lec 18 - nutrient scavenging autophagy (zong)

lysosomes

• characteristics

  • acidic interior → pH = 5

  • contains hydrolytic enzymes (catalyze the breakdown of molecules using water)

  • surrounded by membrane

• lysosomes are analogous to human stomach

  • pH is very acidic

  • enzymes within work effectively in this environment

• pump H+ into lysosome expends energy

  • ATP → ADP

lysosome functions

• intracellular digestion

• recycling cellular organelles

• breakdown of viruses and other cellular invaders

• single celled organisms such as amoebas use lysosomes to digest their food since they have no process for extracellular digestion

lysosomes and cell death

• lysosomes are full of hydrolytic enzymes such as

  • proteases

  • phosphatases

  • lipases

  • nucleases

  • glycosidases

  • sulfatases

• triggers of lysosomal rupture

  • hypoxia

  • lysosomotropic agents

  • DNA damage

  • reactive oxygen species

  • microtubule toxcins

  • photodamage

  • calpain activation

  • hypotonic shock → causes swelling on lysosomal mebranes

• trigger of lysosomal rupture → lysosomal rupture → enzymes released → cathepsin release (proteolytic enzyme that degrades proteins once released in cytosol) → cell death

lysosomal storage diseases

• undegraded material accumulates within the lysosomes of affected individuals

• most of these diseases result from deficiencies in single lysosomal enzymes

  • example = gaucher’s disease

gauchers disease

• most common of lysosomal storage diseases

  • 1 in 50,000 to 100,000 people

• genetic disorder where theres accumulation of lipids in cells and organs

• results from a mutation in the gene that encodes a lysosomal enzyme required for the breakdown of glycolipids → glucocerebrosidase: Gcase

gaucher disease type 1

• anemia

• easy bruising due to low blood platelets

• bone damage

• enlarged liver

• huge spleen

autophagy

• greek

  • auto = self

  • phagein = to eat

• autophagy = the process by which cells recycle cytoplasm and dispose of excess or defective organelles

• autophagy can be induced by cellular stress such as nutrient deprivation

• autophagy promotes cell survival

• excessive autophagy promotes cell death

micro-autophagy

• by invagination of the lysosome membrane, cytosolic components are directly taken up by the lysosome itself

• it may be selective or nonselective

• engulfs material like:

  • proteins

  • lipids

  • organelles

chaperon-mediated autophagy (CMA)

• targeted proteins are translocated across the lysosomal membrane in a complex with chaperone proteins (such as Hsc-70) that are recognize by the lysosomal membrane receptor lysosomal-associated membrane protein 2A (LAMP-2A) → results in their unfolding and degradation

• KFERQ binds to chaperone protein hsc70 → complexbinds to LAMP2A receptor → protein transported into lysosome

macro-autophagy

• delivers cytoplasmic cargo to the lysosome through autophagosome (double membrane bound vesicle)

• autophagosome fuses with the lysosome to form an autolysosome

• could be selective or non selective

• most important type is macroautophagy → referred to as autophagy

types of autophagy

microautophagy

• constitutive → happens regularly under normal conditions

• vesicle-mediated

• proteins/organelles

• selective/nonselective

macroautophagy

• inducible

• vesicle mediated

• proteins/organelles

• selective/nonselective

chaperon-mediated

• inducible

• direct transport

• proteins NO organelles

• selective

autophagy is commonly used to refer to

macroautophagy

macroautophagy diagram

phagospore formation → starts engulfing cytoplasmic material → phagospore closes into a double membraned vesicle called autophagosome → lysosome fuses with autophagosome and forms an autolysosome → material degraded

EM images of autophagosomes

electron microscopy images

• the arrows are pointing to autophagosomes → double membrane

autophagy timeline

• 1955 → christian de duve discovered the lysosome

• 1963 → christian de duve coined the term autophagy

• 1992 → yoshinori ohsumi studied autophagy in yeast and later identified ATG1

• 1999 → beth levine cloned beclin 1 in mammalian cells

• 2002 → protection role of autophagy in huntington disease

• 2003 → tumor suppression role of autophagy in cancer

starved yeast cells increased autophagy

• nutrient deprivation induces autophagy → break down own proteins, lipids and organelles for energy

autophagy related genes

• specific gene related to autophagic activity are named autophagy genes

• table shows some currently known genes involved in various stages of autophagy both in yeast and homologues in humans

LC3 as marker for autophagy

• LC3 → microtubule-associated protein (MAP)-1 light chain 3

  • first identified protein localized in the autophagosome

  • 28% identified as yeast Apg8 which is essential for formation of autophagosome

• LC3-I = cytosolic form

• LC3-II = membrane bound form (phosphatidylethanoamine conjugation of LC1)

LC3 diagram

• LC3-1 + PE → LC3-II → localizes to autophagosomal membrane; serving as marker to visualize autophagy

• western blot

  • shows the effect of treating cells with 2DG (glucose analog that CANNOT be metabolized by mammalian cells) → causes energy stress and activates autophagy

