Journal: Communications Biology

Article Title: Competitive binding of actin and SH3 domains at proline-rich regions of Las17/WASP regulates actin polymerisation

doi: 10.1038/s42003-025-08188-4

Figure Lengend Snippet: A Schematic diagram outlining one possible arrangement of Sla1 SH3 domains bound to the N-terminal PPR of Las17 (amino acids 300-422). SH3 domains of Sla1 are numbered #1, #2 and #3. The PPR-N of Las17 is the region shown to bind SH3 domains. PP in pink boxes indicate poly-proline tracts in this region. Other regions of Las17 are included but not to scale. B Representative pyrene actin assays show inhibition of Las17-Arp2/3 mediated actin polymerisation by Sla1-SH3 domains when added as separate domains (orange #1, green #2 or cyan #3) or on a single peptide (red). Actin; 3 µM, Las17; 300 nM, and Sla1 SH3 domains; 300 nM each. C Biolayer Interferometry measurements of the affinity of Sla1 SH3 domains for Las17 (aa 300-422). Sla1 SH3#1 (aa 3-68), SH3#3 (aa 354-413), SH3#1-2 (aa 5-131), SH3#1-3 (aa 5-413).

Article Snippet: Membrane dot blots carrying overlapping 12-mer peptides of Las17 over the region 300-536 were purchased from Intavis Celluspots.

Techniques: Inhibition

Journal: Communications Biology

Article Title: Competitive binding of actin and SH3 domains at proline-rich regions of Las17/WASP regulates actin polymerisation

doi: 10.1038/s42003-025-08188-4

Figure Lengend Snippet: A Schematic diagram of Las17. The PPR from residues 300-535 is subdivided into PPR-N (300-422) and PPR-C (423-535). The amino acid sequence is shown for PPR-N with three polyproline (PP) motifs indicated. Lines above the sequence are SH3 domain binding consensus sequences with green indicating type-I SH3 domain binding motifs, and blue type-II SH3 domain binding motifs. Underscored arginine residues in red text motifs have previously been identified as important in G-actin binding. Pink text indicates a predicted new ABS region. B A region of the HSQC spectrum from the titration of 15 N-labelled Sla1 SH3 domains 1 and 2 with peptide 3 is shown. For clarity only the titrations with 0, 1, 2, 3, 5 and 8 peptide equivalents are shown (red, orange, yellow, green, blue, purple respectively). C Chemical shift changes in protein signals were fitted to standard equations (Williamson 2013) to estimate binding affinity. The estimated affinities clearly fell into two groups: signals from SH3#1 (top panel) fitted to a common affinity of 24 μM, while signals from SH3#2 (bottom panel) fitted to a common affinity of 190 μM. The signals shown are (top) 15 N shifts for I38 (blue), W42 (red) and W41 (purple) (bottom) 1 H shifts for W108 (blue), G124 (red) and N85 (purple). D Chemical shift changes on addition of each peptide (PP1, PP2, PP3) are shown as the weighted chemical shift changes for 1 H and 15 N, [Δδ H 2 + (0.14Δδ N 2 )] 1/2 . The three peptides bind at similar locations, although peptide 1 binds approximately three times more weakly. The approximate affinities for PP1, PP2 and PP3 obtained from these data are respectively 70, 22 and 24 μM for SH3#1 and 550, 160 and 190 μM for SH3#2. E The binding site on the protein for peptide 3. Chemical shift changes on addition of peptide 3 were used to calculate the mean and standard deviation weighted shift change for each protein residue. Residues with shift changes larger than (mean + sd) are indicated in orange for domain 1 and red for domain 2, and comprise V11, Y12, Y14, S36, I38, D39, W41 and W42 (domain 1), and D105, A106 and W108 (domain 2). The figure shows two views rotated by 180°.

Article Snippet: Membrane dot blots carrying overlapping 12-mer peptides of Las17 over the region 300-536 were purchased from Intavis Celluspots.

