SMURF1 presentation

Question 1

Glioblastoma overview

Answer 1

-It is an aggressive Malignant primary tumor of the brain or spinal cord

-Forms from mutated glial cells called astrocytes

glioblastoma has a high metabolic rate which results in an accumulation of ROS that causes proteins to unfold (abnormal function of ER chaperones) leading to ER STRESS

• resistant to current therapies due to ability to adapt to oxidative and ER stress by exploiting the UPR pathway

Question 2

ER STRESS/ UPR slide

Answer 2

• evidences have indicated that a high level of UPR is frequently found in human tumors including glioblastoma
• UPR attempts to restore normal function by degrading misfolded protein, increasing production of chaperones, and reduce protein synthesis
• Under ER stress, BiP/GRP78 dissociates from the UPR sensors to activate IRE1, PERK and ATF6α to regulate stress response genes

MILD STRESS results in activation of er associated protein degradation (erad), among other mechanism which ultimately lead to an increase in cell survival

and adaptive UPR promotes cancer survival even in adverse environments
This means that even in SEVER er stress, the cell proliferating and surviving; not undergoing UPR mediated apoptosis

Question 3

Materials and Methods Slide

Answer 3

1. Cell Culture:
   - Cells: U251 & LN229 were cultured in a medium w/antibiotics to use as glioblastoma models

• These cells are used as glioblastoma models to test how SMURF1 regulates KEAP1 and NRF2 pathways under stress.

2. Transfection:
   - Mixed with lipofectamine, either flag-SMURF1 or si-SMURF1 was transfected into the glioblastoma model cells
   - Flag-SMURF1 plasmid induces overexpression of SMURF1
   - si-SMURF1 is an siRNA that prevents the production of SMURF1
   - The transfected cells are then used for various assays
3. Western Blotting:
   - Used to measure protein levels and whether SMURF1 degrades KEAP1
   - cells were lysed and gel electrophoresis was performed using SDS-PAGE
   - proteins were separated by size and transferred to a membrane, detected using antibodies
4. Immunofluorescence:
   - Used to visualize the localization of proteins
   - Cells were fixed and stained with primary and secondary antibodies.
   - The secondary antibody was fluorescently labeled and Fluorescent microscopy was used to track the protein of interest
5. Immunodetection:
   In order to visualize where the protein is in the the tissue, tissues were embedded in paraffin, mounted, then deparaffinized
   Antigens were added to the slides before it was washed and stained
   The stained slides are then visualized under a microscope
6. Ubiquitination Assay:
   - Immunoprecipitation of KEAP1 followed by Western blotting to detect ubiquitination (the addition of ubiquitin chains)
   - Shows that SMURF1 promotes KEAP1 ubiquitination and marks it for degradation.
7. Cell Viability Assays:
   CCK-8 assays to assess cell survival under oxidative stress.
   SMURF1 protects cells from stress, but this effect depends on the presence of NRF2.

Question 4

Depleted SMURF1 & NRF2 reduces tumour size & decreases cell proliferation

Answer 4

they transfected a depletion vector of smurf, and NRF2 as well as a control and stained for

results of the immunohistochemistry to visualize expression f SMURF1, KEAP1, NRF2 and Ki67 in tumour tissue slides

Ki67 is a marker for rapidly dividing cells, a good indicator for cell proliferation

it just shows that when their was a knockout of smurf and/or NRf2, cell proliferation was reduced compared to the control shPLKO

tumour know down smurf showed smaller tumour

Question 5

SMURF1 activates NRF2 signalling pathway by promoting its nuclear import:

Answer 5

• SMURF1 indirectly activates NRF2 by allowing it to move from the cytoplasm to the nucleus, the only place it is able to bind to DNA and promote expression of antioxidant genes (critical for survival)
• In unstressed conditions, NRF2 is targeted for degradation when bound inactive by KEAP1, regulating levels of NRF2 to prevent unnecessary activation of the antioxidant pathway
• in Stress, SMURF1 adds ubiquitin molecules to KEAP1 which tags it for degradation by the proteasome, freeing NRF2

-NR2F can now bind to antioxidant response element (ARE) activating transcription of genes that produce antioxidant enzymes like (SOD) and glutathione that neutralize ROS

• but it also helps cancer cells manage oxidative stress and survive because NRF2 inhibits ROS apoptosis pathway and instead promotes cell survival

meaning targeting the SMURF1/NRF2 complex is a potential player in cancer interventions

• either by inhibiting the pathway and promoting apoptosis or to implement it in the protection of normal cells

Question 6

Smurf activates NRF2 proof

Answer 6

bar graph shows levels of NRF2 inn varying levels of SMURF

y-axis: % of cells with nuclear localization of MYC-NRF2 (NRF tagged with MYC for visualization)

x-axis: HA-Vector is control no smurf
HA-SMURF has smurf overexpression

More MYC-NRF in over expression of SMURF conditions compared to controlled aka smurf1 promotes translocation of NRF2 from cytoplasm to the nucleus because higher nuclear NRF2 means activation of the pathway

proof 2

• TG induces ER stress by blocking calcium pumps
• DMSO is the controlled treatment

in x-axis label myc-NRF2 = compared localization of NRF 2 in cells treated with TG (er stressed) or DMSO, with or without expressions of SMURF1

• in HA it shows whether SMURF was overexpressed or not
• DAPI is used to locate the nucleus
• merge shows all fluorescent channels together, if MYC overlaps with DAPI it indicates its been translocated

summary: when under ER stress, overexpression of SMURF sends NRF2 into the nucleus

Question 7

future studies

Answer 7

Targeting SMURF1 & KEAP1/NRF2 signaling as a potential therapeutic cancer treatment & as a glioblastoma biomarker

Role/presence of SMURF1 in other organismal tissues (ie. liver, lungs, kidney) and in various cancer types