Baumeister Autophagy / Stress

• degradation of cytosolic components by lysosomal route

• upregulated during starvation / stress

• quality control (remove protein aggregates, non functional organelles, pathogens)

• evolutionarily conserved

• hand in hand with the Ubiquitin-proteasome system

• protection from DNA damage and chromosomal instability

• Endosomes (peroxisomes) and autophagosomes (from phagophore) fuse with lysosome —> the building blocks for the autolysosomes come from ER, mitos and start at the phagophore assembly site

endoplasmatic reticulum-associated degradation

1. Recognition (of misfolded regions by Chaperones inside ER lumen, ER membrane or in cytosol)

2. Ubiquitination (E1 brings activated ubiquitin, E2 takes it in ATP-dependent process and E3 [ubiquitin ligase] transfers it to the client protein [Lysin residue])

3. Retrotranslocation (Enzyme complex interacts with ubiquitin and foces the protein removal via ATP hydrolysis, removal through channel (Retrotranslocon))

4. Degradation (26 S Proteasome with 3 peptidases cleaves protein, deubiquinating enzymes recycle the ubiquitin)

ERAD is the degradation via proteasomes, the misfolded proteins are “marked” by ubiquitination

ERLAD is the degradation via lysosome, the misfolded protein is glycosylated and then segragated in an ER vesical (with ER phagy receptors) to be degraded

1. IRE1 pathway (regulated mRNA splicing initiated and then mRNA acts as TF and activates chaperone production)

2. PERK (directly reduce more protein production by phosphorylating translation initiation factors and then also activating chaperone transcription)

3. ATF6 (is a TF itself)

All sensors detect in ER lumen and signal cascades out in the cytosol

Hypoxia = lack of oxygen —>triggers dimerization of hypoxia inducible factor 1 which leads to activation of genes (under normoxic conditions that factor is ubiquinated and degraded)

—> it also leads to misfolded proteins because disulphide linkages cant be formed that then triggers UPR because BiP normally binds to the 3 UPR sensors ATF6, PERK and IRE1, it dissociates and activates them when it binds to the mis/unfolded protein

IRE1 and ATF6 both activate transcriptional response whereas PERK also slows down protein translation by phosphorylating translation initiation factor

Hypoxia also inhibits the mTORC1 complex (containing mTOR (target of rapamycin kinase)) which control the whole translation machinery

—> alltogether the hypoxic cells metabolism, autophagy, angiogenese (new blood vessels) and ER homeostasis are altered

• unfolded protein response —> ATF6, PERK, IRE1

• hypoxia —> HiF1

• heat shock —> HSF1

• oxidation stress by ROS (reactive oxigen species) —> Nrf2 (production of antioxidants)

—> because it drives the key energy source: the electron transport chain!

ROS = reactive oxygen species (O²-)

with rising levels more proteins are misfolded and accumulate, the UPRmt is activated if it fails or the ROS level rises too much it leads to mitochondrial damage and then cell death or it leads to mitophagy

ATFS-1 is a TF with a mitochandrial import signal (MTS) [normally dominant] and a nuclear import signal (NLS)

default —> import in Mitos (by TIM and TOM) and degraded (LON protease 1)

stress —> TIM/TOM import blocked so NLS imports it to nucleus and activates UPRmt stress response (as a TF -> genes that help regain mito function and import efficiency…)

TIM and TOM is a mitochondrial channel complex with TOM (translocase of the outer membrane) at the cytosol membrane and TIM (translocase at the inner membrane) at the mitochondrial side of the membrane

ATP dependent import through both membranes

TOM with receptor domain for signal peptide (cleaved after translocation)

The kinase PINK1 binds to damaged Mitochondrium and recuits ubiquitin ligase Parkin

—> ubiquitination of mutiple outher membrane proteins of mito and autophagosomes and lysosomes degrade the Mitochondrium

insert the reporter gene after the promoter that is activated by the TF of the stres response

BiP would be good choice simce IT IS the “First responder” to misfolded Proteins, when IT initiates all the Stress responses by unbinding from them when beimg recruited to the misfolded proteins

e.g. UPRer ATF6 is a direct TF insert the reporter gene after that promotor

or insert it into the transcribed gene for heat shock response hsp-16.2

You need to have a tissue selective promotor, that is only active in neurons or intestine, that promotor will then only allow transcribtion of the reporter gene in the selected tissue

This is problematic in C. elegans because stress in neuron also activates stress response in intestine (i.g. the whole organism is affected by stress)

• you can use tissue specific RNAi / RNAi blockade

• Combination of KO mutant and cell specific promotor expression

macroautophagy —> 2 membranes: an autophagosome transports the material in a vesicle to the lysosome

microautophagy (endosomal)—> 1 membrane: an endosome encloses the material into lysosome

chaperone mediated autophagy (CMA) —> 0 membrane: a chaperone transports e.g. a protein directly to a transloctor at the lysosome

Extra: Mitophagy, Exophagy, piecemeal autophagy of the nucleus

1. Induction

2. Autophagosome formation (ATGs = LC3 in mammels)

3. Vesicle fusion and autophagosome breakdown —> Autolysosome

4. Degradation (in pH 5 level of lysosome via acid hydrolases e.g. cathepsin)

Stress / lack of nutrients or energy / hormones and signaling events all affect (inhibit) the mTORC1 pathway

mTORC1 = mechanistic target of rapamycin 1

mTORC1 as the centre of many signaling pathways in presence of abundant nutrients and growth factors promotes cell growth and metabolic activity and inhibits the ULK1 complex (by binding and phospho inactivating it) and by that autophagy!