Techniques: Sequencing, Binding Assay, Titration, Standard Deviation, Residue

Journal: Communications Biology

Article Title: Competitive binding of actin and SH3 domains at proline-rich regions of Las17/WASP regulates actin polymerisation

doi: 10.1038/s42003-025-08188-4

Figure Lengend Snippet: A A representative pyrene assay comparing the impact of Las17 fragment (300–633) with the minimal fragment (342–392) on actin polymerisation (in the absence of Arp2/3). 300 nM each fragment; 3 µM actin. B MST was used to measure binding of different concentrations of Las17 (wild type or with mutations in the actin binding sites (ABS)) to labelled actin. Error bars are standard error of mean. Green trace shows the impacts of all three sites mutagenized; dark blue is with no sites mutagenized.

Article Snippet: Membrane dot blots carrying overlapping 12-mer peptides of Las17 over the region 300-536 were purchased from Intavis Celluspots.

Techniques: Binding Assay

Journal: Communications Biology

Article Title: Competitive binding of actin and SH3 domains at proline-rich regions of Las17/WASP regulates actin polymerisation

doi: 10.1038/s42003-025-08188-4

Figure Lengend Snippet: All residues are shown as a cartoon ribbon except for arginines that flank the polyproline sequence of the ABS sites, and key interacting actin residues. Yeast G-actin is shown in green (PDB: 1YAG). A Structures for four of the top ten HPEPDOCK predictions of ABS3 docking show interactions in the barbed end groove of the actin monomer. Peptides are shown in different colours. B All four of these structures coordinate actin residue E334 via the double arginine pair (R6 and R7 in the docked peptide). C A structure illustrating one of the top ten structure predictions for ABS1 shows how arginines at each end of the peptide of Las17 may interact simultaneously with acidic residues flanking the barbed end groove of actin (E334, and E361/364). D All three docked peptide structures can be modelled to illustrate binding of three actin monomers along the Las17 peptide. The ABS sequences are shown in red whilst the adjoining sequences that were part of the modelling in pink. The dashed pink line indicates parts of Las17 with no structural information.

Article Snippet: Membrane dot blots carrying overlapping 12-mer peptides of Las17 over the region 300-536 were purchased from Intavis Celluspots.

Techniques: Sequencing, Residue, Binding Assay

Journal: Communications Biology

Article Title: Competitive binding of actin and SH3 domains at proline-rich regions of Las17/WASP regulates actin polymerisation

doi: 10.1038/s42003-025-08188-4

Figure Lengend Snippet: A Microscale thermophoresis showing binding of Las17 to actin in the presence (red) or absence (blue) of Sla1 SH3 domains #1–3. The concentration of actin (50 nM) and Sla1 (4.5 µM) were kept constant throughout the experiment, and the concentration of Las17 300–422 was varied between 0.29 and 9.5 µM. Error bars are standard deviation. B Liposome co-sedimentation assay shows that GST-Sla1 SH3#1-3 co-precipitates with liposomes prepared from bovine brain extract, whereas GST alone does not. C Quantification of liposome co-sedimentation assays. Šídák’s multiple comparisons test P < 0.0001 ( n = 6). D Representative pyrene actin assay showing alleviation of Sla1 inhibition (red) by Sec4 (pink). Actin only (black); Actin + Las17 (blue). E Alphafold prediction of an interaction of Sec4 with a region of Las17 primarily between 327 and 333 which lies at the C-terminal end of the first Las17 PP motif. Shown is surface representation of Sec4 in green and ribbon depiction of Las17 in blue with predicted interacting surface residues within 3.5 Å in red.

Article Snippet: Membrane dot blots carrying overlapping 12-mer peptides of Las17 over the region 300-536 were purchased from Intavis Celluspots.

