scholarly journals A Tale of Two Tails: Efficient Profiling of Protein Degraders by Specific Functional and Target Engagement Readouts

2021 ◽  
pp. 247255522098437
Author(s):  
Alexey L. Chernobrovkin ◽  
Cindy Cázares-Körner ◽  
Tomas Friman ◽  
Isabel Martin Caballero ◽  
Daniele Amadio ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Protein Degradation ◽  
Cellular Protein ◽  
Target Engagement ◽  
Intact Cells ◽  
Binding Characteristics ◽  
Protein Thermal Stability ◽  
The Stability ◽  
Induced Changes ◽  
Small Molecule Therapeutics

Targeted protein degradation represents an area of great interest, potentially offering improvements with respect to dosing, side effects, drug resistance, and reaching “undruggable” proteins compared with traditional small-molecule therapeutics. A major challenge in the design and characterization of degraders acting as molecular glues is that binding of the molecule to the protein of interest (PoI) is not needed for efficient and selective protein degradation; instead, one needs to understand the interaction with the responsible ligase. Similarly, for proteasome targeting chimeras (PROTACs), understanding the binding characteristics of the PoI alone is not sufficient. Therefore, simultaneously assessing the binding to both PoI and the E3 ligase as well as the resulting degradation profile is of great value. The cellular thermal shift assay (CETSA) is an unbiased cell-based method, designed to investigate the interaction of compounds with their cellular protein targets by measuring compound-induced changes in protein thermal stability. In combination with mass spectrometry (MS), CETSA can simultaneously evaluate compound-induced changes in the stability of thousands of proteins. We have used CETSA MS to profile a number of protein degraders, including molecular glues (e.g., immunomodulatory drugs) and PROTACs, to understand mode of action and to deconvolute off-target effects in intact cells. Within the same experiment, we were able to monitor both target engagement by observing changes in protein thermal stability as well as efficacy by simultaneous assessment of protein abundances. This allowed us to correlate target engagement (i.e., binding to the PoI and ligases) and functional readout (i.e., degrader induced protein degradation).

scholarly journals A tale of two tails - efficient profiling of protein degraders by specific functional and target engagement readouts

2020 ◽  
Author(s):  
Alexey L. Chernobrovkin ◽  
Cindy Cázares-Körner ◽  
Tomas Friman ◽  
Isabel Martin Caballero ◽  
Daniele Amadio ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Protein Degradation ◽  
Cellular Protein ◽  
Target Engagement ◽  
Intact Cells ◽  
Binding Characteristics ◽  
Protein Thermal Stability ◽  
The Stability ◽  
Induced Changes ◽  
Small Molecule Therapeutics

AbstractTargeted protein degradation represents an area of great interest, potentially offering improvements with respect to dosing, side effects, drug resistance and reaching ‘undruggable’ proteins compared to traditional small molecule therapeutics. A major challenge in the design and characterization of degraders acting as molecular glues is that binding of the molecule to the protein of interest (PoI) is not needed for efficient and selective protein degradation, instead one needs to understand the interaction with the responsible ligase. Similarly, for proteasome targeting chimeras (PROTACs) understanding the binding characteristics of the PoI alone is not sufficient. Therefore, simultaneously assessing the binding to both PoI and the E3 ligase as well as the resulting degradation profile is of great value. The Cellular Thermal Shift Assay (CETSA) is an unbiased cell-based method, designed to investigate the interaction of compounds with their cellular protein targets by measuring compound-induced changes in protein thermal stability. In combination with mass spectrometry (MS) CETSA can simultaneously evaluate compound induced changes in the stability of thousands of proteins. We have used CETSA MS to profile a number of protein degraders, including molecular glues (e.g. IMiDs) and PROTACs to understand mode of action and to deconvolute off-target effects in intact cells. Within the same experiment we were able to monitor both target engagement by observing changes in protein thermal stability as well as efficacy by simultaneous assessment of protein abundances. This allowed us to correlate target engagement (i.e. binding to the PoI and ligases) and functional readout (i.e. degrader induced protein degradation).

