Traffic Problems: Inherited Disease and Intracellular ... · Traffic Problems: Inherited Disease and Intracellular Trafficking Defect Dr.Paul Gissen The screen versions of these slides

Traffic Problems: Inherited Disease and Intracellular Trafficking Defect

Dr. Paul Gissen

1The screen versions of these slides have full details of copyright and acknowledgements

1


Dr. Paul Gissen

UCL Laboratory for Molecular Cell Biology

and

Great Ormond Street Hospital for Children

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Summary

• Concepts and pathways in intracellular trafficking

• Disorders affecting specific trafficking steps

• ARC syndrome as an example of a trafficking disorder

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1. Protein secretion

– Polypeptides synthesised in the ribosomes

– Folding +/- oligomerisation in the ER

– In ER proteins are packaged into transport vesicles to travel to Golgi

– Post-translational modification and sorting into various routes in Golgi

– Golgi to cell membrane transport via intermediate compartments

2. Endocytosis:

– Internalised proteins

Either recycled to the membrane

Or trafficked to lysosomes

Or transcytosed from basolateral to apical membrane

Intracellular protein trafficking


Dr. Paul Gissen


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Nucleus

ER

Golgi

Lysosome

MVB

EE

LE

Intracellular vesicle traffic

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Clinical syndromes with abnormal intracellular trafficking

• Batten’s disease/neuronal ceroid lipofuscinosis

(accumulation of protein degradation products)

• Niemann Pick type C disease (lipid trafficking defect)

• Mucolipidosis (abnormal transport

of lysosomal enzymes)

• Hermansky-Pudlak syndrome(HPS)

(abnormal biosynthesis of pigment organelles)

• Hereditary spastic paraplegias (defect in trafficking

along axons)

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Donor compartment

Acceptor compartment

Microfilaments

Molecular motors


Dr. Paul Gissen


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• Romanian - US scientist

• Nobel Prize in physiology and medicine 1974

• Identification of cell structure

• Shared with Christian De Duve and Albert Claude

Mitochondria RER Golgi interface

George Palade (1912-2008)

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Membrane Interaction during secretionGolgi apparatus and protein secretion

9 Kallunki et al., Oncogene 2012

• Belgian scientist


• Identification of cell structure: lysosomes, peroxisomes

• Shared with George Palade and Albert Claude

Christian De Duve (1917-2013)


Dr. Paul Gissen


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• US scientist

• 1936- (Washington University, St Louis)

• Biosynthesis, processing, and maturation of N-linked glycan chains

• Phosphorylation of N-glycans on lysosomal enzymes,

which mediates their selective targeting to lysosomes

Stuart Kornfeld

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• US scientist

• 1947- (Yale University)


with Randy Sheckman and Thomas Sudhof

• Developed in vitro assays reproducing intracellular trafficking events

• Identified and cloned the proteins that constitute membrane docking

and fusion machinery

• Introduced SNARE hypothesis

Sollner et al, 1993

James Rothman

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• US scientist 1948- (UC Berkeley)

• Nobel Prize in Physiology and medicine 2013

with James Rothman and Thomas Sudhof

• Isolated secretory (sec) mutants in yeast

• Identified genes

and established biochemical

reactions to reproduce specific

secretory pathway events

Randy Schekman


Dr. Paul Gissen


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Intracellular trafficking machinery

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Cargo recruitment and vesicle biogenesis

• Process requires multisubunit adaptor proteins e.g. AP1

• Coat proteins e.g. Clathrin, COPI, COPII

• Coats, coat-associated proteins, PIPs and Rab proteins

provide cargo specificity

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Disorders of vesicle biogenesis

• Abnormality in COPII vesicle formation

and procollagen trafficking:

– Cranio-lenticulo-sutural dysplasia: SEC23A

• Abnormal biogenesis of melanosomes

and other lysosome related organelles

– Hermansky-Pudlak Syndrome (HPS1-9)


Dr. Paul Gissen


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Defects in Rabs and Rab-associated proteins

• Rab proteins: small GTPases

• Involved in multiple steps of vesicular tafficking:

diverse phenotypes

• Change conformation from active: GTP-bound

to inactive: GDP-bound

• Rabs recruit effector proteins

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Rab cycle

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Rab associated disorders

• Choroideremia: REP1

• Cone-rod dystrophy 18: RAB28

• Griscelli syndrome type 2: RAB27a

• Charcot-Marie-Tooth disease, type 2B: RAB7

• Warburg Micro syndrome: RAB3GAP1, RAB3GAP2, RAB18

• Mental retardation, X-linked 72: RAB39B

• Mental retardation, autosomal dominant 5: SYNGAP1


Dr. Paul Gissen


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Cytoskeleton and associated proteins

• Vesicles are guided to the target membrane by cytoskeleton

• Consists of microtubules and microfilaments

• Microtubules radiate from microtubule

organising centre (centriole)

• Cytoskeletal motors e.g. kinesins and dyneins carry cargos

along the MT

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Dynamic microtubules

T Mitchison

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Mitochondria trafficked along microtubules