Techniques: Microscale Thermophoresis, Binding Assay, Concentration Assay, Standard Deviation, Sedimentation, Liposomes, Pyrene Actin Assay, Inhibition

Journal: Communications Biology

Article Title: Competitive binding of actin and SH3 domains at proline-rich regions of Las17/WASP regulates actin polymerisation

doi: 10.1038/s42003-025-08188-4

Figure Lengend Snippet: A Purified individual GST-tagged Sla1 SH3 domains were used to probe a Celluspot array consisting of a series of 12 amino acid peptides starting with residue 181, and subsequent peptides starting at two amino acid intervals, up to amino acid 540. Binding of GST-Sla1 SH3 to the array was identified by further probing of HRP-tagged anti-GST and subsequent visualisation using chemiluminescence. Peptide spots corresponding to actin binding sites on Las17 (pink), additional spots including P387A (red) and P388A (green) and their corresponding wild type peptide (blue) are also shown. Binding to the peptide containing P387A is strongly reduced or abrogated for each Sla1 SH3 domain. B Microscale Thermophoresis of Las17 300–422 wild type (blue), P387A (red) and P388A (green) showing that neither of these mutations affects Las17 binding to actin. Error bars are standard error of mean.

Article Snippet: Membrane dot blots carrying overlapping 12-mer peptides of Las17 over the region 300-536 were purchased from Intavis Celluspots.

Techniques: Purification, Residue, Binding Assay, Microscale Thermophoresis

Journal: Communications Biology

Article Title: Competitive binding of actin and SH3 domains at proline-rich regions of Las17/WASP regulates actin polymerisation

doi: 10.1038/s42003-025-08188-4

Figure Lengend Snippet: A – C Lifetime of patches from wild type (red) or las17 P387A (blue) expressing cells tagged with fluorescent markers, alongside representative kymographs. A Las17-GFP, ( B ) GFP-Abp1, ( C ) Arc-15 mCherry (describing Arp2/3 complex lifetime). Statistical test Unpaired Mann Whitney test, **** indicates p value < 0.0001. A size bar for each set of kymographs is shown on the upper right of each set. For ( A ) and ( C ) size bar is 100 nm and for B is 200 nm. Error bars are standard deviation. D Time lapse images showing co-localisation of Las17-GFP (green dots) and Arc15-mCherry (red dots) in wild type cells and las17 P387A cells. The initial las17 P387A images contain two adjacent endocytic sites. The site of interest is labelled in the first two time panels with a white arrow. Exposure 0.5 sec with 1 second time lapse. 120 s recorded. E Quantification of lifetime of Las17-GFP alone (green), Arc15-mCherry alone (red) and overlap between Las17-GFP and Arc15-mCherry (yellow) from multiple endocytic patches is shown ( n = 13). Error bars are standard deviation.

Article Snippet: Membrane dot blots carrying overlapping 12-mer peptides of Las17 over the region 300-536 were purchased from Intavis Celluspots.

Techniques: Expressing, MANN-WHITNEY, Standard Deviation

Journal: STAR Protocols

Article Title: Protocol to detect antigen-specific nasal-resident T cells in humans

doi: 10.1016/j.xpro.2022.101995

Figure Lengend Snippet:

Article Snippet: 15-mer SARS-CoV-2 overlapping Spike, Nucleoprotein, Membrane and Non-structural protein 12 (NSP12) peptide pools (Lim et al. ) , Genscript , N/A.

Techniques: Enzyme-linked Immunospot, Recombinant, Membrane, Saline, Selection, Software

