Eucaryotic cell

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

Lysosomes

Lysosomes

Membrane-bound organelles with acidic interior

Degradation of macromolecules

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

Lysosomal („storage“) diseases

Deficiencies of proteins from the lysosomal system lead to storage of material in lysosomes

Lysosomes

Single-membrane vesicles Acidic interior (pH 4.5-5.5)Participate in cellular vesicular transport Interact with endosomes, Golgi, plasma membrane, endoplasmic reticulum

„Classic“ lysosomes are the principal degradative compartment in the cell“

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)

Lysosomes are a part of the cellular flow of membranes and proteins

Golgi

LE

phagocyticvacuole

autophagicvacuole

LY

EE

NC

secretory vesicle

exocytosis

M6PR

endocytosis

chaperone mediated autophagy

EE – early endosomeLE – late endosomeM6PR – mannosa-6-phosphate receptorLY – lysosomeNC – nucleusred arrows – recycling of M6P between trans-Golgi and LEblue arrows - M6PR „scavenging“ pathway

M6PR - „scavenger pathway“

LYSOSOMES that

KILL !!!KILL !!!

… and their relatives

Secretory lysosomes /Lysosome-related 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 T-

lymphocytes)-azurophilic granules -melanosomes -“external“ lysosomes of osteoclasts- delta-granules in platelets

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 Heřmanský-Pudlák syndrome

heatherkirkwood.blogspot.cz

Lysosome-related organelles - osteoclast

bone

sealing zoneruffled border

sealing zone

H+ H+

H+

Protons and hydrolases (mainly proteolytic) enzymes are excreted into the sealed space between the bone and the ruffled border.

Transport of substrates for degradation

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

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

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

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

Synthesis of M6P signal

Endoplasmic reticulum

trans-Golgi

Sorting of proteins containing MP6 signal

cis-Golgi

protein-M6Pprotein

protein-M6P-M6PR lysosome

protein Secretion pathway

Sorting of proteins containing MP6 signal

The majority of soluble (luminal) lysosomal proteins is transported into lysosome via mannose-6-phosphate receptor

Mutations in GlcNAc transferase gene

endoplasmic reticulum

cis-Golgi

Mutations in GlcNAc transferase gene

trans-Golgi

protein

protein-M6P-M6PR lysosome

protein secretion

Proteins transported normally by Proteins transported normally by M6PR are not targeted to lysosomesM6PR are not targeted to lysosomes

... instead, they are secreted out ... instead, they are secreted out of the cell.of the cell.

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“)

I-cell disease

Coarse faciesthickening of gumssmall hepatomegally and splenomegally bone disease - dysostosis multiplexpsychomotor retardationelevated activities of lysosomal hydrolases in plasma, low activities in tissues

Vacuolization of lymphocytes („Inclusion cell“) = storage lysosomes

Copyright ©2001 BMJ Publishing Group Ltd.

van der Meer, W et al. J Clin Pathol 2001;54:724-726

Figure 1 A lymphocyte with many vacuole-like inclusions (original magnification, x900).

Copyright ©2001 BMJ Publishing Group Ltd.

van der Meer, W et al. J Clin Pathol 2001;54:724-726

Figure 3 Electron microscopic image of lymphocytic vacuoles containing round osmiophilic structures (original magnification, x15 000).

Lysosomal hydrolases and their activators

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

N-acetyltransferase activity(deficient in MPS IIIC)

n=5 n=22 n=103

Patients Heterozygotes Controls

R412X/wt

Patients have very low residual activities(less than several percent of controls). The activities in heterozygotes usually overlap withcontrols – enzyme assay is not reliable fordetection of heterozygotes, while it is suitable for patients.

Liver biopsy (HE): ASM deficiency

NPA storage neuron (HE) 75219

ceramidase

arylsulfatase A

sphingomyelinase

a-n

eura

min

idas

e

* a-

Fu

kosi

das

e

tripeptidylpeptidase I

hyaluronidase (hyaluronic acid)

CoA:a-glukosaminid NAc-transf.

iduronosulfat s

ulfatase

lysosomeexpanded by

storage

enzymopathiesmutant

enzyme protein(n=30)

MPS n=10

GLYKOPROTEINOSES n=7

LIPIDOSESn=9

GSD II

NCL1,2,kong. lysosomal storage disorders Ia

**

*

*

2006hydrolases 29transferase 1

kathepin D

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

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

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

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

Mucopolysaccharides

Mucopolysacccharides (glycosaminoglycans) are polysaccharideswith linear chains with repeating disaccharide units (glucosamin, uronic acid), often heavily modified (acetyl, sulfate groups)

common types :

Heparan sulfateDermatan sulfateKeratan sulfateChondroitin sulfateHeparin has structure resembling heparan sulfate

Glycosaminoglycans are a part of proteoglycans

Glycosaminoglycans are degradedby sequential action of lysosomal exoglycosidases

Mucopolysaccharidoses

11 disorders

Most common :MPS I Hurler disease - deficiency of alpha-iduronidase, AR-inheritanceMPS II - Hunter disease - deficiency of iduronate sulfatase , X-linked

Common symptomsProgressive dementia, hepatosplenomegaly, coarse features (gargoylism), bone disease (dysostosis multiplex),corneal opacities, heart disease

Mukopolysacharidosa III, MPS IIIM. 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 15 - 25 yearsof age

Glycoproteinoses are caused bydeficiencies of enzymes participatingin degradation of N-linked glycoproteins.Clinical symptoms resemble mucopolysaccharidoses.

Activators of lysosomal hydrolases

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.

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 activatoractivates hexosaminidase A

Proteolytic processing of prosaposin

Proposed mechanism of actionof GM2 activator, which activatesbeta hexosaminidase A

Lysosomal membrane proteins

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

X-linked inheritance in Danon disease

Lysosomal transporters deficiencies

Cystinosis – cystinosin deficiencyrenal disease with Fanconi syndromerenal failure – renal transplantationcorneal crystals , photophobiagrowth retardationhypothyroidismnormal inteligence

Isolated ocular form

Sialuria – sialin deficiency

cystine

cysteamine

cystin cysteamin

Cystinosis

Cystinosis

Diagnostics and treatment of lysosomal disorders

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 disordersMPS III – no improvement of neurological progressionOther lysosomal disorders

http://www.bmtinfonet.org/bmt/bmt.book/chapter.1.html#p13

Sorting of proteins containing MP6 signal

cis-Golgi

protein-M6Pprotein

protein-M6P-M6PR lysosome

protein Secretion pathway

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 progresionType 1: chronic non-neuronopathicType 2: acute neuronopathicType 3: chronic neuronopathic

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

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, ...

Substrate

Product

Coenzyme

Apoenzyme

b) Inhibition of enzymes in the metabolic pathway proximal to the metabolic block

„ Substrate inhibition (reduction) therapy“

Substrate inhibition therapy

Mutant enzymes have residual activities

N-butyldeoxyjirinomycin (Zavesca)

Inhibitor of glucosylceramide synthase

Gaucher disease, GM1 gangliosidosis

Diagnostics

Measurement of metabolites

Enzyme activity measurement

Mutation analysis

Morphological diagnostics