105 research outputs found
Why Do Some T Cell Receptor Cytoplasmic Domains Associate with the Plasma Membrane?
Based on studies in model systems it has been proposed that the cytoplasmic domains of T cell receptor signaling subunits that have polybasic motifs associate with the plasma membrane, and that this regulates their phosphorylation. Recent experiments in more physiological systems have confirmed membrane association but raised questions as to its function
Influence of lactation on the prolactin secreting cells of the hypophysis of impala (Aepyceros melampus): An immunocytochemical and computer image analysis study
Acute stress in the course of wildlife management has been intensively investigated. Chronic stress,
on the contrary, has not been researched at all, probably due to the difficulty in measuring it as a result
of the overriding effect of the physiological response to the restraining of wild animals. It was therefore
decided to evaluate the use of immunocytochemistry, combined with computer image analysis
to try and determine the magnitude of the structural changes of various hormone-secreting cells of
the hypophysis. Since it was a pilot study to determine whether the combination of immunocytochemistry
with computer image analysis could be of value to distinguish between two normally diverse
groups, it was decided to compare the relative activity of prolactin secreting cells of lactating and nonlactating
impala ewes. After transforming the prolactin immunoreactive area data by log10 to fall inside
the parameters for kurtosis and skewness, a significant difference (P < 0,05, 5-% level, 2-tail)
with the parametric t-test could be shown between the mean prolactin immunoreactive area of lactating
(3,0751 µm2) and non-lactating (3,0467 µm2) ewes. However, the Pearson product moment (r= 0,03)
showed that this difference may not be important for all practical reasons. This may be due to either
sampling errors or limitations of computer image analysis, as it was often difficult to distinguish individual
prolactin immunoreactive areas. Furthermore, a significant increase in the total prolactin immunoreactive
areas of lactating ewes was also established. This technique, however, could distinguish
between the hypophyses of lactating and non-lactating impala ewes, and with further refinement
could be a useful tool in determining chronic stress in wildlife populations.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi.
Adobe Acrobat v.9 was used to OCR the text and also for the merging and conversion to the final presentation PDF-format
A novel adaptor protein orchestrates receptor patterning and cytoskeletal polarity in T-cell contacts.
Recognition of antigen by T cells requires the formation of a specialized junction between the T cell and the antigen-presenting cell. This junction is generated by the recruitment and the exclusion of specific proteins from the contact area. The mechanisms that regulate these events are unknown. Here we demonstrate that ligand engagement of the adhesion molecule, CD2, initiates a process of protein segregation, CD2 clustering, and cytoskeletal polarization. Although protein segregation was not dependent on the cytoplasmic domain of CD2, CD2 clustering and cytoskeletal polarization required an interaction of the CD2 cytoplasmic domain with a novel SH3-containing protein. This novel protein, called CD2AP, is likely to facilitate receptor patterning in the contact area by linking specific adhesion receptors to the cytoskeleton
CD80 (B7-1) Binds Both CD28 and CTLA-4 with a Low Affinity and Very Fast Kinetics
The structurally related T cell surface molecules CD28 and CTLA-4 interact with cell surface ligands CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells (APC) and modulate T cell antigen recognition. Preliminary reports have suggested that CD80 binds CTLA-4 and CD28 with affinities (Kd values ∼12 and ∼200 nM, respectively) that are high when compared with other molecular interactions that contribute to T cell–APC recognition. In the present study, we use surface plasmon resonance to measure the affinity and kinetics of CD80 binding to CD28 and CTLA-4. At 37°C, soluble recombinant CD80 bound to CTLA-4 and CD28 with Kd values of 0.42 and 4 μM, respectively. Kinetic analysis indicated that these low affinities were the result of very fast dissociation rate constants (koff); sCD80 dissociated from CD28 and CTLA-4 with koff values of ⩾1.6 and ⩾0.43 s−1, respectively. Such rapid binding kinetics have also been reported for the T cell adhesion molecule CD2 and may be necessary to accommodate dynamic T cell–APC contacts and to facilitate scanning of APC for antigen
Effects of common mutations in the SARS-CoV-2 Spike RBD domain and its ligand the human ACE2 receptor on binding affinity and kinetics
The interaction between the SARS-CoV-2 virus Spike protein receptor binding domain (RBD) and the ACE2 cell surface protein is required for viral infection of cells. Mutations in the RBD are present in SARS-CoV-2 variants of concern that have emerged independently worldwide. For example, the B.1.1.7 lineage has a mutation (N501Y) in its Spike RBD that enhances binding to ACE2. There are also ACE2 alleles in humans with mutations in the RBD binding site. Here we perform a detailed affinity and kinetics analysis of the effect of five common RBD mutations (K417N, K417T, N501Y, E484K, and S477N) and two common ACE2 mutations (S19P and K26R) on the RBD/ACE2 interaction. We analysed the effects of individual RBD mutations and combinations found in new SARS-CoV-2 Alpha (B.1.1.7), Beta (B.1.351), and Gamma (P1) variants. Most of these mutations increased the affinity of the RBD/ACE2 interaction. The exceptions were mutations K417N/T, which decreased the affinity. Taken together with other studies, our results suggest that the N501Y and S477N mutations enhance transmission primarily by enhancing binding, the K417N/T mutations facilitate immune escape, and the E484K mutation enhances binding and immune escape