Journal: Journal of Virology

Article Title: Characterization of Hepatitis B Precore/Core-Related Antigens

doi: 10.1128/JVI.01695-20

Figure Lengend Snippet: Biogenesis of HBc, HBeAg, and p22cr. (A) Schematic representation of HBcrAg biogenesis. The current HBcrAg assay is thought to detect HBc, HBeAg, and p22cr. HBc, translated from pgRNA, forms the icosahedral capsid inside complete and empty virions. The direct translation product from the precore mRNA is the precore protein (p25), from which HBeAg and p22cr (renamed PreC here) are both derived. Removal of the N-terminal signal peptide (SP) of p25, by the signal peptidase during p25 translocation into the ER lumen, leads to the production of p22, which is further processed at its CTD before being secreted as the dimeric HBeAg (p17). The exact C-terminal processing sites of HBeAg and p22cr appear heterogeneous and not well defined, as indicated by the hashed box. On the other hand, p22cr (starting from Glu −28, in the absence of Met −29) is reported as an incompletely processed product of p25, retaining the N-terminal signal peptide, in contrast to HBeAg, but lacking CTD, similar to HBeAg (14). Another precore-derived protein, similar to p22cr but retaining the initiator Met (−29) in the signal peptide, has also been reported (21). Whereas this remains to be resolved, we use here the nomenclature derived from p22cr, starting from Glu −28, to refer to the PreC proteins we identify here schematically. p22cr was initially reported to form an aberrant capsid inside empty virions, but this has been challenged recently by the detection of HBc in empty virions in the absence of any precore-derived proteins. The diagram shows a comparison of the various components of HBcrAg. Specific epitopes recognized by the following MAbs are indicated with blue braces: MAbs 1A11 and 7E9, specific to precore-derived proteins HBeAg and p22cr (renamed PreC in this study) (the −10 aa region, epitope from aa −10 to 5); MAbs T2221 (epitope from aa 130 to 140), 19C18 (epitope from aa 2 to 5), and 1D8 (epitope from aa 75 to 83), specific to the NTD shared by all HBcrAg components; and MAbs 366-2 (epitope from aa 150 to 164, largely independent of CTD state of phosphorylation), 25-7 (epitope from aa 164 to 182, selective for nonphosphorylated CTD), and 14-2 (epitope from aa 164 to 182, selective for phosphorylated CTD) specific to HBc (CTD). The antibodies HB44 (epitope from aa 31 to 49), HB61 (epitope from aa 131 to 140), HB91 (epitope from aa 1 to 19), H114 (structural epitope from aa 1 to 81, denoted by *), and HB140 (epitope from aa 21 to 40), used in the current HBcrAg commercial assay, and their epitopes are indicated by gray braces and letters (16). (B) Comparison of the CTD sequences retained in the various HBeAg and secreted PreC (p22cr) proteins (as defined in this study) between genotype A and non-A genotypes. Four RXXR motifs, which are potential furin cleavage sites, are underlined (12). Assignment of putative cleavages sites for HBeAg were based on prior publications, except that e2 in genotypes B and C was identified in this study. The arrows indicate the putative cleavage sites to produce HBeAg (referred to as e0, e1, e2, and e3 in this study) and PreC proteins (referred to as PreC0, PreC1, PreC2, and PreC3 in this study), with the preferred cleavage sites indicated by the long arrows.

Article Snippet: A library of 67 15-mer peptides with a 12-residue overlap covering the p25 protein (212 residues, labeled residues −29 to 183) was constructed by Mimotopes, Australia.

Techniques: Derivative Assay, Translocation Assay, Comparison, Phospho-proteomics

Journal: Journal of Virology

Article Title: Characterization of Hepatitis B Precore/Core-Related Antigens

doi: 10.1128/JVI.01695-20

Figure Lengend Snippet: Analysis of HBeAg and PreC proteins in patient sera by sucrose gradient fractionation. Sera from patient 10 (gtC) (A) and patient 12 (gtD) (B) were fractionated by sucrose density gradient centrifugation. The fractions were resolved by high-resolution SDS-PAGE followed by immunoblotting using the precore MAb 1A11, precore/core MAb T2221, and anti-HBs (Virostat) antibodies sequentially on the same membrane. The concentrated supernatant from HBc (p21)- and precore(p25)-transfected Huh7 cells was loaded as a reference for HBc, HBeAg, and PreC proteins. The direction of centrifugation (top to bottom) is indicated.

Article Snippet: A library of 67 15-mer peptides with a 12-residue overlap covering the p25 protein (212 residues, labeled residues −29 to 183) was constructed by Mimotopes, Australia.

