Eucaryotic cell. Alberts et al : Molecular biology of the cell 6th edition

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1 Eucaryotic cell Alberts et al : Molecular biology of the cell 6th edition

2 Lysosomes

3 Lysosomes Membrane-bound organelles with acidic interior Degradation of macromolecules Alberts et al : Molecular biology of the cell 6th edition

4 Lysosomal ( storage ) diseases Deficiencies of proteins from the lysosomal system lead to storage of material in lysosomes

5 Lysosomes Single-membrane vesicles Acidic interior (ph ) Participate in cellular vesicular transport Interact with endosomes, Golgi, plasma membrane, endoplasmic reticulum Classic lysosomes are the principal degradative compartment in the cell

6 Lysosomes Lysosomes contain soluble hydrolases with acidic ph optimum Lysosomal membrane contains glycosylated transmembrane proteins (LAMP family, transporters, and others) Lysosomal membrane is enriched in lysobisphosphatidic acid Acidic ph is mantained by vacuolar ATP-dependent proton pump (vacuolar ATPase)

7 Lysosomes are a part of the cellular flow of membranes and proteins endocytosis phagocytic vacuole EE M6PR scavenger pathway EE early endosome LE late endosome M6PR mannosa-6-phosphate receptor LY lysosome NC nucleus red arrows recycling of M6P between trans-golgi and LE blue arrows - M6PR scavenging pathway chaperone mediated autophagy LE LY exocytosis M6PR NC secretory vesicle Golgi autophagic vacuole

8 LYSOSOMES that KILL!!! and their relatives

9 Secretory lysosomes /Lysosomerelated organelles In some cells (often of haematopoietic origin) there are organelles that have properties of both lysosomes and secretory granules - acidic ph - lysosomal membrane and lumenal proteins - exocytosis in response to a stimulus Lysosome-related organelles (LRO) -lytic granules (NK cells and cytotoxic Tlymphocytes) -azurophilic granules -melanosomes - external lysosomes of osteoclasts - delta-granules in platelets

10 Disorders of lysosome-related organelle biogenesis and function A group of hereditary disorders often associated with - albinism (melanosome dysfunction) - visual impairment (melanosome dysfunction) - bleeding tendency(platelet dysfunction) - inflammatory bowel disease - lung fibrosis - immunodeficiency - huge lysosomes in tissues Heřmanský-Pudlák,Griscelli, Chediak-Higashi syndromes Patient with HeřmanskýPudlák syndrome heatherkirkwood.blogspot.cz

11 Lysosome-related organelles osteoclast ruffled border sealing zone H+ H bone + sealing zone H+ Protons and hydrolases (mainly proteolytic) enzymes are excreted into the sealed space between the bone and the ruffled border.

12 Transport of substrates for degradation Alberts et al : Molecular biology of the cell 6th edition

13 Autophagy Macroautophagy Microautophagy Chaperone-mediated autophagy proteins containing specific signal sequence translocation of proteins driven by binding of chaperones internalization via lamp2a receptor in the lysosomal membrane Lysosomal membrane protein LAMP2 is a receptor involved in fusion of autophagic vacuoles with lysosomes

14 Import of lysosomal proteins into lysosome Soluble lysosomal proteins : mannosa-6 phosphate receptor Lysosomal membrane proteins: - signals in short C-terminal tail ) - signals are recognised by adaptor proteins (AP3..) Other - glucocerebrosidase, lysosomal acid phosphatase - prosaposin - sortilin, LIMPII

15 Transport of soluble lysosomal proteins by mannose-6-phosphate receptors

16 Synthesis of M6P signal trans-golgi Endoplasmic reticulum

17 Sorting of proteins containing MP6 signal protein-m6p-m6pr lysosome protein-m6p protein protein cis-golgi Secretion pathway

18 Sorting of proteins containing MP6 signal The majority of soluble (luminal) lysosomal proteins is transported into lysosome via mannose-6-phosphate receptor

19 Mutations in GlcNAc transferase gene cis-golgi endoplasmic reticulum

20 Mutations in GlcNAc transferase gene lysosome protein-m6p-m6pr Proteins transported normally by M6PR are not targeted to lysosomes protein protein secretion... instead, they are secreted out of the cell. trans-golgi

21 I-cell disease (mucolipidosis II) Very rare disorder of transport of M6P-tagged lysosomal proteins due to mutations in GlcNAC phosphotransferase increased activities of lysosomal proteins in extracellular fluid decreased activities of multiple lysosomal enzymes in lysosomes Lysosomal storage - enlarged lysosomes ( I inclusion cell disease )

22 I-cell disease Coarse facies thickening of gums small hepatomegally and splenomegally bone disease - dysostosis multiplex psychomotor retardation elevated activities of lysosomal hydrolases in plasma, low activities in tissues Vacuolization of lymphocytes ( Inclusion cell ) = storage lysosomes

23 Figure 1 A lymphocyte with many vacuole-like inclusions (original magnification, x900). van der Meer, W et al. J Clin Pathol 2001;54: Copyright 2001 BMJ Publishing Group Ltd.

