A lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB.

The lysosome plays a key role in cellular homeostasis by controlling both cellular clearance and energy production to respond to environmental cues. However, the mechanisms mediating lysosomal adaptation are largely unknown. Here, we show that the Transcription Factor EB (TFEB), a master regulator of lysosomal biogenesis, colocalizes with master growth regulator mTOR complex 1 (mTORC1) on the lysosomal membrane. When nutrients are present, phosphorylation of TFEB by mTORC1 inhibits TFEB activity. Conversely, pharmacological inhibition of mTORC1, as well as starvation and lysosomal disruption, activates TFEB by promoting its nuclear translocation. In addition, the transcriptional response of lysosomal and autophagic genes to either lysosomal dysfunction or pharmacological inhibition of mTORC1 is suppressed in TFEB-/- cells. Interestingly, the Rag GTPase complex, which senses lysosomal amino acids and activates mTORC1, is both necessary and sufficient to regulate starvation- and stress-induced nuclear translocation of TFEB. These data indicate that the lysosome senses its content and regulates its own biogenesis by a lysosome-to-nucleus signalling mechanism that involves TFEB and mTOR.

The expression of soluble and active recombinant Haemophilus influenzae IgA1 protease in E. coli.

Immunoglobulin A1 (IgA1) proteases from Haemophilus influenzae are extracellular proteases that specifically cleave the hinge region of human IgA1, the predominant class of immunoglobulin present on mucosal membranes. The IgA1 proteases may have the potential to cleave IgA1 complexes in the kidney and be a therapeutic agent for IgA1 nephropathy (IgAN), a disease characterized by deposition of the IgA1 antibody in the glomerulus. We have screened for the expression of recombinant H. influenzae IgA1 protease by combining various expression plasmids, IgA1 protease constructs, and E. coli strains under multiple conditions. Using the method we have developed, approximately 20-40 mg/L of soluble and active H. influenzae IgA1 protease can be produced from E. coli strain C41(DE3), a significant increase in yield compared to the yield upon expression in H. influenzae or other related bacteria.

A novel method for the large-scale production of PG-CNP37, a C-type natriuretic peptide analogue.

Achondroplasia is the most common form of human dwarfism caused by a mutation in the fibroblast growth factor receptor 3 (FGFR3), resulting in abnormal endochondral bone formation. C-type natriuretic peptide (CNP) is a potent stimulator of endochondral bone growth and represents a potential therapy for achondroplasia. We have developed a novel, simple and cost effective method to produce a CNP analogue, PG-CNP37, at a large scale from Escherichia coli. A PG-CNP37 fusion protein was over-expressed as inclusion bodies in E. coli, which were purified then cleaved by formic acid to release the PG-CNP37 peptide. Approximately 0.5g of 95% pure, soluble and active PG-CNP37 peptide was produced from 1L of culture using this method and may represent a viable means for large-scale production of other therapeutic peptides.

Lipoprotein receptor binding, cellular uptake, and lysosomal delivery of fusions between the receptor-associated protein (RAP) and alpha-L-iduronidase or acid alpha-glucosidase.

Enzyme replacement therapy for lysosomal storage disorders depends on efficient uptake of recombinant enzyme into the tissues of patients. This uptake is mediated by oligosaccharide receptors including the cation-independent mannose 6-phosphate receptor and the mannose receptor. We have sought to exploit alternative receptor systems that are independent of glycosylation but allow for efficient delivery to the lysosome. Fusions of the human lysosomal enzymes alpha-l-iduronidase or acid alpha-glucosidase with the receptor-associated protein were efficiently endocytosed by lysosomal storage disorder patient fibroblasts, rat C6 glioma cells, mouse C2C12 myoblasts, and recombinant Chinese hamster ovary cells expressing individual members of the low-density lipoprotein receptor family. Uptake of the fusions exceeded that of phosphorylated enzyme in all cases, often by an order of magnitude or greater. Uptake was specifically mediated by members of the low-density lipoprotein receptor protein family and was followed by delivery of the fusions to the lysosome. The advantages of the lipoprotein receptor system over oligosaccharide receptor systems include more efficient cellular delivery and the potential for transcytosis of ligands across tight endothelia, including the blood-brain barrier.