Dr. Paul Gissen


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Disorders of cytoskeletal traffic

• Hereditary spastic paraplegias:

– SPG4 (commonest HSP) +/- cognitive impairment:

SPASTIN (MT associated protein)

– Troyer syndrome: SPARTIN (MT associated protein)

– Charcot Marie Tooth 2a (axonal type): KIF1B

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Kinesin movement along microtubules

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Vesicular tethering and fusion

Neuromuscular junction

Synapse in the brain


Dr. Paul Gissen


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SNAREs and membrane fusion

• SNARE (Soluble NSF attachment

protein REceptor) form tight 4-helix bundles

that pull apposing membranes together

• SNARE-complex formation

• Tethering factors (multisubunit complexes)

bring the two SNAREs to appose each other

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Disorders associated with defective vesicle fusion

• Infantile epileptic encephalopathy: STXBP1

• CEDNIK syndrome: SNAP29

• Arthrogryposis, renal dysfunction and cholestasis syndrome:

VPS33B+ VIPAR

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Arthrogryposis, renal dysfunction, cholestasis syndrome

• Incidence >1:100,000 in the UK

• 1-5% of infantile cholestasis patients

• Autosomal recessive, variable severity

• Panethnic

• Multisystem involvement: neuromuscular,

kidneys, liver, platelets, bone, immune system

• Defects in VPS33B or VIPAR

Gissen et al., Nat Genet 2004

Cullinane et al., Nat Genet 2010


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• Jaundice

• Non-excreting biliary isotope scan

• Deficiency of terminal bile ductules

• Abnormal apical membrane protein localisation

Liver

Mislocalisation of the bile salt export pump

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PatientControl

Kidney

Mislocalisation of CD26

Loop of Henle

Bowman’s capsule

Variable degree

of tubular and glomerular

dysfunction

Proximal tubule

Renal tubule Renal tubule cells

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Control platelet

ARC platelet

ARC platelet

ARC platelet

ARC platelets – a-granule defect


Dr. Paul Gissen


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Osteopenia, fractures, skin laxity

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Fusion

SNAREpinformation

VPS33B

• Sec1/Munc18 (SM) protein family

• SM proteins interact with SNAREs

• Aid SNARE complex formation

• VPS33B possibly interacts with syntaxin 6

(endosomes and TGN)

VIPAR

• Encoded by VIPAS39

• Contains a golgin A5-like domain

• Golgins – structural support for Golgi complex

and involved in membrane tethering events

VPS33B and VIPAR

Südhof T C PNAS 2007;

104: 13541-13542

• VPS33B and VIPAR form a complex and interact with RAB11A

(recycling endosomes)

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What is the intracellular localisation of VIPAR/VPS33B?

• Interaction with Rab11a was detected

• Rab11a is present in apical recycling endosomes

and is involved in apical membrane protein recycling


Dr. Paul Gissen


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Cell model of ARC

• Murine inner medullary collecting duct cells (mIMCD3)

• Stable knockdown of Vps33b and Vipar achieved

• Resistance measured across a monolayer to detect polarisation

• Are the defects seen due to the mislocalisation

of membrane transporters?

• Commonest cause of failure to polarise is the defect in tight junctions

Ω

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Reduced expression of E-cadherin in kd cells

Control shRNA Vipar shRNA Vps33b shRNA

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Reduced E-cadherin expression is due to transcriptional downregulation

E-cadherin

b-actin

b-catenin

Protein expression RNA expression Promoter activity

• No recovery of E-cadherin after protease inhibitor treatment

• Normal exogenous E-cadherin trafficking in kd cells

Control shRNA

Vps33bshRNA

ViparshRNA

Perc

enta

ge E

xpre

ssio

n o

f E

-cadherin r

ela

tive

to c

ontr

ol shR

NA

Lucifera

se a

ctivi

ty r

ela

tive

to

contr

ol shR

NA

Control shRNA

Vps33bshRNA

ViparshRNA


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Zebrafish model of ARC syndrome

Matthews et al., Development, 2005

Vipar ATG MO Injected

Non-Injected control

Brightfield PED-6

NSB

G

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E-cadherin expression in human and zf VIPAR deficiency

Non-injected control

Exon 3 mis-match

Exon 3 MOATG MO

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Conclusions: ARC syndrome

• ARC syndrome is caused by defects in VPS33B and VIPAR

• VPS33B and VIPAR form a complex and are associated

with recycling endosomes

• Still unclear pathway for the loss of apical-basolateral polarity


Dr. Paul Gissen


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Finally

• Many inherited diseases are caused by intracellular

trafficking defects

• Common disorders such as dementia and diabetes

associated with intracellular trafficking abnormalities

• Further research will improve disease understanding

and treatments

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Documents

Traffic Problems: Inherited Disease and Intracellular ... · Traffic Problems: Inherited Disease and Intracellular Trafficking Defect Dr.Paul Gissen The screen versions of these slides