Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 10 de 10
Filter
Add more filters










Publication year range
1.
Angew Chem Int Ed Engl ; 59(36): 15448-15466, 2020 09 01.
Article in English | MEDLINE | ID: mdl-32428344

ABSTRACT

Targeted protein degradation (TPD), the ability to control a proteins fate by triggering its degradation in a highly selective and effective manner, has created tremendous excitement in chemical biology and drug discovery within the past decades. The TPD field is spearheaded by small molecule induced protein degradation with molecular glues and proteolysis targeting chimeras (PROTACs) paving the way to expand the druggable space and to create a new paradigm in drug discovery. However, besides the therapeutic angle of TPD a plethora of novel techniques to modulate and control protein levels have been developed. This enables chemical biologists to better understand protein function and to discover and verify new therapeutic targets. This Review gives a comprehensive overview of chemical biology techniques inducing TPD. It explains the strengths and weaknesses of these methods in the context of drug discovery and discusses their future potential from a medicinal chemist's perspective.


Subject(s)
Proteasome Endopeptidase Complex/metabolism , Proteins/metabolism , Humans , Proteolysis
2.
Curr Opin Neurobiol ; 61: 125-132, 2020 04.
Article in English | MEDLINE | ID: mdl-32199101

ABSTRACT

The deposition of proteins of abnormal conformation is one of the major hallmarks of the common neurodegenerative diseases including Alzheimer's, Parkinson's, amyotrophic lateral sclerosis, frontotemporal dementia, and prion diseases. Protein quality control systems have evolved to protect cells and organisms against the harmful consequences of abnormally folded proteins that are constantly produced in small amounts. Mutations in rare inherited forms of neurodegenerative diseases have provided compelling evidence that failure of protein quality control systems can drive neurodegeneration. With extensive knowledge of these systems, and the notion that protein quality control may decline with age, many laboratories are now focussing on manipulating these evolutionarily optimized defence mechanisms to reduce the protein misfolding burden for therapeutic benefit.


Subject(s)
Neurodegenerative Diseases , Amyotrophic Lateral Sclerosis , Humans , Proteins
3.
Drug Discov Today Technol ; 37: 73-82, 2020 Dec.
Article in English | MEDLINE | ID: mdl-34895657

ABSTRACT

Small molecule induced protein degradation has created tremendous excitement in drug discovery within recent years. Not being confined to target inhibition and being able to remove disease-causing protein targets via engagement and subsequent ubiquitination has provided scientists with a powerful tool to expand the druggable space. At the center of this approach sits the ternary complex formed between an E3 ubiquitin ligase, the small molecule degrader, and the target protein. A productive ternary complex is pivotal for a ubiquitin to be transferred to a surface lysine of the target protein resulting in poly-ubiquitination which enables recognition and finally degradation by the proteasome. As understanding the ternary complex means understanding the degradation process, many efforts are put into obtaining structural information of the ternary complex and getting a snapshot of the underlying conformations and molecular contacts. Locking this transient trimeric intermediate in a crystalline state has proven to be very demanding but the obtained results have tremendously improved our understanding of small molecule degraders. This review discusses target protein degradation from a structural perspective and highlights the evolution of certain degraders based on the obtained structural insights.


Subject(s)
Proteasome Endopeptidase Complex , Ubiquitin-Protein Ligases , Proteasome Endopeptidase Complex/metabolism , Proteolysis , Ubiquitin , Ubiquitin-Protein Ligases/metabolism , Ubiquitination
4.
Nat Struct Mol Biol ; 25(3): 261-269, 2018 03.
Article in English | MEDLINE | ID: mdl-29483652

ABSTRACT

The survival rate of cancer patients is steadily increasing, owing to more efficient therapies. Understanding the molecular mechanisms of chemotherapy-induced premature ovarian insufficiency (POI) could identify targets for prevention of POI. Loss of the primordial follicle reserve is the most important cause of POI, with the p53 family member p63 being responsible for DNA-damage-induced apoptosis of resting oocytes. Here, we provide the first detailed mechanistic insight into the activation of p63, a process that requires phosphorylation by both the priming kinase CHK2 and the executioner kinase CK1 in mouse primordial follicles. We further describe the structural changes induced by phosphorylation that enable p63 to adopt its active tetrameric conformation and demonstrate that previously discussed phosphorylation by c-Abl is not involved in this process. Inhibition of CK1 rescues primary oocytes from doxorubicin and cisplatin-induced apoptosis, thus uncovering a new target for the development of fertoprotective therapies.


