Final Report Summary - SCOC AND FEZ (Functional analysis of SCOC and FEZ proteins in autophagy using mammalian cell models and zebrafish)
Functional analysis of SCOC and FEZ proteins in autophagy using mammalian cell models and zebrafish
Autophagy is a conserved and highly regulated catabolic pathway, which allows cells to survive stress such as starvation. In response to nutrient deprivation cytoplasmic proteins and organelles are sequestered in double-membrane bound vesicles called autophagosomes. After fusion with lysosomes the autophagosomal content gets degraded and recycled back to the cytosol to restore intracellular nutrients and molecular building blocks. Next to this important stress survival function autophagy is also an important quality control mechanism for cells that ensures cellular health by removing toxic macromolecules and damaged organelles such as mitochondria.
In multicellular organisms autophagy is essential for embryonic development, cell lineage differentiation and tissue homeostasis and needs to be tightly regulated since both elevated and reduced autophagy levels can have deleterious effects. Deregulation of autophagy has been implicated in a broad range of diseases including cancer and neurodegenerative disorders and manipulation of the autophagic pathway may provide promising novel therapeutic strategies to treat these diseases. However, this requires a comprehensive understanding of the molecular protein machinery and membrane trafficking events mediating and regulating autophagy and there is also still a genuine need to learn more about the physiological and pathological role of autophagy in vertebrate organisms.
In a genome wide siRNA screen we identified SCOC (short coiled-coil protein), a small Golgi protein, as a novel positive regulator of starvation-induced autophagosome formation. SCOC interacts through FEZ1 (Fasciculation and elongation protein zeta 1), a kinesin-1 adapter molecule implicated axonal transport with ULK1 (Unc-51-like kinase 1) and UVRAG (UV irradiation resistance gene). The ULK1 protein kinase complex and the UVRAG-Beclin1-PI3K lipid kinase complex are key signalling complexes regulating autophagosome formation and maturation.
Interestingly, in vertebrates, both SCOC and FEZ1 are highly expressed in the developing nervous system. Worms mutant for SCOC, FEZ1 or ULK1 exhibit an “uncoordinated phenotype” caused by defects in axonal elongation. Depletion of ULK1 kinase in mouse brain also impairs axon outgrowth and autophagy seems to be essential for nervous system development and neuronal health.
In our project substantial progress has been made towards understanding the biological function of SCOC and FEZ1 in autophagy using a multidisciplinary approach including biochemical, cell biological, and biophysical approaches as well as zebrafish as a vertebrate model system.
SCOC and FEZ1 interact directly with each other through coiled-coil interactions. To better understand the interaction we employed structural analyses. We solved the crystal structure of SCOC at a resolution of 2.1Å and can show that SCOC forms a parallel dimer. Biochemical and biophysical analyses allowed us to map the minimal interacting of FEZ1 for SCOC binding and we were able to learn more about amino acid residues that are critical for the interaction and the stoichiometry of the complex. Currently we are also trying to solve the crystal structure of SCOC in complex with FEZ1.
Our biochemical and cell biological analyses on the ULK1 protein kinase complex and Beclin1-PI3K lipid kinase complexes suggests a role of SCOC and FEZ1 in regulating recruitment and trafficking of these signalling complexes to autophagosome formation sites and we are further characterizing this.
Zebrafish has proven to be an excellent vertebrate model system to study development and human disease. Although zebrafish has emerged as a promising alternative model system to mammals for drug screening, it has been hardly used for autophagy research so far. Moreover, there haven’t been genetic mutants of autophagy genes available because of the lack of targeted genome editing tools in zebrafish. This has changed recently and we have been generating zebrafish lines mutant for scoc, fez1 and other autophagy genes using the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system. Gene expression analyses showed that scoc, fez1 and many autophagy genes are already present in early embryonic developmental stages (starting from the one cell stage embryo) and we are therefore currently focusing on phenotypes that will affect early zebrafish development and will test whether these are caused by defects in autophagy and neuronal development. My work will therefore be one of the first providing insights into the function of autophagy and the novel autophagy regulators SCOC and FEZ1 in development and physiology of zebrafish using genetic mutants. This will also help to discover more about the process of autophagy in health and disease.
