Self-organization of intrinsically disordered proteins with folded N-termini

Thousands of human proteins lack recognizable tertiary structure in most of their chains. Here we hypothesize that some use their structured N-terminal domains (SNTDs) to organise the remaining protein chain via intramolecular interactions, generating partially structured proteins. This model has several attractive features: as protein chains emerge, SNTDs form spontaneously and serve as nucleation points, creating more compact shapes. This reduces the risk of protein degradation or aggregation. Moreover, an interspersed pattern of SNTD-docked regions and free loops can coordinate assembly of sub-complexes in defined loop-sections and enables novel regulatory mechanisms, for example through posttranslational modifications of docked regions

Self-Organization and Regulation of Intrinsically Disordered Proteins with Folded N-Termini

How do mostly disordered proteins coordinate the specific assembly of very large signal transduction protein complexes? A newly emerging hypothesis may provide some clues towards a molecular mechanism

Structural studies of the RAF kinase inhibitor protein family

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Structural insights into engineering a T-cell receptor targeting MAGE-A10 with higher affinity and specificity for cancer immunotherapy

Background T-cell receptor (TCR) immunotherapy is becoming a viable modality in cancer treatment with efficacy in clinical trials. The safety of patients is paramount, so innovative cell engineering methods are being employed to exploit adaptive immunity while controlling the factors governing antigen receptor (ie, TCR) specificity and cross-reactivity. We recently reported a TCR engineering campaign and selectivity profiling assay (X-scan) targeting a melanoma antigen gene (MAGE)-A10 peptide. This helped to distinguish between two well-performing TCRs based on cross-reactivity potential during preclinical drug evaluation, allowing one to be advanced to T-cell immunotherapeutic clinical trials. Here, we present three-dimensional structural information on those TCRs, highlighting engineering improvements and molecular mechanisms likely underpinning differential selectivity.Methods Parental and engineered TCRs were purified and crystallized either alone or complexed to human leucocyte antigen (HLA)-A*02:01 presenting the MAGE-A10 9-mer peptide, GLYDGMEHL (pHLA/MAGE-A10-9). Using X-ray diffraction, we solved four high-resolution crystal structures and evaluated them relative to previously reported functional results.Results The unligated parental TCR displayed similar complementarity-determining region (CDR) loop conformations when bound to pHLA/MAGE-A10-9; a rigid-body movement of TCR beta chain variable domain (TRBV) relative to TCR alpha chain variable domain helped optimal pHLA engagement. This first view of an HLA-bound MAGE-A10 peptide revealed an intrachain non-covalent ‘staple’ between peptide Tyr3 and Glu7. A subtle Glu53-Asp mutation in βCDR2 of the parental TCR generated a high-affinity derivative. Its pHLA-complexed structure shows that the shorter Asp leans toward the pHLA with resulting rigid-body TRBV shift, creating localized changes around the peptide’s C-terminus. Structural comparison with a less selective TCR indicated that differential cross-reactivity to MAGE-A10 peptide variants is most readily explained by alterations in surface electrostatics, and the size and geometry of TCR-peptide interfacial cavities.Conclusions Modest changes in engineered TCRs targeting MAGE-A10 produced significantly different properties. Conformational invariance of TCR and antigen peptide plus more space-filling CDR loop sequences may be desirable properties for clinically relevant TCR–pHLA systems to reduce the likelihood of structurally similar peptide mimics being tolerated by a TCR. Such properties may partially explain why the affinity-enhanced, in vitro-selected TCR has been generally well tolerated in patients

Hypertension and small vessel disease: do the drugs work?

