Nanobodies restore stability to cancer-associated mutants of tumor suppressor protein p16INK4a

2021 ◽  
Author(s):  
Owen Burbidge ◽  
Martyna W Pastok ◽  
Samantha L Hodder ◽  
Grasilda Zenkeviciute ◽  
Martin EM Noble ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Critical Role ◽  
Tumor Suppressor Protein ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Pharmacological Chaperones ◽  
Binding Interface ◽  
Familial Cancers ◽  
Single Domain Antibodies ◽  
The Stability

We describe the generation and characterization of camelid single-domain antibodies (nanobodies) raised against tumor suppressor protein p16INK4a (p16). p16 plays a critical role in the cell cycle by inhibiting cyclin-dependent kinases CDK4 and CDK6, and it is inactivated in sporadic and familial cancers. The majority of the p16 missense mutations cause loss of function by destabilizing the protein structure. We show that the nanobodies bind p16 with nanomolar affinities and restore the stability of a range of different cancer-associated p16 mutations located at sites throughout the protein. The nanobodies also bind and stabilize p16 in a cellular setting. The crystal structure of a nanobody-p16 complex reveals that the nanobody binds to the opposite face of p16 to the CDK-binding interface permitting formation of a ternary complex. These findings indicate that nanobodies could be used as pharmacological chaperones to determine the consequences of restoring the function of p16 in the cell.

scholarly journals Argyrin A Reveals a Critical Role for the Tumor Suppressor Protein p27kip1 in Mediating Antitumor Activities in Response to Proteasome Inhibition

Cancer Cell ◽  
2008 ◽  
Vol 14 (1) ◽  
pp. 23-35 ◽  
Author(s):  
Irina Nickeleit ◽  
Steffen Zender ◽  
Florenz Sasse ◽  
Robert Geffers ◽  
Gudrun Brandes ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Critical Role ◽  
Proteasome Inhibition ◽  
Tumor Suppressor Protein ◽  
Antitumor Activities

scholarly journals Tumor suppressor protein Pdcd4 interacts with Daxx and modulates the stability of Daxx and the Hipk2-dependent phosphorylation of p53 at serine 46

Oncogenesis ◽  
2013 ◽  
Vol 2 (1) ◽  
pp. e37-e37 ◽  
Author(s):  
N Kumar ◽  
N Wethkamp ◽  
L C Waters ◽  
M D Carr ◽  
K-H Klempnauer
Keyword(s):  
Tumor Suppressor ◽  
Tumor Suppressor Protein ◽  
The Stability

scholarly journals Distinct effects of tubulin isotype mutations on neurite growth in Caenorhabditis elegans

2017 ◽  
Author(s):  
Chaogu Zheng ◽  
Margarete Diaz-Cuadros ◽  
Ken C.Q. Nguyen ◽  
David H. Hall ◽  
Martin Chalfie
Keyword(s):  
Function Analysis ◽  
Critical Role ◽  
Neurite Growth ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Post Translational Modification ◽  
Exterior Surface ◽  
Tubulin Isotypes ◽  
C Elegans ◽  
Tubulin Isotype

AbstractTubulins, the building block of microtubules (MTs), play a critical role in both supporting and regulating neurite growth. Eukaryotic genomes contain multiple tubulin isotypes, and their missense mutations cause a range of neurodevelopmental defects. Using the C. elegans touch receptor neurons, we analyzed the effects of 67 tubulin missense mutations on neurite growth. Three types of mutations emerged: 1) loss-of-function mutations, which cause mild defects in neurite growth; 2) antimorphic mutations, which map to the GTP binding site and intradimer and interdimer interfaces, significantly reduce MT stability, and cause severe neurite growth defects; and 3) neomorphic mutations, which map to the exterior surface, increase MT stability, and cause ectopic neurite growth. Structure-function analysis reveals a causal relationship between tubulin structure and MT stability. This stability affects neuronal morphogenesis. As part of this analysis, we engineered several disease-associated human tubulin mutations into C. elegans genes and examined their impact on neuronal development at the cellular level. We also discovered an α-tubulin (TBA-7) that appears to destabilize MTs. Loss of TBA-7 led to the formation of hyperstable MTs and the generation of ectopic neurites; the lack of potential sites for polyamination and polyglutamination on TBA-7 may be responsible for this destabilization.Table of Content (TOC) Highlight SummaryDifferent tubulin isotypes perform different functions in the regulation of MT structure and neurite growth, and missense mutations of tubulin genes have three types of distinct effects on MT stability and neurite growth. One α-tubulin isotype appears to induce relative instability due to the lack of potential post-translational modification sites.