—> under stress inhibition of mTORC1 —> suppression of cell growth (energy saving) and induction of autophagy by releasing of ULK1 complex to enable stress adaptaion and suvival

Upstream signaling factors:

• AMPK —> when high levels of AMP = low energy (AMPK can also phospho activate ULK1) (drug metformin mimics starvation and upregulates AMPK)

• HIFs —> hypoxia inducable factors

TOR = Taget of rapamycin —> rapamycin (immunsuppressive drug)

Autophagy control by VPS34 is a positive regulation by membrane recruitment for phagophore assembly:

it recruits ATG complex (=LC3), Bcl-2, Beclin 1 —> phagophore assembly site

—> then the Autophagophore matures and fuses with lysosome —> autolysosome

1. neurodegeneration

   • positive: prevent intracellular proteins from accumulation to toxic levels

   • negative: inefficient lysosomal clearance results in accumulation of autophagosomes —> amyloid precursor into toxic forms

2. cancer

   • pos: tumor suppression by removing damaged organelles, growth factors and stabalize chromosomes

   • neg: protects cancer cells from treatments by helping them survive low nutrient supply

3. infection and immunity

   • pos: bacteria and virus removal

   • neg: microbes have evolved to use autophagy as their niche

Deregulation of autophagy has been linked to cancer, aging, neurodegeneration, metabolic disorders, fibrotic diseases — increasing autophagy is not always beneficial

1. Choose a UPRER-responsive promoter: Identify and select a promoter region that is known to be responsive to ER stress. The promoter region is crucial for controlling the expression of the reporter gene in response to ER stress. Commonly used promoters for UPRER reporters include the promoters of genes such as BiP (Binding immunoglobulin Protein) or CHOP (C/EBP homologous protein).

2. Select a reporter gene: Choose a reporter gene that is easy to detect and quantitate. Commonly used reporter genes include Green Fluorescent Protein (GFP), Luciferase, or beta-galactosidase. The choice of reporter gene depends on the specific requirements of your experiment.

3. Design the reporter construct: Clone the selected UPRER-responsive promoter upstream of the chosen reporter gene. This construct should also include any necessary regulatory elements, such as enhancers and terminators, to ensure proper expression.

Atg8/LC3 (Autophagy-related protein 8/microtubule-associated protein 1A/1B-light chain 3) is a protein involved in autophagy, a cellular process that plays a crucial role in the degradation and recycling of cellular components. The key distinction between Atg8/LC3-I and Atg8/LC3-II lies in their post-translational modifications, which are indicative of different stages of autophagic activity.

1. Atg8/LC3-I:

   • LC3-I is the cytoplasmic form of the protein.

   • It is synthesized in the cytoplasm and remains soluble.

   • LC3-I is typically found in the early stages of autophagy and is not associated with autophagosomal membranes.

2. Atg8/LC3-II:

   • LC3-II is the lipidated and membrane-bound form.

   • During the process of autophagy, LC3-I is conjugated to phosphatidylethanolamine (PE) to form LC3-II.

   • LC3-II is associated with the autophagosomal membrane and is involved in autophagosome elongation and closure.

Why Atg8/LC3 is Particularly Interesting in Cell Biological Research: Atg8/LC3 has become a widely used marker in autophagy research and is often employed to monitor and quantify autophagic activity. Its distinct transition from LC3-I to LC3-II is a reliable indicator of autophagosome formation and autophagic flux. Monitoring LC3 levels and the LC3-I to LC3-II conversion helps researchers study various aspects of autophagy, including its regulation, dynamics, and the impact of different cellular conditions or stimuli on the autophagic process.

a. Main Substrate of mTORC1 in the Control of Autophagy: The main substrate of mTORC1 in the regulation of autophagy is ULK1 (Unc-51-like autophagy-activating kinase 1). ULK1 is a serine/threonine kinase that plays a crucial role in the initiation of autophagy. When mTORC1 is active (under nutrient-rich conditions), it phosphorylates ULK1 at multiple sites, inhibiting its kinase activity. This phosphorylation prevents the activation of the autophagic machinery, leading to suppression of autophagy. In contrast, when mTORC1 is inhibited (under nutrient-deprived or stress conditions), ULK1 is dephosphorylated and activated, promoting autophagy initiation.

b. Phosphorylation Target of mTORC1: 4E-BP (Eukaryotic Initiation Factor 4E-Binding Protein): Phosphorylation of 4E-BP by mTORC1 affects gene expression at the level of translation initiation. 4E-BP normally inhibits translation initiation by binding to eIF4E (eukaryotic initiation factor 4E), a key factor involved in the initiation of cap-dependent translation. When 4E-BP is phosphorylated by mTORC1, its affinity for eIF4E decreases, leading to the release of eIF4E. Released eIF4E can then associate with other initiation factors and the mRNA 5' cap, promoting the assembly of the translation initiation complex.

Consequences of Phosphorylated 4E-BP on Gene Expression:

1. Enhanced Translation Initiation: Phosphorylated 4E-BP releases eIF4E, allowing it to participate in the formation of the eIF4F complex. The eIF4F complex facilitates the binding of mRNA to the ribosome, promoting translation initiation. This leads to increased translation of mRNAs involved in cell growth, proliferation, and survival.

2. Global Impact on Gene Expression: Phosphorylation of 4E-BP by mTORC1 has a general impact on translation initiation, influencing the expression of a wide range of genes. The increased availability of eIF4E can lead to the translation of mRNAs with complex 5' UTR structures or those containing highly structured internal ribosome entry sites (IRES), which may be less dependent on eIF4E for initiation.

3. Cell Growth and Proliferation: The phosphorylation of 4E-BP by mTORC1 is often associated with increased protein synthesis, promoting cell growth and proliferation. This molecular mechanism plays a crucial role in coordinating cellular responses to nutrient availability and growth factor signaling.