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Lysosomal pH is (acidic/basic/neutral)
What protects the lysosomal membrane from degradation?
Thick glycocalyx composed of the highly glycosylated lysosomal integral membrane proteins (LIMPS) and lysosomal associated membrane proteins (LAMPS)
How are lysosomal hydrolases marked to be sent to lysosomes?
- Theory of folding-dependent "patch" that is recognized
- Following N-glycosylation of asparagine residues in the ER, GlcNAc phosphotransferase in the Golgi catalyzes transfer of GlcNAc-1-phosphate to the C6 position of selected mannose residues on high mannose-type oligosacchiarides
- Mannose-6-phosphate marker is exposed by removal of N-acetylglucosamine in trans Golgi
- M6P receptor in TGN recognizes the M6P, goes in clathrin coated vesicle, released in low pH of endosome, transferred to lysosome.
4 receptors involved in targeting soluble lysosomal proteins and what they do
- MPR300: bind M6P hydrolases to endosome, get recycled back to plasma membrane
- MPR46: same
- LIMP-2: bind βGC in M6P-independent manner in TGN to bring to endosome, does NOT get recycled
- Sortilin: binds certain things in TGN (neurotensin, LPL, ProSAP, e.g.) to bring to endosome, gets recycled back to plasma membrane
What is a hallmark of lysosomal storage disorders?
CNS pathology (most common cause of pediatric neurodegenerative disease)
Most common cause of pediatric neurodegenerative disease?
Lysosomal storage diseases
Glycosphingolipid with one or more sialic acids linked on the sugar chain
Describe the β-hexosaminidase system, including the genes, the polypeptides encoded by those genes, the functions of the polypeptides, and the pathologies caused by mutations in the genes
- System of enzymes to degrade GM2
- 3 Genes:
- HEXA: encodes alpha subunit
- HEXB: encodes beta subunit
- GM2A: encodes GM2 activator protein, that binds to GM2 and presents it to hexosaminidase enzyme
- Hex S: 2 alpha subunits (not very stable)
- Hex A: one alpha (from HEXA), one beta (from HEXB)
- Hex B: 2 beta subunits
- GM2 Activator proteinPathologies:
- Tay-Sachs: mutation of HEXA, leading to deficient Hex A protein but not Hex B
- Sandhoff Disease: mutation in HEXB, leading to deficient Hex A and Hex B
- AB variant: mutation in GM2A, functional Hex A + B but still store GM2
Mutation in HEXA gene, leading to deficient Hex A protein, which normally would break down GM2 ganglioside
Mutation in HEXB, leading to deficient Hex A and Hex B enzymes, no GM2 breakdown
Mutation in GM2A, functional Hex A and Hex B but no breakdown of GM2 in vivo
Membranous cytoplasmic bodies in neuronal cell bodies is typical of what disease?
Mechanism of GM2 activator
Has a hydrophobic pocket that plucks GM2 out of membrane and allows Hex A enzyme to reach it
Golgi enzyme that transfers M6P to lysosomal hydrolases
GlcNAc phosphotransferase mutation
- Causes mucolipidoses II and III
- Lysosomal hydrolases are not marked for lysosome, so undigested material accumulates in lysosome since there are no lysosomal hydrolases there.
Formylglycine generating enzyme (FGE)
- Catalyzes oxidation of a cysteine residue on inactive sulfatases, activating them.
- Mutation in FGE causes multiple sulfatase deficiency (MSD)
- Brings LDL-derived cholesterol out of lysosome
- Mutation causes lysosomal storage of cholesterol
Metabolic cross-correction to treat lysosomal storage disorders
- Diseased cells can take up exogenous enzyme, which can be delivered to the lysosome and function
- Can be infused directly into blood, or can use gene therapy or stem cells (because rarely in normal cells M6P not added to the enzyme, which is released from plasma membrane and can be taken up by neighboring cells, in this case the diseased cells)
- Not every cell needs to be corrected!
Substrate reduction therapy to treat lysosomal storage disorders
- Oral medication that inhibits the first committed step in glycosphingolipid biosynthesis
- Reduction of biosynthesis of glycosphingolipids offsets defect in catabolizing glycosphingolipids
- Potential to treat LSDs with CNS pathology, as drug in clinical use can cross blood-brain barrier
Pharmacological chaperone therapy to treat lysosomal storage disorders
Application of small molecules that enhance folding or prevent premature degradation of defective enzyme
Identification of CLEAR gene network
- Coexpression analysis of lysosomal genes using public microarray data reveals that lysosomal genes are highly coexpressed
- Promoter analysis reveals that lysosomal gene promoters contain E-boxes
Transcriptional control of lysosome homeostasis via mTORC1 signaling and TFEB
- Fed state:
- TFEB interacts with LYNUS machinery
- mTOR-dependent phosphorylation of TFEB triggers binding of 14-3-3 proteins to TFEB, sequestering TFEB in cytoplasm
- Starved state:
- m-TORC1 released from LYNUS machinery, can no longer phosphorylate TFEB
- Inhibition of lysosomal function reduced mTOR-dependent phosphorylation of TFEB, leading to lower binding of TFEB to 14-3-3, leading to TFEB going to nucleus to stimulate transcription of genes involved in lysosomal biogenesis
- Thus, TFEB controls starvation response by activating lipophagy and fatty acid beta oxidation