scholarly journals Second-Generation Pharmacological Chaperones: Beyond Inhibitors

Molecules ◽  
2020 ◽  
Vol 25 (14) ◽  
pp. 3145 ◽  
Author(s):  
My Lan Tran ◽  
Yves Génisson ◽  
Stéphanie Ballereau ◽  
Cécile Dehoux
Keyword(s):  
Protein Misfolding ◽  
First Generation ◽  
Second Generation ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Pharmacological Chaperone ◽  
Conformational Diseases ◽  
Pharmacological Chaperones ◽  
Drug Candidates ◽  
Binding Pockets

Protein misfolding induced by missense mutations is the source of hundreds of conformational diseases. The cell quality control may eliminate nascent misfolded proteins, such as enzymes, and a pathological loss-of-function may result from their early degradation. Since the proof of concept in the 2000s, the bioinspired pharmacological chaperone therapy became a relevant low-molecular-weight compound strategy against conformational diseases. The first-generation pharmacological chaperones were competitive inhibitors of mutant enzymes. Counterintuitively, in binding to the active site, these inhibitors stabilize the proper folding of the mutated protein and partially rescue its cellular function. The main limitation of the first-generation pharmacological chaperones lies in the balance between enzyme activity enhancement and inhibition. Recent research efforts were directed towards the development of promising second-generation pharmacological chaperones. These non-inhibitory ligands, targeting previously unknown binding pockets, limit the risk of adverse enzymatic inhibition. Their pharmacophore identification is however challenging and likely requires a massive screening-based approach. This review focuses on second-generation chaperones designed to restore the cellular activity of misfolded enzymes. It intends to highlight, for a selected set of rare inherited metabolic disorders, the strategies implemented to identify and develop these pharmacologically relevant small organic molecules as potential drug candidates.

scholarly journals Distinct effects of tubulin isotype mutations on neurite growth in Caenorhabditis elegans

2017 ◽  
Vol 28 (21) ◽  
pp. 2786-2801 ◽  
Author(s):  
Chaogu Zheng ◽  
Margarete Diaz-Cuadros ◽  
Ken C. Q. Nguyen ◽  
David H. Hall ◽  
Martin Chalfie
Keyword(s):  
Caenorhabditis Elegans ◽  
Neuronal Development ◽  
Function Analysis ◽  
Critical Role ◽  
Neurite Growth ◽  
Cellular Level ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Exterior Surface ◽  
C Elegans

Tubulins, the building block of microtubules (MTs), play a critical role in both supporting and regulating neurite growth. Eukaryotic genomes contain multiple tubulin isotypes, and their missense mutations cause a range of neurodevelopmental defects. Using the Caenorhabditis elegans touch receptor neurons, we analyzed the effects of 67 tubulin missense mutations on neurite growth. Three types of mutations emerged: 1) loss-of-function mutations, which cause mild defects in neurite growth; 2) antimorphic mutations, which map to the GTP binding site and intradimer and interdimer interfaces, significantly reduce MT stability, and cause severe neurite growth defects; and 3) neomorphic mutations, which map to the exterior surface, increase MT stability, and cause ectopic neurite growth. Structure-function analysis reveals a causal relationship between tubulin structure and MT stability. This stability affects neuronal morphogenesis. As part of this analysis, we engineered several disease-associated human tubulin mutations into C. elegans genes and examined their impact on neuronal development at the cellular level. We also discovered an α-tubulin (TBA-7) that appears to destabilize MTs. Loss of TBA-7 led to the formation of hyperstable MTs and the generation of ectopic neurites; the lack of potential sites for polyamination and polyglutamination on TBA-7 may be responsible for this destabilization.

scholarly journals Messing up disorder: how do missense mutations in the tumor suppressor protein APC lead to cancer?

2011 ◽  
Vol 10 (1) ◽  
pp. 101 ◽  
Author(s):  
David P Minde ◽  
Zeinab Anvarian ◽  
Stefan GD Rüdiger ◽  
Madelon M Maurice
Keyword(s):  
Tumor Suppressor ◽  
Tumor Suppressor Protein ◽  
Missense Mutations
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