24 Figure 3 Electron microscopic image of lymphocytic vacuoles containing round osmiophilic structures (original magnification, x15 000). van der Meer, W et al. J Clin Pathol 2001;54: Copyright 2001 BMJ Publishing Group Ltd.

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26 Lysosomal hydrolases and their activators

27 Lysosomal enzymes 30 enzymes hereditary deficiencies of which cause human diseases lipids lipidoses, including sphingolipidoses glycosaminoglycans mucopolysaccharidoses N-glycans, oligosacharides glycoproteinoses glycogen glycogenosis type II (Pompe) proteins proteinoses

28 N-acetyltransferase activity (deficient in MPS IIIC) n=5 n=22 n=103 Patients have very low residual activities (less than several percent of controls). The activities in heterozygotes usually overlap with controls enzyme assay is not reliable for detection of heterozygotes, while it is suitable for patients. R412X/wt Patients Heterozygotes Controls

29 Liver biopsy (HE): ASM deficiency

30 NPA storage neuron (HE) 75219

31 enzymopathies p tri D in ep s e I th ka tida ep ylp tid ep * se a n eli y om e A g as hin t p a s lf u s yl ar LIPIDOSES n= tas a f ul ts e MPS n=10 lfa u os nsf. n a r t o c r d NA i idu n i sam o k u :a-gl A o C expanded by storage hy*al * uronidas e (hy * alur onic acid ) * a-fukosidase e ceramidas lysosome minid ase GSD II lysosomal storage disorders Ia mutant enzyme protein (n=30) a-neu ra NCL1,2,kong. GLYKOPROTEINOSES n=7 hydrolases 29 transferase 1

32 Lipidoses 9 types Gaucher disease glucocerebrosidase deficiency Fabryho disease alpha-galactosidase A deficiency Niemann-Pick disease type A/B acid sphingomyelinase deficiency Niemann-Pick disease type C - deficit of proteins involved in intracellular transport of unesterified cholesterol Krabbe disease - beta-galactosylceramidase deficiency Metachromatic leukodystrophy arylsulfatase A deficiency

33 Sugar moiety of glycosphingolipids is sequentially degraded by a set of highly specific lysosomal exoglycosidases.

34 Fabry disease alpha-galactosidase A deficiency X-linked disease lysosomal storage of glycolipids with terminal alpha-galactose, predominantly globotriaosylceramide storage in vessel endothel, smooth muscle of the vessels, cardiomyocytes, glomerules and tubules and other cell types

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38 Fabry disease clinical picture hypertrophic cardiomyopathy, arythmias chronic progressive renal disease leading to renal failure TIA, parestesias angiokeratomas, cornea verticilata X-linked disease In females the severity of phenotype depends on X-inactivation

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41 Mucopolysaccharides Mucopolysacccharides (glycosaminoglycans) are polysaccharides with linear chains with repeating disaccharide units (glucosamin, uronic acid), often heavily modified (acetyl, sulfate groups) common types : Heparan sulfate Dermatan sulfate Keratan sulfate Chondroitin sulfate Heparin has structure resembling heparan sulfate

42 Glycosaminoglycans are a part of proteoglycans

43 Glycosaminoglycans are degraded by sequential action of lysosomal exoglycosidases

44 Mucopolysaccharidoses 11 disorders Most common : MPS I Hurler disease - deficiency of alpha-iduronidase, ARinheritance MPS II - Hunter disease - deficiency of iduronate sulfatase, Xlinked Common symptoms Progressive dementia, hepatosplenomegaly, coarse features (gargoylism), bone disease (dysostosis multiplex), corneal opacities, heart disease

45 Mukopolysacharidosa III, MPS III M. Sanfilippo In the first years of life normal development At 2 6 years of age prominent hyperactivity, sleep disorders, slowly progressive dementia Coarse facies, coarse hair small hepatosplenomegaly Spasticity, dementia, death usually between years of age

46 Glycoproteinoses are caused by deficiencies of enzymes participating in degradation of N-linked glycoproteins. Clinical symptoms resemble mucopolysaccharidoses.