Optimization of Organic Fibres%[Kevlar/Arobocel/Acrylic] in NAO Brake Pad Application and its Effect on Thermal Stability & Friction Characteristics

2012 ◽  
Vol 531-532 ◽  
pp. 8-12
Author(s):  
M.A. Sai Balaji ◽  
K. Kalaichelvan
Keyword(s):  
Thermal Stability ◽  
Weight Loss ◽  
Minimum Weight ◽  
Friction Material ◽  
Brake Pad ◽  
Temperature Zone ◽  
Good Thermal Stability ◽  
Friction Testing ◽  
Brake Application ◽  
The Stability

Organic fibres (Kevlar/ Arbocel / Acrylic) have good thermal stability, higher surface area and bulk density. The optimization of organic fibres percentage for thermal behaviour is considered using TGA. The temperature raise during brake application will be between 150-4000 C and this temperature zone is very critical to determine the fade characteristics during friction testing. Hence, three different friction composites are developed with the same formulation varying only the Kevlar, Arbocel and Acrylic fibres which are compensated by the inert filler namely the barites and are designated as NA01, NA02 and NA03 respectively. After the fabrication, the TGA test reveals that the composite NA03 has minimum weight loss. The friction coefficient test rig is then used to test the friction material as per SAE J661a standards. The results prove that the brake pad with minimum weight loss during TGA has higher friction stability. Thus, we can correlate the thermal stability with the stability of friction.

scholarly journals Identification of phosphosites that alter protein thermal stability

2021 ◽  
Author(s):  
Ian R. Smith ◽  
Kyle N. Hess ◽  
Anna A. Bakhtina ◽  
Anthony S. Valente ◽  
Ricard A. Rodríguez-Mias ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Protein Thermal Stability

Intrinsic Thermal Stability of Anisotropic Thin-Film Superconductors

1990 ◽  
Vol 112 (1) ◽  
pp. 10-15 ◽  
Author(s):  
M. I. Flik ◽  
C. L. Tien
Keyword(s):  
Thin Film ◽  
Thermal Stability ◽  
Stability Criterion ◽  
High Temperature Superconductors ◽  
Stability Criteria ◽  
Anisotropic Thermal Conductivity ◽  
Operating Current ◽  
Released Energy ◽  
The Stability ◽  
Thermal Stability Of

Intrinsic thermal stability denotes a situation where a superconductor can carry the operating current without resistance at all times after the occurrence of a localized release of thermal energy. This novel stability criterion is different from the cryogenic stability criteria for magnets and has particular relevance to thin-film superconductors. Crystals of ceramic high-temperature superconductors are likely to exhibit anisotropic thermal conductivity. The resultant anisotropy of highly oriented films of superconductors greatly influences their thermal stability. This work presents an analysis for the maximum operating current density that ensures intrinsic stability. The stability criterion depends on the amount of released energy, the Biot number, the aspect ratio, and the ratio of the thermal conductivities in the plane of the film and normal to it.

scholarly journals Electro-responsive polyelectrolyte-coated surfaces

2017 ◽  
Vol 199 ◽  
pp. 335-347 ◽  
Author(s):  
V. Sénéchal ◽  
H. Saadaoui ◽  
J. Rodriguez-Hernandez ◽  
C. Drummond
Keyword(s):  
Electric Field ◽  
Smart Materials ◽  
Electrical Stimulus ◽  
Colloidal Dispersions ◽  
Polymer Chains ◽  
Molecular Response ◽  
Coated Surfaces ◽  
The Stability ◽  
Induced Changes ◽  
Applied Fields

The anchoring of polymer chains at solid surfaces is an efficient way to modify interfacial properties like the stability and rheology of colloidal dispersions, lubrication and biocompatibility. Polyelectrolytes are good candidates for the building of smart materials, as the polyion chain conformation can often be tuned by manipulation of different physico-chemical variables. However, achieving efficient and reversible control of this process represents an important technological challenge. In this regard, the application of an external electrical stimulus on polyelectrolytes seems to be a convenient control strategy, for several reasons. First, it is relatively easy to apply an electric field to the material with adequate spatiotemporal control. In addition, in contrast to chemically induced changes, the molecular response to a changing electric field occurs relatively quickly. If the system is properly designed, this response can then be used to control the magnitude of surface properties. In this work we discuss the effect of an external electric field on the adhesion and lubrication properties of several polyelectrolyte-coated surfaces. The influence of the applied field is investigated at different pH and salt conditions, as the polyelectrolyte conformation is sensitive to these variables. We show that it is possible to fine tune friction and adhesion using relatively low applied fields.

scholarly journals Thermal Stability of Residual Stresses in Differently Deep Rolled Surface Layers of Steel SAE 1045

2021 ◽  
Author(s):  
Stephanie Saalfeld ◽  
Thomas Wegener ◽  
Berthold Scholtes ◽  
Thomas Niendorf
Keyword(s):  
Thermal Stability ◽  
Elevated Temperature ◽  
Residual Stresses ◽  
Elevated Temperatures ◽  
Fatigue Behavior ◽  
Decisive Role ◽  
Deep Rolling ◽  
Material Conditions ◽  
The Stability ◽  
Thermal Stability Of