  • increasing LC3-I levels over time indicate autophagic activity

  • tubulin is shown as loading control to confirm equal protein loading

• untreated vs 2DG

  • untreated → minimal LC3 dots

  • treated with 2DG → more LC3 staining → accumulating of autophagosomes due to activated autophagy

signaling pathway and autophagy

nutrient abundance

• activates mTORc1 → inhibits ULK complex which suppresses autophagy

nutrient depletion and stress

• activates AMPK → inhibits mTORc1 → since mTORc1 is NO LONGER SUPRESSES ULK complex, it activates autophagy directly or activates PI3K-III complex which also activates autophagy

growth factors & insulin

• activates PI3K-1 → activates PDK1 → activates AKT → activates mTORc1 which suppresses autophagy

PI3K

• PI3K = lipid kinases that phosphoryate the 3-’OH group of phosphatidylinositol and phosphoinositides → they are critical signaling lipid

PIs

• kinases add phosphate groups to specific positions

• phosphatases remove phosphate groups

• class III PI3K converts PI → PI(3)P → essential step in forming autophagosomal membranes → recruits autophagic machinery and contributes to me,brane trafficking

PI3K and autophagy

growth factors cross into cell → activates class IA PI3Ks → converts PI(4,5)P₂ to PI(3,4,5)P₃ → activates AKT → activates mTOR → promotes translation, proliferation, survival and inhibits autophagy

class III PI3Ks convert PI → PI(3)P → autophagy

so class IA = inhibits autophagy

class III = induces autophagy

vps34 deficiency

• vps34 deficiency leads to enlarged fatty liver

• deficient H&E staining → clear structural disruption

• oil red O staining → significant increase in red lipid drops → confirming fatty liver

• western blot → VPS34 deficient show increased p62 and ubiquinated proteins → they are markers that are degraded via autophagy but since VPS34 is not really there they arent getting degraded

• liver to body mass comparison in fed vs fasted

  • normally, fasting decreases liver mass slightly due to increased autophagy mediated breakdown

  • VPS34-deficient animals fail to signifiantly reduce liver mass → impaired autophagy

• VPS34 = class III PI3K critical for starting autophagy → deficiency = NO autophagy

autophagy has important biological functions

• autophagy activated during fasting or nutrient deprivation

• recycling role

• cellular garbage disposal

• clearance of intracellular pathogens

atg5

• atg5 knockout pups do NOT survive during neonatal nutrient storage

• atg5 is required for autophagosome formation → without it neonatals CANNOT adapt to early nutrient deprivation

autophagy sequesters pathogens

• in starvation → increased LC3

• BCG = pathogen

  • yellow shows where LC3 surrounds BCG

  • limited autophagic response as theres not much yellow in the control but more in starvation → starvation enhances autophagic sequestrationa of bacteria

autophagy inducers

• physiological stress stimuli

  • starvation

• hormonal stimuli

  • glucagon

• pharmacological agents

  • rapamycin, torin 1

• various disease

  • cancer

the role of autophagy in human diseases

• involves in diseases such as:

  • developmental defects

  • crohn’s disease

  • infection and immunity

  • neurodegenerative disease

  • cancer

  • heart disease

  • myopathies

  • ageing

  • metabolic disorders

pro-tumourigenic vs anti-tumourigenic

role of autophagy in cancer

• pro-tumourigenic → PRO TUMOR

  • sustain the elevated metabolic needs of established tumors by degrading and recycling cellular components biosynthetic and energy generation

  • prevents cell death

• anti-tumourigenic

  • buffering metabolic and oxidative stress by preventing toxic buildup of misfolded protein and damaged organelles

  • inhibit cell growth and proliferation

  • promotes non-apoptotic cell death

  • protects genomic integrity

• chloroquine inhibits lysosome acidification and fusion with the autophagosome, blocking the final step → accumulation of undegraded material and blocked autophagy

autophagy can be induced by genotoxins

• Eto increases LC3-II (membrane bound form)→ confirming autophagy induction

• Eto treated cells show many brigh LC3-puncta → autophagosomes forming in response to DNA damage

• eto + 3-MA → fewer LC3 puncta → 3MA inhibits autophagy by blocking class III PI3K

chloroquine (CQ) and hydroxychloroquine (HCQ)

• discovered in 1934 at bayer

• trialed for treating malaria by the US army during WWII → approved in 1947

• deprotonated in the cytosol (physiological pH) → permeable to membrane → trapped in lysosomes where it is protonated at low pH

• when protonated, CQ forms a toxic complex with heme (food for malaria parasites in RBCs → NO food b/c toxic so parasite death)

• CQ and HCQ raise lysosomal pH (they are weak bases so when they are protonated they TAKE an H+ from lysosome → makes it more basic) which inhibits lysosomal activity → blocks autophagy which is good b/c disease can use autophagy to recycle nutrients and avoid cell death (NO autophagy → no nutrients → die)