Subject(s)
Casein Kinase I/metabolism , Checkpoint Kinase 2/metabolism , DNA Damage , Oocytes/enzymology , Transcription Factors/metabolism , Tumor Suppressor Proteins/metabolism , Animals , Casein Kinase I/antagonists & inhibitors , Cell Line, Tumor , Cisplatin/toxicity , Doxorubicin/toxicity , Humans , Mice , Oocytes/drug effects , Oocytes/metabolism , Phosphorylation , Protein Multimerization
5.
Nat Protoc ; 12(7): 1451-1457, 2017 Jul.
Article in English | MEDLINE | ID: mdl-28686587

ABSTRACT

Phenotypic analysis of mouse models of human diseases is essential to understanding the underlying disease mechanisms and to developing therapeutics. Many models of neurodegenerative diseases are associated with motor dysfunction, a powerful readout for the disease. We describe here a set of measures to quantitatively monitor early disease onset and progression. We named this set of rules qMotor because it enables sensitive, robust and quantitative measurement of motor performance in 3 d. qMotor can be used to assess early disease onset, before paralysis, as well as disease progression in diverse mouse models, and can be exploited to define robust and humane experimental end points, thereby reducing animal suffering. As an example, we apply qMotor to SOD1G93A transgenic mice. Early studies with the original transgenic SOD1G93A mice in the hybrid background (B6SJL-Tg(SOD1-G93A) have been criticized because of high noise in this mixed background and because of inadequate study designs. We applied qMotor in SOD1G93A transgenic mice in an inbred C57BL/6J background, hereafter called iSOD1G93A mice, and show a remarkably robust and consistent phenotype in this line that we use to evaluate a therapeutic approach. qMotor is a protocol generically applicable to different mouse models.


Subject(s)
Motor Activity , Neurodegenerative Diseases/pathology , Animals , Disease Models, Animal , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mutation, Missense , Superoxide Dismutase-1/genetics
6.
Cell Death Differ ; 23(12): 1930-1940, 2016 12.
Article in English | MEDLINE | ID: mdl-27716744

ABSTRACT

Members of the p53 tumor-suppressor family are expressed as multiple isoforms. Isoforms with an N-terminal transactivation domain are transcriptionally active, while those ones lacking this domain often inhibit the transcriptional activity of other family members. In squamous cell carcinomas, the high expression level of ΔNp63α inhibits the tumor-suppressor function of TAp73ß. This can in principle be due to blocking of the promoter or by direct interaction between both proteins. p63 and p73 can hetero-oligomerize through their tetramerization domains and a hetero-tetramer consisting of two p63 and two p73 molecules is thermodynamically more stable than both homo-tetramers. Here we show that cells expressing both p63 and p73 exist in mouse epidermis and hair follicle and that hetero-tetramer complexes can be detected by immunoprecipitation in differentiating keratinocytes. Through structure determination of the hetero-tetramer, we reveal why this hetero-tetramer is the thermodynamically preferred species. We have created mutants that exclusively form either hetero-tetramers or homo-tetramers, allowing to investigate the function of these p63/p73 hetero-tetramers. Using these tools, we show that inhibition of TAp73ß in squamous cell carcinomas is due to promoter squelching and not direct interaction.


Subject(s)
Phosphoproteins/chemistry , Phosphoproteins/metabolism , Protein Multimerization , Trans-Activators/chemistry , Trans-Activators/metabolism , Tumor Protein p73/chemistry , Tumor Protein p73/metabolism , Amino Acid Sequence , Animals , Humans , Mice , Mutation/genetics , Promoter Regions, Genetic/genetics , Protein Binding , Salts/chemistry , Transcription, Genetic
7.
Angew Chem Int Ed Engl ; 53(39): 10300-14, 2014 Sep 22.
Article in English | MEDLINE | ID: mdl-25070284

ABSTRACT

The dream of cell biologists is to be able to watch biological macromolecules perform their duties in the intracellular environment of live cells. Ideally, the observation of both the location and the conformation of these macromolecules with biophysical techniques is desired. The development of many fluorescence techniques, including superresolution fluorescence microscopy, has significantly enhanced our ability to spot proteins and other molecules in the crowded cellular environment. However, the observation of their structure and conformational changes while they attend their business is still very challenging. In principle, NMR and EPR spectroscopy can be used to investigate the conformation and dynamics of biological macromolecules in living cells. The development of in-cell magnetic resonance techniques has demonstrated the feasibility of this approach. Herein we review the different techniques with a focus on liquid-state in-cell NMR spectroscopy, provide an overview of applications, and discuss the challenges that lie ahead.


