- MS platform
Project 1. Reorganization of membrane-bound organelles during proteostasis stress
We discovered that the chaperone-interacting ubiquitin ligase CHIP docks on cellular membranes during acute stress. HSP70 and lipids compete for mutually exclusive binding to the TPR domain of CHIP. At new cellular locations, access to compartment-specific substrates enables CHIP to participate in the reorganization of the respective organelles, as exemplified by the fragmentation of the Golgi apparatus. Currently, we are analyzing how the oligomerization of CHIP contributes to its association with lipids. Other TPR domain-containing proteins from the proteostasis network (HOP, DnaJC7, FKBP51, FKBP52) will be tested for the involvement in similar regulatory loops. The organelle adaptation driven by CHIP and other cochaperones might explain the diversification of the cellular stress response during adaptation or disease development.
Kopp Y., Lang W.H., Schuster T.B., Martínez-Limón A., Hofbauer H.F., Ernst R., Calloni G., Vabulas R.M. (2017) CHIP as a membrane-shuttling proteostasis sensor. eLIFE. 6: e29388.
Project 2. Stress compartmentalization without membranes (RNA-protein assemblies)
RNA in the cell is almost always associated with proteins. An exception represents the polysome disassembly during stress when naked RNA is massively released into the cytosol. We identified the mRNA methylation machinery which enhances sequestration of transcripts in stress granules to protect them during stress. Unexpectedly, inhibition of the machinery affected clearance of an amyloidogenic protein and increased bystander protein and RNA coaggregation. Our results revealed a tight link between protein and RNA homeostasis. Currently, we are seeking to identify methyl-adenine reader proteins responsible for the mRNA sequestration during acute and chronic stress. Furthermore, we are looking for similar mechanisms operating in human cells during RNA virus infection.
Alriquet M., Calloni G., Martínez-Limón A., Delli Ponti R., Hanspach G., Hengesbach M., Tartaglia G.G., Vabulas R.M. (2018) RNA Methyltransferase Safeguards mRNAs during Proteostasis Stress and Amyloidogenesis. (Under revision)
Project 3. Targeted disruption of human flavoproteome
Riboflavin (vitamin B2) is the precursor of FMN and FAD cofactors, which define a small, but metabolically highly important flavoproteome encoded by ca. 100 genes in humans. Using quantitative mass spectrometry and biochemical assays, we uncovered the destabilization of a significant fraction of flavoproteins during vitamin B2 deficiency. The destabilization resulted in increased protein aggregation. Our data suggest that relative insufficiency of apoprotein degradation caused by vitamin shortage can aggravate protein aggregation disorders. Stability analysis of flavoproteins, such as NADPH oxidases, NO synthases or respiratory chain complexes, bears the exciting possibility to connect nutrition and gut microbiome with chronic diseases, inflammation and aging-related conditions. From the other side, we are investigating the possibility to use local and transient shortage of vitamin B2 to increase vulnerability of tumor cells for chemotherapy.
Martínez-Limón A., Alriquet M., Lang W.H., Calloni G., Wittig I., Vabulas R.M. (2016) Recognition of enzymes lacking bound cofactor by protein quality control. PNAS USA. 113(43):12156-61.
Mass spectrometry platform at the BMLS Institute
In 2016, our group established a proteomics platform at the BMLS Institute. The system is based on the Q Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer from Thermo Scientific. The spectrometer combines high-performance quadrupole precursor selection with high resolution, accurate mass (HR/AM) Orbitrap detection. In 2017, the platform was enhanced with a column oven to increase the stability of the measurements. Dr. Giulia Calloni has been in charge of its maintenance, operation and training of other users.
We routinely perform quantitative (label-free and labelling-based) proteomics analyses aimed at identifying whole-proteome changes, organelle proteomes and interactomes of individual proteins of interest.