Studies on immunoreceptor activation and SARS-CoV-2 RBD/ACE2 interactions
<p>The immune system must maintain a very fine balance recognising threats without damaging healthy cells and it uses cell surface non-catalytic tyrosine phosphorylated receptors (NTRs) to achieve this. In order to study this complex behaviour this work has taken a reductionist approach, using...
| Egile nagusia: | |
|---|---|
| Beste egile batzuk: | |
| Formatua: | Thesis |
| Hizkuntza: | English |
| Argitaratua: |
2022
|
| Gaiak: |
| _version_ | 1826310757502943232 |
|---|---|
| author | Barton, MI |
| author2 | van der Merwe, A |
| author_facet | van der Merwe, A Barton, MI |
| author_sort | Barton, MI |
| collection | OXFORD |
| description | <p>The immune system must maintain a very fine balance recognising threats without damaging healthy cells and it uses cell surface non-catalytic tyrosine phosphorylated receptors (NTRs) to achieve this. In order to study this complex behaviour this work has taken a reductionist approach, using artificial systems in order to control as many variables as possible. The use of a generic cell surface ligand system to activate NTRs has suggested that they share a common mechanism for activation and shed light on specific characteristics of this mechanism. While NTRs are significantly different in terms of sequence homology they share similar structural characteristics which make them sensitive to receptor ligand dimensions. I was also able to show that altering the valency of the ligand could improve activation, but this was not due to clustering of the receptor. The mobility of the ligand was shown not to influence activation. Finally, changing the affinity of the ligand suggests the presence of a kinetic proofreading step during receptor activation.</p>
<p>This system was then applied to the accessory immune receptors CD5 and CD6. Until now, they have been very difficult to understand as they have been reported to both increase and decrease TCR activation. Using a combination of the generic ligand system and mutagenesis I was able to identify and characterise activatory and inhibitory effects of CD5 and CD6. The activatory effect was dependent on ligand engagement and expression of the TCR and comprised of contributions from both the extracellular domain through adhesion and signalling through the cytoplasmic tail. The inhibitory effects were independent of ligand engagement and, in the case of CD6, required the cytoplasmic tyrosine residue 489. CD5 was also suggested to exhibit a dual role with the extracellular domain inducing activation and the cytoplasmic tail responsible for inhibition.</p>
<p>Lastly, due to the emergence of the SARS-CoV-2 virus, the focus of my study shifted from receptor-ligand interactions in the immune response to characterise the effect of common mutations on the interaction of the SARS-CoV-2 Spike protein and its cell surface receptor on human cells ACE2. I purified and obtained detailed affinity data for nine ACE2 missense variants which are present in the human population plus one found through in silico saturation mutagenesis. I then purified and obtained detailed affinity and kinetic data for nineteen RBD constructs which contain mutations found in the common SARS-CoV-2 variants of concern (Alpha, Beta, Gamma, Delta, and Omicron).</p> |
| first_indexed | 2024-03-07T07:58:10Z |
| format | Thesis |
| id | oxford-uuid:de1a7457-a4bd-476e-b23e-d1c2ee3e8eb1 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T07:58:10Z |
| publishDate | 2022 |
| record_format | dspace |
| spelling | oxford-uuid:de1a7457-a4bd-476e-b23e-d1c2ee3e8eb12023-08-29T08:41:07ZStudies on immunoreceptor activation and SARS-CoV-2 RBD/ACE2 interactionsThesishttp://purl.org/coar/resource_type/c_db06uuid:de1a7457-a4bd-476e-b23e-d1c2ee3e8eb1ImmunologyEnglishHyrax Deposit2022Barton, MIvan der Merwe, ASattentau, Q<p>The immune system must maintain a very fine balance recognising threats without damaging healthy cells and it uses cell surface non-catalytic tyrosine phosphorylated receptors (NTRs) to achieve this. In order to study this complex behaviour this work has taken a reductionist approach, using artificial systems in order to control as many variables as possible. The use of a generic cell surface ligand system to activate NTRs has suggested that they share a common mechanism for activation and shed light on specific characteristics of this mechanism. While NTRs are significantly different in terms of sequence homology they share similar structural characteristics which make them sensitive to receptor ligand dimensions. I was also able to show that altering the valency of the ligand could improve activation, but this was not due to clustering of the receptor. The mobility of the ligand was shown not to influence activation. Finally, changing the affinity of the ligand suggests the presence of a kinetic proofreading step during receptor activation.</p> <p>This system was then applied to the accessory immune receptors CD5 and CD6. Until now, they have been very difficult to understand as they have been reported to both increase and decrease TCR activation. Using a combination of the generic ligand system and mutagenesis I was able to identify and characterise activatory and inhibitory effects of CD5 and CD6. The activatory effect was dependent on ligand engagement and expression of the TCR and comprised of contributions from both the extracellular domain through adhesion and signalling through the cytoplasmic tail. The inhibitory effects were independent of ligand engagement and, in the case of CD6, required the cytoplasmic tyrosine residue 489. CD5 was also suggested to exhibit a dual role with the extracellular domain inducing activation and the cytoplasmic tail responsible for inhibition.</p> <p>Lastly, due to the emergence of the SARS-CoV-2 virus, the focus of my study shifted from receptor-ligand interactions in the immune response to characterise the effect of common mutations on the interaction of the SARS-CoV-2 Spike protein and its cell surface receptor on human cells ACE2. I purified and obtained detailed affinity data for nine ACE2 missense variants which are present in the human population plus one found through in silico saturation mutagenesis. I then purified and obtained detailed affinity and kinetic data for nineteen RBD constructs which contain mutations found in the common SARS-CoV-2 variants of concern (Alpha, Beta, Gamma, Delta, and Omicron).</p> |
| spellingShingle | Immunology Barton, MI Studies on immunoreceptor activation and SARS-CoV-2 RBD/ACE2 interactions |
| title | Studies on immunoreceptor activation and SARS-CoV-2 RBD/ACE2 interactions |
| title_full | Studies on immunoreceptor activation and SARS-CoV-2 RBD/ACE2 interactions |
| title_fullStr | Studies on immunoreceptor activation and SARS-CoV-2 RBD/ACE2 interactions |
| title_full_unstemmed | Studies on immunoreceptor activation and SARS-CoV-2 RBD/ACE2 interactions |
| title_short | Studies on immunoreceptor activation and SARS-CoV-2 RBD/ACE2 interactions |
| title_sort | studies on immunoreceptor activation and sars cov 2 rbd ace2 interactions |
| topic | Immunology |
| work_keys_str_mv | AT bartonmi studiesonimmunoreceptoractivationandsarscov2rbdace2interactions |