Eucaryotic cell
Transcript of Eucaryotic cell
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