Missense variants in human ACE2 strongly affect binding to SARS-CoV-2 Spike providing a mechanism for ACE2 mediated genetic risk in Covid-19:A case study in affinity predictions of interface variants
SARS-CoV-2 Spike (Spike) binds to human angiotensin-converting enzyme 2 (ACE2) and the strength of this interaction could influence parameters relating to virulence. To explore whether population variants in ACE2 influence Spike binding and hence infection, we selected 10 ACE2 variants based on affinity predictions and prevalence in gnomAD and measured their affinities and kinetics for Spike receptor binding domain through surface plasmon resonance (SPR) at 37°C. We discovered variants that reduce and enhance binding, including three ACE2 variants that strongly inhibited (p.Glu37Lys, ΔΔG = –1.33 ± 0.15 kcal mol(-1) and p.Gly352Val, predicted ΔΔG = –1.17 kcal mol(-1)) or abolished (p.Asp355Asn) binding. We also identified two variants with distinct population distributions that enhanced affinity for Spike. ACE2 p.Ser19Pro (ΔΔG = 0.59 ± 0.08 kcal mol(-1)) is predominant in the gnomAD African cohort (AF = 0.003) whilst p.Lys26Arg (ΔΔG = 0.26 ± 0.09 kcal mol(-1)) is predominant in the Ashkenazi Jewish (AF = 0.01) and European non-Finnish (AF = 0.006) cohorts. We compared ACE2 variant affinities to published SARS-CoV-2 pseudotype infectivity data and confirmed that ACE2 variants with reduced affinity for Spike can protect cells from infection. The effect of variants with enhanced Spike affinity remains unclear, but we propose a mechanism whereby these alleles could cause greater viral spreading across tissues and cell types, as is consistent with emerging understanding regarding the interplay between receptor affinity and cell-surface abundance. Finally, we compared mCSM-PPI2 ΔΔG predictions against our SPR data to assess the utility of predictions in this system. We found that predictions of decreased binding were well-correlated with experiment and could be improved by calibration, but disappointingly, predictions of highly enhanced binding were unreliable. Recalibrated predictions for all possible ACE2 missense variants at the Spike interface were calculated and used to estimate the overall burden of ACE2 variants on Covid-19
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Ligand-induced segregation from large cell-surface phosphatases is a critical step in γδ TCR triggering
Gamma/delta (γδ) T cells are unconventional lymphocytes that recognize diverse ligands via somatically recombined T cell antigen receptors (γδ TCRs). The molecular mechanism by which ligand recognition initiates γδ TCR signaling, a process known as TCR triggering, remains elusive. Unlike αβ TCRs, γδ TCRs are not mechanosensitive and do not require co-receptors or typical binding-induced conformational changes for triggering. Here, we show that γδ TCR triggering by nonclassical MHC class Ib antigens, a major class of ligands recognized by γδ T cells, requires steric segregation of the large cell-surface phosphatases CD45 and CD148 from engaged TCRs at synaptic close-contact zones. Increasing access of these inhibitory phosphatases to sites of TCR engagement, by elongating MHC class Ib ligands or truncating CD45/148 ectodomains, abrogates TCR triggering and T cell activation. Our results identify a critical step in γδ TCR triggering and provide insight into the core triggering mechanism of endogenous and synthetic tyrosine-phosphorylated immunoreceptors
Effects of common mutations in the SARS-CoV-2 Spike RBD and its ligand the human ACE2 receptor on binding affinity and kinetics
The interaction between the SARS-CoV-2 virus Spike protein receptor binding domain (RBD) and the ACE2 cell surface protein is required for viral infection of cells. Mutations in the RBD are present in SARS-CoV-2 variants of concern that have emerged independently worldwide. For example, the B.1.1.7 lineage has a mutation (N501Y) in its Spike RBD that enhances binding to ACE2. There are also ACE2 alleles in humans with mutations in the RBD binding site. Here we perform a detailed affinity and kinetics analysis of the effect of five common RBD mutations (K417N, K417T, N501Y, E484K, and S477N) and two common ACE2 mutations (S19P and K26R) on the RBD/ACE2 interaction. We analysed the effects of individual RBD mutations and combinations found in new SARS-CoV-2 Alpha (B.1.1.7), Beta (B.1.351), and Gamma (P1) variants. Most of these mutations increased the affinity of the RBD/ACE2 interaction. The exceptions were mutations K417N/T, which decreased the affinity. Taken together with other studies, our results suggest that the N501Y and S477N mutations enhance transmission primarily by enhancing binding, the K417N/T mutations facilitate immune escape, and the E484K mutation enhances binding and immune escape
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