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Western University Biochemistry 3385A Final Exam Study Guide
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BIOCHEM3385 FINAL EXAM REVIEWTopic 3 Lecture 1: COVID-19 Tissue/Organ Damage
SARS-CoV-2 (positive-sense single strand RNA virus)
o Structure: RNA genome with 6 ORFs (+ 7 ORFs for accessory proteins)
Envelope, Membrane, Nuclear Capsid, Spike (host binding), & Replicase
o Infection: primarily spreads through respiratory droplets
Binds to ACE2 (angiotensin converting enzyme 2 – ectopeptidase)
S1 region of the spike protein binds to this
Cleavage at S1/S2 and S2 regions of spike protein by TMPSS2 –
priming step, truncated protein to fuse viral and cell membranes
o Extra PRRAR motif = allows more serine proteases to
cleave and prime it = potential reason for more
transmissible
ACE2 normally produces angiotensin that signals through MAS protein –
signals for anti-inflammatory/vasodilation/vascular protection AND
inhibits pro-inflammatory/vasoconstriction/oxidative stress
Virus may inactivate it? Cannot function properly
o Infection & Replication: priming & fusion
Uncoating, translation of replicase
Proteolytic activation
RNA replication
Transcription/translation
Assembly, budding, release of new virions
o Tissue/Organ Damage of COVID-19:
Lungs are primary site like SARS-CoV
Can also impact kidneys, liver, heart, skin, eyes, brain, GI tract
Impact on organs may correlate with where ACE2 receptors are
most abundant
o Sepsis: organ dysfunction due to a dysregulated response to infection
Excessive inflammatory response (but NOT increased inflammatory cells
– patients were actually leukopenic), multi-system changes that can cause
multi-organ failure
Topic 3 Lecture 2: Inflammatory Responses to COVID-19 (Part 1: Transcriptomes)
Study was to identify the gene transcription of leukocytes in COVID+ and COVID-
patients in the ICU with sepsis (COVID+ patients were leukopenic)
o Blood samples from 7 COVID- and 7 COVID+ patients (BUFFY COAT –
immune cells and platelets), extracted RNA from buffy coat
PCA Analysis of RNA Transcripts:
o Clustering of COVID-19 transcripts = similar transcription responses
“Volcano” Plot:
o 2 sets of genes = different gene expressions b/w COVID- and + patients
“Heat Map” of Gene Expression:
o COVID+ Patients: mostly gene shutdown, but some upregulation
o COVID- Patients: mostly gene upregulation, but some downregulation
Found 1,311 differentially expressed genes b/w COVID- and + patients
, 254 upregulated, 1057 downregulated
Similar transcriptomes b/w COVID+ patients
Metascape Functional & Reactome Pathway Analyses: clusters and colours based on
intensity of change; identified relations b/w the genes that were differentially expressed
o 3 main pathways affected by COVID-19:
Interferon Signalling – upregulated
Cell Cycle – downregulated
Protein Translation – downregulated (dramatic – trying to limit viral
protein translation too)
Interferons (IFNs): cytokines associated with viral infections (innate antiviral response)
o Type 1 (alpha, beta, omega) – secreted by fibroblasts/platelets/monocytes, signal
through IFN-alpha/beta receptors (IFNAR)
o Type 2 (gamma) – secreted by cytotoxic T cells (NK – natural killer), signal
through IFN-gamma receptors (IFNGR)
Interferon Signalling: IFN binds to receptor, signals through JAK1/JAK2
and TYK2, recruits and phosphorylates STAT1/STAT2
Type I/III: STAT1/STAT2 then recruit IRF9, complex binds to
ISREs on DNA, transcription of ISGs (IFN-stimulated genes)
Type II: STAT1/STAT2 bind to GAS on DNA, transcribe ISGs
Signalling Continued: transcription of ISGs stimulates shutdown of
protein translation and cell cycle
Also have RNA degradation, upregulation of E2F/pRb (cell cycle),
TLRs (toll-like receptors that detect viral pathogens)
o Acute Anti-Viral Responses: recruitment of pro-inflammatory monocytes,
NK/cytotoxic T cells (to clean up)
o Chronic Anti-Viral Responses: inflammatory cytokine persistence, immune cell
suppression and promote apoptosis
Topic 3 Lecture 3: Inflammatory Responses to COVID-19 (Part 2: Cytokines)
Study plasma cytokines/inflammatory analytes over time compared to the leukocyte
transcriptomes of COVID+ patients (now looking at proteins)
o Blood samples from 10 COVID- and 10 COVID+ patients (& 10 controls)
PLASMA (excluded buffy coat)
tSNE Analyses and Disease Association Data:
o Top 6 analytes: TNF, Granzyme B, HSP70, IL-18, IP-10, Elastase 2
Change in Analyte [] Over Time:
o Found significant differences b/w COVID- and + between 2-3 days (levels in
COVID+ were higher)
Tumor Necrosis Factor (TNF): protein secreted by macrophages/other
leukocytes/fibroblasts, major pro-inflammatory mediator of septic shock (signals through
caspases, MAPK, and NF-kB to induce apoptosis, inflammation and survival)
o Plasma TNF levels are usually elevated temporarily in response to tissue
damage/sepsis/ARDS
BUT in COVID-19+ patients, elevated levels were sustained (despite low
leukocyte #)
Granzyme B: serine protease secreted by cytotoxic T/NK cells alongside perforin (pore-
forming protein) to induce apoptosis in tumors/virally-infected cells
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