Periodic Reporting for period 1 - CarboPore (Deciphering the mechanism of cellular aging: interaction of oxidatively damaged proteins with the cellular membranes)
Reporting period: 2020-04-01 to 2022-03-31
Aging is a physiological process characterized by a progressive deterioration of organismal functions. As such, aging is a major risk factor for death and chronic conditions such as neurodegeneration, cancer, diabetes, arthritis, and cardiovascular diseases. Since the portion of elderly individuals in the human population has been rapidly increasing, healthy aging has become one of the most important goals and challenges of the contemporary society. However, despite extensive research, etiology and the mechanisms of aging remain some of the greatest mysteries in biology.
In this project we have explored the molecular mechanisms of aging with the ultimate goal to set basis for development of treatments that would attenuate aging and delay the onset of age-related diseases. Our work was based on a concept that cumulative oxidative damage to proteins leads to age-associated cellular dysfunction. In particular, our goal was to test a hypothesis that the oligomers and aggregates formed by the oxidized proteins compromise the integrity of cellular membranes. The objectives of the project were the following: 1) to characterize the aggregates formed by the oxidized proteins; 2) to assess their interaction with the biological membranes and their ability to perforate the membranes; 3) to evaluate their cellular toxicity; and 4) to identify molecules that could either prevent the formation of the toxic aggregates, or protect the membranes from such aggregates. To that end, we used interdisciplinary approaches that included advanced imaging techniques – atomic force microscopy (AFM), stimulated emission depletion microscopy (STED) and Fourier transform infrared (FTIR) microscopy and spectroscopy, mass spectrometry (MS) and various biochemical techniques.
In this project we have explored the molecular mechanisms of aging with the ultimate goal to set basis for development of treatments that would attenuate aging and delay the onset of age-related diseases. Our work was based on a concept that cumulative oxidative damage to proteins leads to age-associated cellular dysfunction. In particular, our goal was to test a hypothesis that the oligomers and aggregates formed by the oxidized proteins compromise the integrity of cellular membranes. The objectives of the project were the following: 1) to characterize the aggregates formed by the oxidized proteins; 2) to assess their interaction with the biological membranes and their ability to perforate the membranes; 3) to evaluate their cellular toxicity; and 4) to identify molecules that could either prevent the formation of the toxic aggregates, or protect the membranes from such aggregates. To that end, we used interdisciplinary approaches that included advanced imaging techniques – atomic force microscopy (AFM), stimulated emission depletion microscopy (STED) and Fourier transform infrared (FTIR) microscopy and spectroscopy, mass spectrometry (MS) and various biochemical techniques.
During the project we have identified bacterial and mammalian oxidation-sensitive subset of the proteome by MS. Furthermore, the aggregates formed by the oxidized proteins within the mammalian cells were characterized using FTIR microscopy, AFM and cell fractionation experiments. Interaction with the membranes was analyzed for the total cellular aggregates and aggregates formed by a recombinant protein using STED microscopy and biochemical assays based on cell fractionation and floatation on sucrose density gradients. The recombinant protein used in this work was superoxide dismutase 1 (SOD1), a protein implicated in amyotrophic lateral sclerosis (ALS) which represents a typical age-related neurodegenerative disease. While the oxidized and non-oxidized cellular aggregates bound membranes in a similar manner, oxidized recombinant SOD1 had different affinity for the artificial membranes relative to its non-oxidized counterpart. Moreover, aggregates formed by the oxidized and non-oxidized SOD1 differed in their ability to perforate membranes, as well as in their cellular toxicity. In search for the agents that could protect the cells from the toxic oxidized protein aggregates, we found that the presence of certain lipids in the artificial membranes was protective against all kinds of aggregates, and some other lipids were protective against oxidative damage of the cells. Finally, we identified antioxidants as the most efficient in preventing protein oxidation in the cells.
Various parts of these results were presented to the scientific community in one peer-reviewed publication, as well as at one local (Life Science Club in Split, Croatia) and three international conferences (IMCAS in Paris, France; Advances in Biomedical Research in Split, Croatia; and ABMBBIH in Sarajevo, BiH) in the form of oral or poster presentations. Furthermore, the results were communicated at two smaller meeting with other institutions, during one invited lecture in Slovenia, and at numerous internal meetings. The main messages of our work were also shared with broader audience during three local outreach events, and the basic project information were featured on social media, project website and within an interview at an internet portal of a Serbian radio station. Future exploitation of the results was secured through establishing connections with a French cosmetic company.
Various parts of these results were presented to the scientific community in one peer-reviewed publication, as well as at one local (Life Science Club in Split, Croatia) and three international conferences (IMCAS in Paris, France; Advances in Biomedical Research in Split, Croatia; and ABMBBIH in Sarajevo, BiH) in the form of oral or poster presentations. Furthermore, the results were communicated at two smaller meeting with other institutions, during one invited lecture in Slovenia, and at numerous internal meetings. The main messages of our work were also shared with broader audience during three local outreach events, and the basic project information were featured on social media, project website and within an interview at an internet portal of a Serbian radio station. Future exploitation of the results was secured through establishing connections with a French cosmetic company.
The results of this project suggest that the oxidative damage to proteins may represent a mechanistic link between aging and the onset of age-related diseases. In particular, we found that oxidation modulates aggregation properties of ALS-implicated recombinant SOD1, as well as its cellular toxicity and the ability to perforate membranes. Protein oxidation could be prevented by specific antioxidants, and the resistance of membranes to protein aggregates could be increased by enrichment of specific lipids. Thus, our project results may open new avenues for development of novel strategies to delay the onset of ALS and perhaps other age-related diseases.
During the fellowship, the awardee has been promoted into an independent group leader, as well as appointed to Research associate scientific grade and Assistant professor scientific-teaching grade. Moreover, the fellowship has led to creation of several new sustainable international collaborations for MedILS - with Northwestern University in the USA, Jozef Stefan Institute in Slovenia, and Split University in Croatia.
During the fellowship, the awardee has been promoted into an independent group leader, as well as appointed to Research associate scientific grade and Assistant professor scientific-teaching grade. Moreover, the fellowship has led to creation of several new sustainable international collaborations for MedILS - with Northwestern University in the USA, Jozef Stefan Institute in Slovenia, and Split University in Croatia.