Associations of hypertension with ischaemic stroke and intracerebral haemorrhage, particularly when attributed to cerebral small vessel disease, are well established. While it seems plausible that treating hypertension should prevent small vessel disease from developing or progressing, there is limited evidence demonstrating this. This article critically appraises the evidence answering this clinical question. Hypertension is also closely associated with chronic kidney disease, with anatomical and functional similarities between the vasculature of the brain and kidneys leading to the hypothesis that shared multi-system pathophysiological processes may be involved. Therefore, the article also summarises data on prevention of progression of chronic kidney disease. Evidence supports a target blood pressure of <130/80 mmHg to optimally prevent progression of both small vessel disease and chronic kidney disease. However, future studies are needed to determine long-term effects of more intensive blood pressure treatment targets on small vessel disease progression and incident dementia

Structural and Functional Analysis of Hepatitis C Virus Strain JFH1 Polymerase▿

The hepatitis C virus (HCV) isolate JFH1 represents the only cloned wild-type sequence capable of efficient replication in cell culture, as well as in chimpanzees. Previous reports have pointed to the viral polymerase NS5B as a major determinant for efficient replication of this isolate. To understand the underlying mechanisms, we expressed and purified NS5B of JFH1 and of the closely related isolate J6, which replicates below the limit of detection in cell culture. The JFH1 enzyme exhibited a 5- to 10-fold-higher specific activity in vitro, consistent with the polymerase activity itself contributing to efficient replication of JFH1. The higher in vitro activity of the JFH1 enzyme was not due to increased RNA binding, elongation rate, or processivity of the polymerase but to higher initiation efficiency. By using homopolymeric and heteropolymeric templates, we found that purified JFH1 NS5B was significantly more efficient in de novo initiation of RNA synthesis than the J6 counterpart, particularly at low GTP concentrations, probably representing an important prerequisite for the rapid replication kinetics of JFH1. Furthermore, we solved the crystal structure of JFH1 NS5B, which displays a very closed conformation that is expected to facilitate de novo initiation. Structural analysis shows that this closed conformation is stabilized by a sprinkle of substitutions that together promote extra hydrophobic interactions between the subdomains “thumb” and “fingers.” These analyses provide deeper insights into the initiation of HCV RNA synthesis and might help to establish more efficient cell culture models for HCV using alternative isolates

Alanine scanning mutagenesis reveals amino acids of LOX-PP required for CIN85 binding and c-Cbl competition.

<p>(<b>A</b>) To begin to identify the critical amino acids in LOX-PP, vectors expressing individual point mutants in which residues aa 111 to aa 120 were replaced with alanine, or WT LOX-PP protein were co-transfected with FLAG-CIN85 WT in HEK293T cells. (right panel) The mutated proteins compared with the WT LOX-PP for their ability to interact with CIN85 using GST-pull down assays and WB with FLAG and GST antibodies. (left panel) Input, 4%. EV, empty vector DNA. (<b>B</b>) To confirm the interaction of CIN85 and endogenous c-Cbl, GFP or GFP-CIN85 proteins were expressed in HEK293T cells and TX-100 extracts prepared. Following immunoprecipitation with a GFP antibody, the precipitated proteins subjected to WB with antibodies against c-Cbl (upper panel) and GFP (lower panel). (<b>C</b>) To test whether LOX-PP mutants unable to interact with CIN85 compete for its binding with c-Cbl, GFP-tagged-full-length CIN85 was co-expressed with GST, or GST-tagged LOX-PP-WT (PP-WT-G), LOX-PP-P111A (PP-P111A-G), or LOX-PP-R116A (PP-R116A-G) in HEK293T cells. TX-100 extracts were immunoprecipitated with GFP antibody and the precipitated proteins detected with antibodies against c-Cbl, GFP and GST. Input, 4%. (<b>D</b>) To test the specificity of the changes in binding of the mutant LOX-PP proteins, vectors expressing GST tagged LOX-PP-WT (WT), or mutants LOX-PP-P111A (P111A) or LOX-PP-R116A (R116A) or GST (EV) were transfected into ZR-75 cells and their ability to interact with c-Raf, which maps to aa 26-100, monitored by GST-pull down assays. WB for CIN85 (Upstate: clone 84), c-Raf and GST was performed.</p