A5102848969- Plant Gene Expression Analysis
- Fungal Plant Pathogen Control
- Phytochemistry and biological activities of Ficus species
- Parkinson's Disease Mechanisms and Treatments
- Mitochondrial Function and Pathology
- Fungal and yeast genetics research
- Endoplasmic Reticulum Stress and Disease
- Plant biochemistry and biosynthesis
- Biotin and Related Studies
- Protein Kinase Regulation and GTPase Signaling
- Advanced Fluorescence Microscopy Techniques
- RNA and protein synthesis mechanisms
- Genomics and Chromatin Dynamics
- Cellular transport and secretion
- Autophagy in Disease and Therapy
- RNA regulation and disease
- Glycosylation and Glycoproteins Research
- Bacterial biofilms and quorum sensing
- Plant Reproductive Biology
- Vibrio bacteria research studies
- Transgenic Plants and Applications
- Histone Deacetylase Inhibitors Research
- Ubiquitin and proteasome pathways
- Cellular Mechanics and Interactions
- Catalytic C–H Functionalization Methods
Goethe University Frankfurt
2021-2024
Research Network (United States)
2023-2024
Aligning Science Across Parkinson's
2023
Align Technology (United States)
2023
Structural Genomics Consortium
2023
DKFZ-ZMBH Alliance
2017-2021
Heidelberg University
2017-2021
Harvard University
2017
University of Stuttgart
2016
During terminal differentiation, the global protein complement is remodeled, as epitomized by erythrocytes, whose cytosol ~98% globin. The erythroid proteome undergoes a rapid transition at reticulocyte stage; however, mechanisms driving programmed elimination of preexisting cytosolic proteins are unclear. We found that mutation in murine
Tail-anchored (TA) proteins insert post-translationally into the endoplasmic reticulum (ER), outer mitochondrial membrane (OMM) and peroxisomes. Whereas GET pathway controls ER-targeting, no dedicated factors are known for OMM insertion, posing question of how accuracy is achieved. The AAA-ATPase Msp1 removes mislocalized TA from OMM, but it unclear, clients targeted degradation. Here we screened involved in degradation to mitochondria. We show that ER-associated (ERAD) E3 ubiquitin ligase...
Loss-of-function mutations in PTEN-induced kinase 1 (PINK1) are a frequent cause of early-onset Parkinson’s disease (PD). Stabilization PINK1 at the translocase outer membrane (TOM) complex damaged mitochondria is critical for its activation. The mechanism how activated TOM unclear. Here, we report that co-expression human and all seven subunits Saccharomyces cerevisiae sufficient We use this reconstitution system to systematically assess role each subunit toward unambiguously demonstrate...
Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of familial Parkinson’s disease (PD) and risk factor for the sporadic form. Increased activity was shown patients with both PD, making LRRK2 inhibitors major focus drug development efforts. Although much progress has been made understanding structural biology LRRK2, there no available structures inhibitor complexes. To this end, we solved cryo–electron microscopy wild-type PD-linked mutants, bound to LRRK2-specific type I...
Pseudomonas aeruginosa has emerged as an important opportunistic human pathogen that is often highly resistant to eradication strategies, mediated in part by the formation of multicellular aggregates. Cellular aggregates may occur attached a surface (biofilm), at air-liquid interface (pellicle), or suspended Compared communities, knowledge about regulatory processes involved cell still limited. We have recently described SiaA/D signal transduction module regulates macroscopic aggregation...
The Rab GTPase switch 2 region is a hotspot for post-translational modifications. Its phosphorylation can determine whether individuals develop Parkinson's disease or not. Other modifications of the same are catalysed by enzymes from bacterial pathogens when they infect human cells. Here, we profiled set kinases including LRRK1, LRRK2, DYRK1A, MST1, and TBK1 their capability phosphorylating GTPases. We identified several novel kinase:Rab pairs, such as LRRK1:Rab43 TBK1:Rab29. Further,...