Autophagy is a conserved and highly regulated catabolic pathway, which allows cells to survive stress such as starvation. In response to nutrient deprivation cytoplasmic proteins and organelles are sequestered in double-membrane bound vesicles called autophagosomes. After fusion with lysosomes the autophagosomal content gets degraded and recycled back to the cytosol to restore intracellular nutrients and molecular building blocks. Next to this important stress survival function autophagy is also an important quality control mechanism for cells that ensures cellular health by removing toxic macromolecules and damaged organelles such as mitochondria.
In multicellular organisms autophagy is essential for embryonic development, cell lineage differentiation and tissue homeostasis and needs to be tightly regulated since both elevated and reduced autophagy levels can have deleterious effects. Deregulation of autophagy has been implicated in a broad range of diseases including cancer and neurodegenerative disorders and manipulation of the autophagic pathway may provide promising novel therapeutic strategies to treat these diseases. However, this requires a comprehensive understanding of the molecular protein machinery and membrane trafficking events mediating and regulating autophagy and there is also still a genuine need to learn more about the physiological and pathological role of autophagy in vertebrate organisms.
In a genome wide siRNA screen we identified SCOC (short coiled-coil protein), a small Golgi protein, as a novel positive regulator of starvation-induced autophagosome formation. SCOC interacts through FEZ1 (Fasciculation and elongation protein zeta 1), a kinesin-1 adapter molecule implicated axonal transport with ULK1 (Unc-51-like kinase 1) and UVRAG (UV irradiation resistance gene). The ULK1 protein kinase complex and the UVRAG-Beclin1-PI3K lipid kinase complex are key signalling complexes regulating autophagosome formation and maturation.
Interestingly, in vertebrates, both SCOC and FEZ1 are highly expressed in the developing nervous system. Worms mutant for SCOC, FEZ1 or ULK1 exhibit an “uncoordinated phenotype” caused by defects in axonal elongation. Depletion of ULK1 kinase in mouse brain also impairs axon outgrowth and autophagy seems to be essential for nervous system development and neuronal health.
In our project substantial progress has been made towards understanding the biological function of SCOC and FEZ1 in autophagy using a multidisciplinary approach including biochemical, cell biological, and biophysical approaches as well as zebrafish as a vertebrate model system.
SCOC and FEZ1 interact directly with each other through coiled-coil interactions. To better understand the interaction we employed structural analyses. We solved the crystal structure of SCOC at a resolution of 2.1Å and can show that SCOC forms a parallel dimer. Biochemical and biophysical analyses allowed us to map the minimal interacting of FEZ1 for SCOC binding and we were able to learn more about amino acid residues that are critical for the interaction and the stoichiometry of the complex. Currently we are also trying to solve the crystal structure of SCOC in complex with FEZ1.
Our biochemical and cell biological analyses on the ULK1 protein kinase complex and Beclin1-PI3K lipid kinase complexes suggests a role of SCOC and FEZ1 in regulating recruitment and trafficking of these signalling complexes to autophagosome formation sites and we are further characterizing this.
Zebrafish has proven to be an excellent vertebrate model system to study development and human disease. Although zebrafish has emerged as a promising alternative model system to mammals for drug screening, it has been hardly used for autophagy research so far. Moreover, there haven’t been genetic mutants of autophagy genes available because of the lack of targeted genome editing tools in zebrafish. This has changed recently and we have been generating zebrafish lines mutant for scoc, fez1 and other autophagy genes using the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system. Gene expression analyses showed that scoc, fez1 and many autophagy genes are already present in early embryonic developmental stages (starting from the one cell stage embryo) and we are therefore currently focusing on phenotypes that will affect early zebrafish development and will test whether these are caused by defects in autophagy and neuronal development. My work will therefore be one of the first providing insights into the function of autophagy and the novel autophagy regulators SCOC and FEZ1 in development and physiology of zebrafish using genetic mutants. This will also help to discover more about the process of autophagy in health and disease.