Subject(s)
Electron Spin Resonance Spectroscopy , Nuclear Magnetic Resonance, Biomolecular , Proteins/chemistry , Animals , Escherichia coli/metabolism , Humans , Nucleic Acids/chemistry , Saccharomyces cerevisiae/metabolism
8.
J Am Chem Soc ; 135(37): 13796-803, 2013 Sep 18.
Article in English | MEDLINE | ID: mdl-23968199

ABSTRACT

Proteins and nucleic acids maintain the crowded interior of a living cell and can reach concentrations in the order of 200-400 g/L which affects the physicochemical parameters of the environment, such as viscosity and hydrodynamic as well as nonspecific strong repulsive and weak attractive interactions. Dynamics, structure, and activity of macromolecules were demonstrated to be affected by these parameters. However, it remains controversially debated, which of these factors are the dominant cause for the observed alterations in vivo. In this study we investigated the globular folded peptidyl-prolyl isomerase Pin1 in Xenopus laevis oocytes and in native-like crowded oocyte extract by in-cell NMR spectroscopy. We show that active Pin1 is driven into nonspecific weak attractive interactions with intracellular proteins prior to substrate recognition. The substrate recognition site of Pin1 performs specific and nonspecific attractive interactions. Phosphorylation of the WW domain at Ser16 by PKA abrogates both substrate recognition and the nonspecific interactions with the endogenous proteins. Our results validate the hypothesis formulated by McConkey that the majority of globular folded proteins with surface charge properties close to neutral under physiological conditions reside in macromolecular complexes with other sticky proteins due to molecular crowding. In addition, we demonstrate that commonly used synthetic crowding agents like Ficoll 70 are not suitable to mimic the intracellular environment due to their incapability to simulate biologically important weak attractive interactions.


Subject(s)
Macromolecular Substances/chemistry , Peptidylprolyl Isomerase/chemistry , Protein Folding , Amino Acid Sequence , Animals , Humans , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Oocytes/enzymology , Peptidylprolyl Isomerase/genetics , Proteins , Substrate Specificity , Xenopus
9.
Cell ; 144(4): 566-76, 2011 Feb 18.
Article in English | MEDLINE | ID: mdl-21335238

ABSTRACT

TAp63α, a homolog of the p53 tumor suppressor, is a quality control factor in the female germline. Remarkably, already undamaged oocytes express high levels of the protein, suggesting that TAp63α's activity is under tight control of an inhibitory mechanism. Biochemical studies have proposed that inhibition requires the C-terminal transactivation inhibitory domain. However, the structural mechanism of TAp63α inhibition remains unknown. Here, we show that TAp63α is kept in an inactive dimeric state. We reveal that relief of inhibition leads to tetramer formation with ∼20-fold higher DNA affinity. In vivo, phosphorylation-triggered tetramerization of TAp63α is not reversible by dephosphorylation. Furthermore, we show that a helix in the oligomerization domain of p63 is crucial for tetramer stabilization and competes with the transactivation domain for the same binding site. Our results demonstrate how TAp63α is inhibited by complex domain-domain interactions that provide the basis for regulating quality control in oocytes.


Subject(s)
Oocytes/metabolism , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Trans-Activators/chemistry , Trans-Activators/metabolism , Animals , DNA/metabolism , Dimerization , Female , Gamma Rays , Mice , Models, Molecular , Phosphorylation , Protein Multimerization , Tumor Suppressor Protein p53/metabolism
10.
J Struct Biol ; 172(1): 94-106, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20460156

ABSTRACT

G-protein coupled receptors still represent one of the most challenging targets in membrane protein research. Here we present a strategic approach for the cell-free synthesis of these complex membrane proteins exemplified by the preparative scale production of the human endothelin A receptor. The versatility of the cell-free expression system was used to modulate sample quality by alteration of detergents hence presenting different solubilization environments to the synthesized protein at different stages of the production process. Sample properties after co-translational and post-translational solubilization have been analysed by evaluation of homogeneity, protein stability and receptor ligand binding competence. This is a first quality evaluation of a membrane protein obtained in two different cell-free expression modes and we demonstrate that both can be used for the production of ligand-binding competent endothelin A receptor in quantities sufficient for structural approaches. The presented strategy of cell-free expression protocol development could serve as basic guideline for the production of related receptors in similar systems.


Subject(s)
Cell-Free System/metabolism , Receptor, Endothelin A/biosynthesis , Receptors, G-Protein-Coupled/biosynthesis , Binding, Competitive , Blotting, Western , Chromatography, Affinity , Circular Dichroism , Detergents/chemistry , Fluorescence Polarization , Humans , Ligands , Protein Stability , Proteomics/methods , Radioligand Assay , Receptor, Endothelin A/chemistry , Receptor, Endothelin A/metabolism , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/metabolism , Reproducibility of Results , Solubility
SELECTION OF CITATIONS
SEARCH DETAIL
...