From opportunistic protist Cryptosporidium parvum we identified and functionally assayed a fatty acyl-CoA-binding protein (ACBP) gene. The CpACBP1 gene encodes a protein of 268 aa that is three times larger than typical ~10 KD ACBPs of humans and animals. Sequence analysis indicated that the CpACBP1 protein consists of an N-terminal ACBP domain (approximately 90 aa) and a C-terminal ankyrin repeat sequence (approximately 170 aa). The entire CpACBP1 open reading fragment (ORF) was engineered into a maltose-binding protein fusion system and expressed as a recombinant protein for functional analysis. Acyl-CoA-binding assays clearly revealed that the preferred binding substrate for CpACBP1 is palmitoyl-CoA. RT-PCR, Western blotting and immunolabelling analyses clearly showed that the CpACBP1 gene is mainly expressed during the intracellular developmental stages and that the level increases during parasite development. Immunofluorescence microscopy showed that CpACBP1 is associated with the parasitophorous vacuole membrane (PVM), which implies that this protein may be involved in lipid remodelling in the PVM, or in the transport of fatty acids across the membrane. We also identified two distinct oxysterol binding protein (OSBP)-related proteins (ORPs) from this parasite (CpORP1 and CpORP2). The short-type CpOPR1 contains only a ligand binding (LB) domain, while the long-type CpORP2 contains Pleckstrin homology (PH) and LB domains. Lipid-protein overlay assays using recombinant proteins revealed that CpORP1 and CpORP2 could specifically bind to phosphatidic acid (PA), various phosphatidylinositol phosphates (PIPs), and sulfatide, but not to other types of lipids with simple heads. Cholesterol was not a ligand for these two proteins. CpOPR1 was found mainly on the parasitophorous vacuole membrane (PVM), suggesting that CpORP1 is probably involved in the lipid transport across this unique membrane barrier between parasites and host intestinal lumen. Although Cryptosporidium has two ORPs, other apicomplexans, including Plasmodium, Toxoplasma, and Eimeria, possess only a single long-type ORP, suggesting that this family of proteins may play different roles among apicomplexans.

From opportunistic protist Cryptosporidium parvum we identified and functionally assayed a fatty acyl-CoA-binding protein (ACBP) gene. The CpACBP1 gene encodes a protein of 268 aa that is three times larger than typical ~10 KD ACBPs of humans and animals. Sequence analysis indicated that the CpACBP1 protein consists of an N-terminal ACBP domain (approximately 90 aa) and a C-terminal ankyrin repeat sequence (approximately 170 aa). The entire CpACBP1 open reading fragment (ORF) was engineered into a maltose-binding protein fusion system and expressed as a recombinant protein for functional analysis. Acyl-CoA-binding assays clearly revealed that the preferred binding substrate for CpACBP1 is palmitoyl-CoA. RT-PCR, Western blotting and immunolabelling analyses clearly showed that the CpACBP1 gene is mainly expressed during the intracellular developmental stages and that the level increases during parasite development. Immunofluorescence microscopy showed that CpACBP1 is associated with the parasitophorous vacuole membrane (PVM), which implies that this protein may be involved in lipid remodelling in the PVM, or in the transport of fatty acids across the membrane. We also identified two distinct oxysterol binding protein (OSBP)-related proteins (ORPs) from this parasite (CpORP1 and CpORP2). The short-type CpOPR1 contains only a ligand binding (LB) domain, while the long-type CpORP2 contains Pleckstrin homology (PH) and LB domains. Lipid-protein overlay assays using recombinant proteins revealed that CpORP1 and CpORP2 could specifically bind to phosphatidic acid (PA), various phosphatidylinositol phosphates (PIPs), and sulfatide, but not to other types of lipids with simple heads. Cholesterol was not a ligand for these two proteins. CpOPR1 was found mainly on the parasitophorous vacuole membrane (PVM), suggesting that CpORP1 is probably involved in the lipid transport across this unique membrane barrier between parasites and host intestinal lumen. Although Cryptosporidium has two ORPs, other apicomplexans, including Plasmodium, Toxoplasma, and Eimeria, possess only a single long-type ORP, suggesting that this family of proteins may play different roles among apicomplexans.

A mutation in ER membrane protein VAPB is responsible for causing a familial form of ALS (ALS8). The VAPB-P56S mutation causes protein aggregation and a nuclear envelope defect, where retrograde transport is disrupted. Over-expression of a FFAT peptide from OSBP1 reduces the size of VAPB-P56S aggregates and restores retrograde transport. A screen was performed on FFAT-motif containing ORPs to determine if any could rescue the mutant phenotype. ORP3 successfully reduced aggregate size and restored transport to the nuclear envelope. ER membrane protein Sac1, a PI4P phosphatase cycles between the ER and Golgi and becomes trapped in expanded ERGIC compartments with VAPB-P56S. Loss of Sac1 in the ER leads to an increase in intracellular PI4P. ORP3 may increase Sac1 phosphatase activity by acting as a lipid sensor. We propose that VAPB, Sac1 and ORP3 are interacting partners that together modulate levels of PI4P. Disruptions in the gradient of PI4P may result in the vesicle trafficking defects observed in VAPB-P56S cells.

A mutation in ER membrane protein VAPB is responsible for causing a familial form of ALS (ALS8). The VAPB-P56S mutation causes protein aggregation and a nuclear envelope defect, where retrograde transport is disrupted. Over-expression of a FFAT peptide from OSBP1 reduces the size of VAPB-P56S aggregates and restores retrograde transport. A screen was performed on FFAT-motif containing ORPs to determine if any could rescue the mutant phenotype. ORP3 successfully reduced aggregate size and restored transport to the nuclear envelope. ER membrane protein Sac1, a PI4P phosphatase cycles between the ER and Golgi and becomes trapped in expanded ERGIC compartments with VAPB-P56S. Loss of Sac1 in the ER leads to an increase in intracellular PI4P. ORP3 may increase Sac1 phosphatase activity by acting as a lipid sensor. We propose that VAPB, Sac1 and ORP3 are interacting partners that together modulate levels of PI4P. Disruptions in the gradient of PI4P may result in the vesicle trafficking defects observed in VAPB-P56S cells.

Mitochondria is no longer viewed as merely a powerhouse of the cell. It is now apparentthat mitochondria play a central role in signaling, maintaining cellular homeostasis and cell fate.Mitochondrial dysfunction has been linked to many human diseases caused by cellular metabolicderegulation, such as obesity, diabetes, neurodegenerative disease, cardiovascular disease andcancer. Eukaryotic organisms have evolved an efficient way in sensing, communicating andresponding to cellular stress and regulating mitochondrial activity correspondingly through acomplex network of intercommunicating protein kinases and their downstream effectors. Thisdissertation focuses on the interplay of two of the master metabolic regulators in the cell: AMPKand PASK, and characterization of the functions of their downstream substrates: OSBP andMED13. AMPK is an energy sensing kinase that maintains energy homeostasis in the cell,whereas PASK is a nutrient sensing kinase that regulates glucose partitioning and respiration inthe cell. Both kinases play important roles in mitochondrial function and regulation, anddeficiency in either kinase has been found to associate with various human pathologies. Furthercharacterization of the cross-talk and molecular mechanisms of both kinases in controllingmitochondrial health and function may aid in the identification of new targets for treatingmetabolic diseases.