Techniques: Fractionation, Gradient Centrifugation, SDS Page, Western Blot, Membrane, Transfection, Centrifugation

Journal: Journal of Virology

Article Title: Characterization of Hepatitis B Precore/Core-Related Antigens

doi: 10.1128/JVI.01695-20

Figure Lengend Snippet: Separation of HBeAg and PreC proteins from HBV virion particles secreted by HBV-infected PHH culture by CsCl density gradient fractionation. (A) Concentrated culture supernatant from gtD HBV-infected PHHs harvested 10 days postinfection (dpi) was fractionated by CsCl density gradient centrifugation. The virion particles and antigens in the fractions were analyzed by native agarose gel electrophoresis. HBV DNA and core protein were detected, as described in the legend to Fig. 6, using the indicated NTD- or CTD-specific MAbs. Ca, capsid, containing DNA or RNA or empty; e1/PreC1, secreted HBeAg and PreC protein from HBV genotype D-infected PHHs. (B) Concentrated culture supernatant from mock-infected or gtD HBV-infected PHHs harvested 10 days postinfection, as well as selected fractions from the CsCl gradient shown in panel A, were resolved by high-resolution SDS-PAGE, along with the serum from the gtD HBV-infected patient (no.12) and chimpanzee (no. 1616, week 22) (5; Hong et al., submitted). (C) Epitope mapping for MAbs 1D8 and 7E9 by ELISA using an overlapping peptide library. The linear epitope recognition of the anti-HBV precore/core MAbs 1D8 and 7E9 was interrogated against an overlapping, biotinylated peptide library, numbered 1 to 69, of the linear precore precursor protein (p25, residues −29 to 183 [Table 1]) epitopes, immobilized on a streptavidin-coated ELISA plate. Precore- and HBc-specific regions covered by the overlapping peptide library are indicated. Recombinant HBeAg and HBc were included as assay binding controls. The direction of centrifugation (top to bottom) is indicated in panels A and B.

Article Snippet: A library of 67 15-mer peptides with a 12-residue overlap covering the p25 protein (212 residues, labeled residues −29 to 183) was constructed by Mimotopes, Australia.

Techniques: Infection, Fractionation, Gradient Centrifugation, Agarose Gel Electrophoresis, SDS Page, Enzyme-linked Immunosorbent Assay, Recombinant, Binding Assay, Centrifugation

Journal: Journal of Virology

Article Title: Characterization of Hepatitis B Precore/Core-Related Antigens

doi: 10.1128/JVI.01695-20

Figure Lengend Snippet: Failure of precore-related proteins to form capsids or virions in human hepatoma cell culture. HepG2 cells were cotransfected with pSVB45H or pSVHBV1.5Core-, along with pCI-HBc (p21), pCI-p17, pCI-p22, pCI-p22cr, or pCI-precore (p25), as indicated. Cytoplasmic lysate and culture supernatant were harvested at day 5 posttransfection. (A) Cytoplasmic lysate (top two panels) and concentrated supernatant (bottom two panels) were resolved by SDS-PAGE, followed by immunoblotting using the HBeAg/PreC-specific MAb 1A11 or anti-precore/core (NTD) MAb T2221. (B) Cytoplasmic lysate (top two panels) and concentrated cell culture supernatant (bottom three panels) were resolved by native agarose gel electrophoresis. Following transfer to nitrocellulose membrane, HBV RNA associated with intracellular capsids was detected by a plus-strand-specific HBV riboprobe and HBV DNA in extracellular virions and naked capsids was detected by a HBV DNA probe, followed by immunoblotting using the anti-precore/core (NTD) MAb T2221 and anti-HBs polyclonal antibody (in the case of supernatant only). The different viral and subviral particles are represented schematically to the right. The wavy and straight lines inside the capsid (hexagon) indicate the pgRNA and single-stranded DNA, respectively. The small and large circles denotes the HBsAg spheres and HBV virions, respectively. p17*, artificially expressed (not processed from p25 or p22cr); S, HBs subviral particles. *, unassembled precore or core proteins.

Article Snippet: A library of 67 15-mer peptides with a 12-residue overlap covering the p25 protein (212 residues, labeled residues −29 to 183) was constructed by Mimotopes, Australia.

Techniques: Cell Culture, SDS Page, Western Blot, Agarose Gel Electrophoresis, Membrane