Journal: bioRxiv

Article Title: Phosphorylation-State Modulated Binding of HSP70: Structural Insights and Compensatory Protein Engineering

doi: 10.1101/2025.02.17.637997

Figure Lengend Snippet: A, schematics of HSP70, HSC70, and CHIP depicting key domains and amino acid position: Nucleotide-binding domain (NBD), substrate binding domain (SBD), C-terminal domain (CTD), tetratricopeptide repeat (T), coiled-coil (CC), and U-box (U). B , C , in silico structures of WT CHIP, B , and G132N CHIP, C , interacting with phosphorylated threonine (pThr-636) of HSP70. D, schematic of NanoBiT protein interaction assay used to assess interactions between LgBiT-HSP70 T636A/D and SmBiT-CHIP variants in HEK-293 and COS-7 cells. E , F , Percent association of NanoBiT fragments with various CHIP mutants combined with HSP70-T636A or HSP70-T636D in HEK-293 cells, E, and COS-7 cells, F . Data represented by dot plot and summarized by the mean ± SD from replicates across three independent transfections analyzed via two-way ANOVA: main effects of SmBiT- CHIP, F( , ) = 80.93, P < 0.001 and LgBiT-HSP70, F( , ) = 63.45, P < 0.001, and an interaction effect between SmBiT-CHIP and LgBiT- HSP70, F( , ) = 4.647, P = 0.014. SmBiT-CHIP accounted for 56.08% of the total variation, while the LgBiT-HSP70 accounted for 21.98%. Post-test results within SmBiT-CHIP conditions are included on the plot, * or † indicate P < 0.05 or < 0.001 vs. WT SmBiT. G, cell counts of HEK-293 and COS-7 cells transfected with the indicated HSP70 and CHIP vectors at 24 h and 48 h most post-transfection, represented by heatmap from three independent experiments, results of three-way ANOVA are in Table 1. H, representative images of the conditions in G . I, representative immunoblots of MYC (CHIP constructs) and Flag (HSP70 constructs) levels in COS-7 cells detected after immunoprecipitation (IP) of MYC or the cell lysate input. The co-precipitated Flag was quantified via densitometry with relative levels and levels normalized to the Flag input represented by a heatmap. J, representative immunoblots of HSP70 steady-state levels with different amino acids at position 636 following the addition of cycloheximide (CHX) or MG132 with the percent change from time 0 at each 6 h time point. K, representative gel image of cell-free ubiquitination of fluorescently-labeled HSC70 by CHIP-WT or -G132N (upper) and quantification of the relative ubiquitination of HSC70 represented by dot-plot summarized by the mean ± SD from three experiments analyzed via t-test.

Article Snippet: The amino-terminal biotin-labeled HSP70 tail peptide (biotin-GSGSGPTIEEVD, termed as biotin-EEVD) and the amino-terminal biotin-labeled pT-HSP70 tail peptide (biotin-GSGSGP(pT)IEEVD, termed as biotin-pEEVD) were purchased (Lifetein).

Techniques: Binding Assay, In Silico, Protein Interaction Assay, Transfection, Western Blot, Construct, Immunoprecipitation, Ubiquitin Proteomics, Labeling

Journal: bioRxiv

Article Title: Phosphorylation-State Modulated Binding of HSP70: Structural Insights and Compensatory Protein Engineering

doi: 10.1101/2025.02.17.637997

Figure Lengend Snippet: A, left , fluorescent polarization of HSP70 peptide tracers (EEVD upper , pEEVD lower ) were used to measure binding affinity to recombinant proteins in a cell-free system, with decreased polarization (lower) indicating less interaction. Right , CHIP domains, coded by color: TPR (dark green), coiled-coil (CC, yellow), and the U-box (purple) and locations of key amino acids. B, polarization (mP) is represented by the percentage of max binding of EEVD or pEEVD with increasing full-length versions of recombinant CHIP protein: left -WT, center -G132N, and right -K30A. Data are represented by a scatterplot of the mean ± SD from three independent experiments and the resulting curve fit to determine the K d . C, polarization (mP) is represented by the percentage of max binding of EEVD or pEEVD with increasing CHIP or CHIP domain fragments. Data are represented by a scatterplot of the mean ± SD from three independent experiments and the resulting curve fit to determine the K d . D, upper , schematic of CHIP and HOP TPR constructs used for biolayer interferometry analysis with biotin-tagged EEVD and pEEVD peptides, lower . E, binding affinity of isolated TPR domains and EEVD or pEEVD represented by dot plot and summarized by the mean ± SD analyzed via two-way ANOVA: main effects of TRP construct, F(5, 108) = 69.54, P < 0.001 and EEVD peptide, F(1, 108) = 4.584, P = 0.035, and an interaction effect between TPR construct and EEVD peptide, F(5, 108) = 10.95, P < 0.001. TPR construct accounted for 68% of the total variation, while the EEVD peptide accounted for 1%. Post-test results of pairwise comparisons of interest are included in the plot.

