Within the Leibniz Graduate School on Aging in Jena the Membrane Trafficking Group invites applications for a PhD student position (TV-L E13/2) for the project:
Functional characterization of AGE modifications in aging .
The homeostasis of the proteome of cells is required to maintain the function of organs and it was shown to decline during aging. Advanced glycation end products (AGEs) are a family of non-enzymatic posttranslational modifications that have been shown to accumulate in aging tissues. Although a handful of specific AGE-modified proteins have been identified, a detailed characterization of the targets of AGEs and the relationship between this modification and protein function is still missing. We have devised a mass spectrometry based approach to identify specific sites of carboxymethyl lysine (CML, one of the most abundant AGEs) modifications in proteins. Using this approach, we were able to identify over 1000 CML sites in cells treated with glycating agents and over 800 sites in primary tissues. Our data revealed that in vivo CML modifications occur in a specific set of proteins with members of the mitochondrial respiratory chain being particularly affected.
In this project, we would like to investigate the functional role of these modifications and their relationships to the aging process. In particular, our aims are: (1) to develop targeted proteomics assays for the identified CML sites to investigate their value as biomarkers of aging; (2) to assess the consequences of CML modifications in terms of protein stability, interactions and turn-over; (3) to investigate the relationship between mitochondrial dysfunction and CML modification of respiratory chain complexes.
(1) We will establish targeted proteomic assays based on isotopycally labeled peptide standards derived from our list of identified CML sites. Priority will be given to high confidence CML sites identified in vivo and across multiple tissues. The gain in sensitivity provided by the targeted measurements (approx. one order of magnitude) and the accuracy of quantification guaranteed by using spike-in standard will enable us to quantify the endogenous CML modified peptides without prior enrichment. This will drastically increase our throughput and enable us to monitor the abundance of CML sites across multiple tissues and age groups. First, we will address which CML sites are indeed correlated with chronological aging. Second, we will investigate whether age-related conditions (e.g., diabetes) and anti-aging interventions (e.g., dietary restriction) have a quantitative impact on the extent of CML modification in specific proteins. Our measurements will be complemented by evaluation of total CML levels as well as other AGEs. (2) In preliminary experiments, we have demonstrated that addition of glycating agents to cells induces both changes of protein abundance and thermal stability. We will continue this work by exploring whether AGEs are sufficient to alter protein stability of specific proteins using in vitro assay such as differential scanning fluorimetry. In addition, we will investigate the relationship between AGEs and protein turnover by performing proteome-wide measurement of protein turnover using mass spectrometry. Finally, to gain complementary information regarding the status of the proteostasis machinery, we will assess the activity of the proteasome and autophagy upon treatment of cells with glycating agents. (3) Since our preliminary work points to mitochondrial proteins are primary targets of AGEs, we will try to establish a mechanistic link between mitochondrial dysfunction and AGEs. We will induce mitochondrial dysfunction, reactive oxygen species or lipid peroxidation in mammalian cell culture systems and quantitatively measure the abundance of CML sites. In parallel, we will assess the impact of CML modification on the assembly state of respiratory chain complexes by native gel activity assays and investigate mitochondrial function upon induction of AGEs in multiple cell types.
The ideal candidate will be a motivated and independent thinking person willing to undertake the challenge of working across disciplines’ boundaries. She/he should hold a Master degree or equivalent in biology/biochemistry/bioinformatics/medicine or related field.
A position in a well-equipped research group of a high quality institute for age research, which harbors several state-of-the-art facilities. Our work is embedded in the Beutenberg Campus, an interdisciplinary base for innovative research.
The PhD position will be integrated in our Leibniz Graduate School on Aging and Age Related Diseases
The contract conditions and the salary will be according to the collective labor agreement for public service employees of the Federal State of Germany (TV-L E13 50-65%).
Please acquaint yourself with the LGSA Procedures and with the application procedure on our website lgsa.leibniz-fli.de, fill in the application form and send it electronically to the LGSA.
Please note that your application will be made available to the LGSA selection committee for assessment. Please check here for FLI data protection (www.leibniz-fli.de/dataprotection) regulation and the processing of personal information according to the EU-GDPR.