaction to the COVID-19 crisis, to ”offer grantees the flexibility to adjust their research project” and to address COVID-19 related research, we decided to exploit our expertise in membrane trafficking to fill some of these knowledge gaps. We uncovered the biological activity of a protein of SARS-CoV-2 and of other coronaviruses (NSP6) and identified it as a potential target for anti-viral therapy. Our results were published in Nature this year (Ricciardi et al., PMID: 35551511).
Inflammation Inflammasome complexes drive the innate immune response to pathogens and other danger signals. We demonstrated that inflammasome activators induce an increase in PI4P levels on endosomes, and that this increase in endosomal PI4P is required for the activation of the inflammasome component NRLP3. Our datahave been accepted for publication in Nature Immunology.
Based on the co-expression of membrane trafficking genes we identified 23 consensus Membrane Trafficking Modules and we experimentally validated some of them. We studied the ER and the Golgi complex contact sites (Venditti et al., PMID: 30659100), and showed that they control the Golgi levels of PI4P, a lipid governing the function of the Golgi complex (Venditti et al., PMID: 30659099). We identified the protein FAPP1 as a PI4P sensor at the ER-Golgi contact sites. FAPP1 belongs to module 14 and it shows, in this module, a high degree of co-expression with GOLPH3, a PI4P effector and an oncogene. Indeed, we have obtained results indicating that FAPP1 controls GOLPH3 activity in promoting cell migration and invasion by regulating PI4P at the Golgi complex, thus showing that the definition of the membrane trafficking modules is highly informative and instrumental in identifying biologically relevant processes.
Membrane trafficking in human disease
Inherited Diseases We obtained evidence that: 1. OCRL (mutated in OculoCerebroRenal Lowe syndrome) is involved in the lysosome cargo response, a novel signalling pathway that we have uncovered on lysosomes (De Leo et al. PMID: 27398910); 2. TRAPPC2 (mutated in spondyloepiphyseal dysplasia tarda, SEDT) plays a pivotal role the biogenesis of stress granules (Zappa et al., PMID: 31429971); and 3. VAPB (mutated in Amyotrophic Lateral Sclerosis 8) is required to maintain the PI4P balance in endo-lysosomes (Genevini et al., PMID: 30745341) and controls a bidirectional relationship between the ER and mitochondria.
We developed a Medaka fish model, which reproduces the SEDT phenotype and which is amenable to drug screening. We have developed a pipeline for high content screening, identified hits which are able to correct the endocytic phenotype in Lowe syndrome cell and models (that we contributed to characterize in Festa et al., PMID: 30590522). One compound (CpdA) was particularly effective in rescuing the function of proximal tubular cells in these mice. The CpdA producer company is in advanced clinical studies for CpdA, in which it demonstrated to have efficacy in a number of clinical indications and also showed a remarkable safety profile. Prompted by our results the company decided that a patent application and an Orphan Drug Designation request for CpdA in Lowe syndrome should be submitted in the next months. Importantly a Phase II exploratory study will be launched in 2023 in collaboration with the company.
COVID19 The COVID-19 pandemic exposed our ignorance about many of the basic mechanisms, including those driving membrane remodeling, at the coronavirus-host interface. Thus, also prompted by the availability of the ERC, in reaction to the COVID-19 crisis, to ”offer grantees the flexibility to adjust their research project” and to address COVID-19 related research, we decided to exploit our expertise in membrane trafficking to fill some of these knowledge gaps. We uncovered the biological activity of a protein of SARS-CoV-2 and of other coronaviruses (NSP6) and identified it as a potential target for anti-viral therapy. Our results were published in Nature this year (Ricciardi et al., PMID: 35551511).
Inflammation Inflammasome complexes drive the innate immune response to pathogens and other danger signals. We demonstrated that inflammasome activators induce an increase in PI4P levels on endosomes, and that this increase in endosomal PI4P is required for the activation of the inflammasome component NRLP3. Our datahave been accepted for publication in Nature Immunology.
We have achieved the first demonstration that membrane trafficking genes are organized in modules, we have obtained demonstration that these co-expression modules have a functional correlate.
We have highlighted a novel signalling pathway taking place on lysosomal membrane: the lysosome cargo response is the first demonstration of a lumen-to-cytosol signalling occurring in lysosomes and controlling lysosome composition and membrane dynamics.
We have developed new technologies for the study of membrane contact sites and applied it to dissect the organization and regulation of ER-Golgi membrane contact sites. We identified in FAPP1, a PI4P-sensor: first demonstration of a sensor controlling phosphoinositide interconversion.
We provided a new paradigm for the activation of the inflammasome and identified in the altered composition of endosome a “damage” signal that activates innate response.
Lowe syndrome: we have implemented a complete pipeline starting from basic discoveries of disease mechanism in cells, to identification through high content screening of FDA-approved drugs of correctors of cell phenotype and validation of one of them in animal model up to the design of a clinical trial.
We have uncovered the function of a beta coronavirus protein nsp6 and delineated a novel mechanism by which viruses exploit the continuity of their replication organelle with the ER to derive lipid constituents needed for the expansion of the replication organelle but at the same time preventing the arrival of luminal ER sensors that may be detrimental for viral RNA replication. We will exploit our findings to identify small molecules that can counteract the zippering and act as pan anti-coronavirus agents.
We have highlighted a novel signalling pathway taking place on lysosomal membrane: the lysosome cargo response is the first demonstration of a lumen-to-cytosol signalling occurring in lysosomes and controlling lysosome composition and membrane dynamics.
We have developed new technologies for the study of membrane contact sites and applied it to dissect the organization and regulation of ER-Golgi membrane contact sites. We identified in FAPP1, a PI4P-sensor: first demonstration of a sensor controlling phosphoinositide interconversion.
We provided a new paradigm for the activation of the inflammasome and identified in the altered composition of endosome a “damage” signal that activates innate response.
Lowe syndrome: we have implemented a complete pipeline starting from basic discoveries of disease mechanism in cells, to identification through high content screening of FDA-approved drugs of correctors of cell phenotype and validation of one of them in animal model up to the design of a clinical trial.
We have uncovered the function of a beta coronavirus protein nsp6 and delineated a novel mechanism by which viruses exploit the continuity of their replication organelle with the ER to derive lipid constituents needed for the expansion of the replication organelle but at the same time preventing the arrival of luminal ER sensors that may be detrimental for viral RNA replication. We will exploit our findings to identify small molecules that can counteract the zippering and act as pan anti-coronavirus agents.