AbstractThe stability of compressive residual stresses generated by deep rolling plays a decisive role on the fatigue behavior of specimens and components, respectively. In this regard, deep rolling at elevated temperature has proven to be very effective in stabilizing residual stresses when fatigue analysis is conducted at ambient temperature. However, since residual stresses can be affected not only by plastic deformation but also when thermal energy is provided, it is necessary to analyze the influence of temperature and time on the relaxation behavior of residual stresses at elevated temperature. To evaluate the effect of deep rolling at elevated temperatures on stability limits under thermal as well as combined thermo-mechanical loads, the present work introduces and discusses the results of investigations on the thermal stability of residual stresses in differently deep rolled material conditions of the steel SAE 1045.

scholarly journals Combining mutations that modulate inter-subunit interactions and proteolytic inactivation enhance the stability of factor VIIIa

2014 ◽  
Vol 112 (07) ◽  
pp. 43-52 ◽  
Author(s):  
Hironao Wakabayashi ◽  
Jennifer M. Wintermute ◽  
Philip J. Fay
Keyword(s):  
Thermal Stability ◽  
Thrombin Generation ◽  
Resistant Mutant ◽  
Decay Rates ◽  
Single Type ◽  
Wild Type ◽  
Subunit Interactions ◽  
Factor Viiia ◽  
The Stability ◽  
A3 Subunit

SummaryFVIIIa is labile due to the dissociation of A2 subunit. Previously, we introduced hydrophobic mutations at select A1/A2/A3 subunit interfaces yielding more stable FVIII(a) variants. Separately we showed that altering the sequence flanking the primary FXa cleavage site in FVIIIa (Arg336) yielded reduced rates of proteolytic inactivation of FVIIIa. In this study we prepared the FXa-cleavage resistant mutant (336(P4-P3’)562) combined with mutations of Ala108Ile, Asp519Val/ Glu665Val or Ala108Ile/Asp519Val/Glu665Val and examined the effects of these combinations relative to FVIII thermal stability, rates of FVIIIa decay and proteolytic inactivation of FVIIIa by FXa. Thermal decay rates for 336(P4-P3’)562/Ala108Ile, 336(P4-P3’)562/Asp519Val/ Glu665Val, and 336(P4-P3’)562/Ala108Ile/Asp519Val/Glu665Val variants were reduced by ∼2– to 5-fold as compared with wild-type (WT) primarily reflecting the effects of the A domain interface mutations. FVIIIa decay rates for 336(P4-P3’)562/Asp519Val/Glu665Val and 336(P4-P3’)562/Ala108Ile/Asp519Val/Glu665Val variants were reduced by ∼25 fold, indicating greater stability than the control Asp519Val/Glu665Val variant (∼14-fold). Interestingly, 336(P4-P3’)562/Asp519Val/Glu665Val and 336(P4-P3’)562/Ala108Ile/ Asp519Val/Glu665Val variants showed reduced FXa-inactivation rates compared with the 336(P4-P3’)562 control (∼4-fold), suggesting A2 subunit destabilisation is a component of proteolytic inactivation. Thrombin generation assays using the combination variants were similar to the Asp519Val/Glu665Val control. These results indicate that combining multiple gain-of-function FVIII mutations yields FVIII variants with increased stability relative to a single type of mutation.

scholarly journals A glycine-specific N-degron pathway mediates the quality control of protein N-myristoylation

Science ◽  
2019 ◽  
Vol 365 (6448) ◽  
pp. eaaw4912 ◽  
Author(s):  
Richard T. Timms ◽  
Zhiqian Zhang ◽  
David Y. Rhee ◽  
J. Wade Harper ◽  
Itay Koren ◽  
...  
Keyword(s):  
Quality Control ◽  
Protein Stability ◽  
Protein Degradation ◽  
E3 Ligase ◽  
Cleavage Sites ◽  
Caspase Cleavage ◽  
E3 Ligases ◽  
Ring E3 Ligase ◽  
The Stability ◽  
Ring E3

The N-terminal residue influences protein stability through N-degron pathways. We used stability profiling of the human N-terminome to uncover multiple additional features of N-degron pathways. In addition to uncovering extended specificities of UBR E3 ligases, we characterized two related Cullin-RING E3 ligase complexes, Cul2ZYG11B and Cul2ZER1, that act redundantly to target N-terminal glycine. N-terminal glycine degrons are depleted at native N-termini but strongly enriched at caspase cleavage sites, suggesting roles for the substrate adaptors ZYG11B and ZER1 in protein degradation during apoptosis. Furthermore, ZYG11B and ZER1 were found to participate in the quality control of N-myristoylated proteins, in which N-terminal glycine degrons are conditionally exposed after a failure of N-myristoylation. Thus, an additional N-degron pathway specific for glycine regulates the stability of metazoan proteomes.

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