Abstract Selectivity for closely related isoforms of protein kinases is a major challenge in the design drugs and chemical probes. Covalent targeting unique cysteines potential strategy to achieve selectivity highly conserved binding sites. Here, we used pan-LIMK inhibitor selectively probe LIMK1 over LIMK2 by LIMK1-specific cysteine C349 located glycine-rich loop region. Binding kinetics both non-covalent covalent LIMK inhibitors were investigated, fast on-rate small size type-I inhibitor....
The pyrimidine core has been utilized extensively to construct kinase inhibitors, including eight FDA-approved drugs. Because the hinge-binding motif is accommodated by many human kinases, kinome-wide selectivity of resultant molecules can be poor. This liability was seen as an advantage since it well tolerated understudied kinases. We hypothesized that nonexemplified aminopyrimidines bearing side chains from well-annotated pyrimidine-based inhibitors with off-target activity on kinases...
Purified, recombinant proteins are essential for biochemical characterization and structural studies. However, every protein is unique in its vitro behaviour thus, establishing a stable expression system purification protocol can be challenging. Therefore, testing several constructs parallel may increase the chances of getting high quality prep while minimizing laborious work. In this we providing an overview main steps order to clone test with varying N-and C-terminal boundaries human PTEN...
Size-exclusion chromatography (SEC) is a method for separating proteins according to their size. To achieve this, protein sample applied column that tightly packed with porous beads.The size separation determined by the thickness of hydration shell in solvent. Small molecules can penetrate pores, while large cannot and are eluted first from column. Smaller follow, whereby elution time inversely proportional We used this investigate whether form stable complex. The individual complex partners...
Expression and purification of human LRRK2 its variants from insect cells.
Mutations in Leucine Rich Repeat Kinase 2 (LRRK2) are a common cause of familial Parkinson’s Disease (PD), and risk factor for the sporadic form. Increased kinase activity has been shown both PD patients, making LRRK2 inhibitors major focus drug development efforts PD. Although significant progress made understanding structural biology LRRK2, there no available structures inhibitor complexes. To this end, we solved cryo-EM wild-type PD-linked mutants, bound to LRRK2-specific type-I MLi-2...
Abstract Leucine rich repeat kinase 2 (LRRK2) is a large multidomain protein containing two catalytic domains, and GTPase, as well interactions including WD40 domain. The association of increased LRRK2 activity with both the familial sporadic forms Parkinson’s disease (PD) has led to intense interest in determining its cellular function. However, small molecule probes that can bind report on or affect are needed. Here, we identified series high-affinity LRRK2-binding designed ankyrin-repeat...
Here we describe a C-SWAT library for high-throughput tagging of Saccharomyces cerevisiae ORFs. It consists 5661 strains with an acceptor module inserted after each ORF, which can be efficiently replaced tags or regulatory elements. We validate the targeted sequencing and demonstrate its use by yeast proteome bright fluorescent proteins, determining how sequences downstream ORFs influence protein expression localizing previously undetected proteins.
This protocol can be used in this form or with small adjustments regarding concentration and reaction time to determine vitro substrate phosphorylation for any purified kinase pair. It inhibition curves by addition of series inhibitors. Reaction is carried out 50 µL mix containing final nM 5 mM substrate.
This protocol can be used in this form or with small adjustments regarding concentration and reaction time to determine vitro substrate phosphorylation for any purified kinase pair. It inhibition curves by addition of series inhibitors. Reaction is carried out 50 µL mix containing final nM 5 µM substrate.
Thermal shift assay or differential scanning fluorimetry analyzes the effect of small molecules on thermostability a protein by gradual heat denaturation and monitoring absorption fluorescent dye SYPR Orange at 488 nm.
Abstract Loss of function mutations in PTEN-induced kinase 1 (PINK1) are a frequent cause early-onset Parkinson’s disease (PD). Stabilisation PINK1 at the Translocase Outer Membrane (TOM) complex damaged mitochondria is critical step for its activation. To date mechanism how activated TOM unclear. Herein we report co-expression human and all seven subunits Saccharomyces cerevisiae sufficient We use this reconstitution system to systematically assess role each subunit towards unambiguously...