47 Activators of lysosomal hydrolases

48 Glycosidases, which act on glycolipid substrates with shorter oligosaccharide moieties (<3 sugar residues), require activator proteins for their action. The activators make the short oligosaccharide chains of glycolipids, which are embedded in the membranes, accessible to the catalytic proteins.

49 Activators of lysosomal hydrolases Saposins A,B,C,D Arise by proteolytic processing of precursor protein - prosaposin. Saposin deficits lead to variant forms of disorders due to deficiencies of hydrolases activated by respective saposins (e.g. metachromatic leukodystrophy is caused by arylsulfatase A deficiency. Arylsulfatase A is activated by saposin B. Deficiency of saposin B leads to variant form of metachromatic leukodystrophy ) GM2 activator activates hexosaminidase A Proteolytic processing of prosaposin

50 Proposed mechanism of action of GM2 activator, which activates beta hexosaminidase A

51 Lysosomal membrane proteins

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53 Danon disease LAMP2 deficiency Lamp 2 participates in fusion of lysosomes with autophagic vacuoles Cardiomyopathy - usually hypertrophic, but can be also dilated Arrythmia - typically preexcitation syndrome - WPW syndrome Intelectual disability in males Other symptoms X-linked disease females have usually milder phenotype Accumulation of autophagic vacuoles predominantly in cardiac and skeletal muscle

54 X-linked inheritance in Danon disease

55 Lysosomal transporters deficiencies Cystinosis cystinosin deficiency renal disease with Fanconi syndrome renal failure renal transplantation corneal crystals, photophobia growth retardation hypothyroidism normal inteligence Isolated ocular form Sialuria sialin deficiency

56 cystine cysteamine

57 Cystinosis cystin cysteamin

58 Cystinosis

59 Diagnostics and treatment of lysosomal disorders

60 Bone marrow transplantation Haematopoetic stem cell transfer Pro: In contrast to enzyme replacement therapy can influence CNS disease Con: High morbidity and mortality Lysosomal disorders Mucopolysacharidosis I Modifies natural course of the disease Early treatment can prevent neurological disease Residual disease Other MPS disorders MPS III no improvement of neurological progression Other lysosomal disorders book/chapter.1.html#p13

61 Sorting of proteins containing MP6 signal protein-m6p-m6pr lysosome protein-m6p protein protein cis-golgi Secretion pathway

62 Gaucher disease Lysosomal storage disorder Deficiency of glucocerebrosidase (acid beta glucosidase ) Accumulation of glucosylceramide preferentially in cells of macrophage origin (Gaucher cells) Multisystem disorder Hepatomegaly, splenomegaly, bone disease, trombocytopenia, anemia, lung infiltration In type 2 and 3 Gaucher disease: CNS disease Clinical variability, chronic progresion Type 1: chronic non-neuronopathic Type 2: acute neuronopathic Type 3: chronic neuronopathic

63 Enzyme supplementation therapy in Gaucher disease Receptor-mediated endocytosis Macrophage targeted glucocerebrosidase - treatment with exoglycosidases Mannose receptor (macrophages, endothelia, liver) Regular infusions Originally glucocerebrosidase isolated from human placentas (Ceredase, Genzyme) Recombinant enzyme Cerezyme (Genzyme) Cho cells Does not cross haematoencephalic barrier High costs

64 Enzyme supplementation therapy Supplementation of deficient enzyme in regular infusions Gaucher disease (glucocerebrosidase) Fabry disease (alpha galactosidase A) Pompe disease (acid alpha glucosidase) MPS I (alpha iduronidase) MPS II (alpha iduronate sulfatase) MPS VI, Maroteaux-Lamy (arylsulfatase B) Niemann-Pick disease B (acid sphingomyelinase) MPS IVA, Morquio A,... Production of recombinant enzymes Genzyme, TKT, Biomarin, Shire, Inotech,...

65 b) Inhibition of enzymes in the metabolic pathway proximal to the metabolic block Substrate inhibition (reduction) therapy Coenzyme Apoenzyme Substrate Product

66 Substrate inhibition therapy Mutant enzymes have residual activities N-butyldeoxyjirinomycin (Zavesca) Inhibitor of glucosylceramide synthase Gaucher disease, GM1 gangliosidosis

67 Diagnostics Measurement of metabolites Enzyme activity measurement Mutation analysis Morphological diagnostics