Article Snippet: The amino-terminal biotin-labeled HSP70 tail peptide (biotin-GSGSGPTIEEVD, termed as biotin-EEVD) and the amino-terminal biotin-labeled pT-HSP70 tail peptide (biotin-GSGSGP(pT)IEEVD, termed as biotin-pEEVD) were purchased (Lifetein).

Techniques: Binding Assay, Recombinant, Construct, Isolation

Journal: bioRxiv

Article Title: Phosphorylation-State Modulated Binding of HSP70: Structural Insights and Compensatory Protein Engineering

doi: 10.1101/2025.02.17.637997

Figure Lengend Snippet: A, overlay of CHIP-TPR/HSP70 tail peptide structures with the previously solved structure of CHIP-TPR in complex with the helical lid+tail domain of HSP70. PDB IDs and coloring: 4KBQ: CHIP in light grey with HSP70 helical lid+tail domain in dark grey; 9DYA: CHIP in light purple with EEVD in yellow; 9DYB: CHIP in light pink with pEEVD in cyan. Throughout, CHIP-TPR residues interacting with only the HSP70 tail are colored forest green, CHIP-TPR residues interacting with only the HSP70 helical lid domain are colored magenta, CHIP-TPR residues interacting with both the HSP70 helical lid and HSP70 tail are colored orange. B, structure of CHIP-TPR in complex with the helical lid+tail domain of HSP70. C, structure of CHIP-TPR in complex with pEEVD. D, structure of CHIP-TPR in complex with EEVD. E, structure of CHIP-TPR with the in silico-modeled G132N mutation in complex with pEEVD with the N132 side chain colored lime green. F, comparison of RMSF for C atoms by residue for each trajectory of complexes formed by EEVD (purple) or pEEVD (blue) with CHIP-TPR (solid lines) or CHIP- TPR-G132N (dotted lines). G, comparison of intermolecular H-bond profiles for each complex formed by EEVD (purple) or pEEVD (blue) with CHIP-TPR (solid lines) or CHIP-TPR-G132N (dotted lines). Additionally, the contribution of N132 to the total H-bond profile between CHIP-TPR-G132N and pEEVD is plotted (To, green: ten-frame-average number of H-bonds). The MD simulation videos can be found at http://hdl.handle.net/2374.MIA/6854 .

Article Snippet: The amino-terminal biotin-labeled HSP70 tail peptide (biotin-GSGSGPTIEEVD, termed as biotin-EEVD) and the amino-terminal biotin-labeled pT-HSP70 tail peptide (biotin-GSGSGP(pT)IEEVD, termed as biotin-pEEVD) were purchased (Lifetein).

Techniques: In Silico, Mutagenesis, Comparison, Residue

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: Cell death in glioblastoma and the central nervous system

doi: 10.1007/s13402-024-01007-8

Figure Lengend Snippet: Direct apoptotic pathway targeting agents in preclinical GBM

Article Snippet: U87MG (H, Imm) LN18 (H, Imm) LN229 (H, Imm) SMA560 (M, Imm) , Smac Mimetic : Smac peptides containing AVPIAQK N-terminal amino acids of endogenous Smac , , TRAIL αCD95 antibody (apo1) Doxorubicin , • Smac overexpression sensitizes GBM cells to TRAIL, CD95 engagement and doxorubicin, even when GBM cells overexpress BCL-2 • TRAIL alone induces significant apoptosis only in GBM cells (murine and human). No effect on astrocytes, Schwann cells, or other normal tissue. Enhanced by Smac cotreatment • In vivo, Smac + TRAIL treatment induced significant U87MG apoptosis, reduced tumor growth and extended mouse survival with long-term cures. Smac alone had no impact. , [ ] .

Techniques: Inhibition, Ex Vivo, Expressing, In Vivo, Knockdown, Mouse Assay, Phospho-proteomics, In Vitro, Activity Assay, Irradiation, Activation Assay, Over Expression, Liposomes, Virus