Monitoring changes in the cellular content of biomolecules during ageing with FTIR spectroscopy

Abstract

Dietary regimens have proven to promote longevity in several eukaryotic model organisms including the budding yeast Saccharomyces cerevisiae. These interventions are effective strategies for preventing ageing and diseases and many of them are linked to amino acid and protein levels. The aim of this work was to better understand how the age-related TOR1 and SCH9 genes and the presence of amino acids affect cell metabolomes and to establish their impact on the ageing process. Cellular metabolic profiles were determined by FTIR spectroscopy. We demonstrated that metabolic signatures of cells deficient in SCH9, the major TORC1 effector, were very different to those of wild type and TOR1 deficient cells. In cells lacking Sch9 we also observed changes in other processes related to ageing such as endoplasmic reticulum stress and autophagy. We identified several anti-ageing biomarkers being the most relevant the intracellular content of pyruvate, glucose, ribose/deoxyribose associated compounds, and the presence of special protein conformational structures. The very sensitive FTIR technique allowed us to highlight important changes that occur along ageing in the metabolomes of the cells deficient in the key nutrient-sensitive Tor1-Sch9 pathway, even though slight differences on chronological lifespan were detected in our conditions.

Introduction

Ageing is a multifactorial and complex process characterized by a progressive damage of cellular functions that inevitably leads to death. Since ever extension of lifespan has been a human challenge. In the last decades ageing research has exploded and the progress in this area opens the possibility of reducing incidence of age-related diseases such as cancer, diabetes, cardiovascular and neurodegenerative diseases, or simply of increasing overall lifespan [[1], [2], [3]].

The unicellular yeast Saccharomyces cerevisiae is an excellent eukaryotic model vastly used in basic and applied research, and this organism has already been used to study the molecular mechanisms of cell ageing and death [4]. Advantages of using yeast cells are their short doubling time, strictly well-known culture media, easy genetic manipulation, and ample availability of omics databases. Furthermore, there are many yeast proteins that show strong functional homology with human proteins. So, it makes yeast suitable to model different biological processes and human diseases [5].

In model organisms ranging from yeast to mammals, dietary restriction without malnutrition is an intervention that can effectively prolong lifespan [6,7]. Dietary restriction means a regimen in which one specific nutrient or a group of nutrients are reduced or removed from the diet, whereas caloric restriction, a particular dietary restriction, is a reduced intake of calories [7].

S. cerevisiae cells divide by asymmetric budding, with the daughter cell that is produced being smaller than the mother from which it is derived [8]. Ageing research in this yeast has been carried out using two different paradigms: replicative life span (RLS) and chronological life span (CLS). RLS indicates the number of daughter cells a single mother cell can produce and CLS is defined as the time it takes for non-dividing cells to survive in stationary phase [8]. Both yeast model systems, RLS and CLS, parallel the ageing of mammalian cells, such as fibroblasts and lymphocytes, which undergo a fixed number of population doublings when maintained in culture, and mammalian cells that do not divide, such as neurons, or have long non-mitotic resting phases [4].

TOR (Target Of Rapamycin) nutrient-sensing pathway plays important roles in the ageing process in different organisms [6]. Budding yeast have two TOR genes, TOR1 and TOR2; instead, all other eukaryotic cells possess only one. Two functionally and structurally distinct multiprotein complexes have been described: TORC1, which is sensitive to rapamycin, and TORC2, which is insensitive to rapamycin. TORC1 contains either Tor1 or Tor2 proteins as the catalytic subunit, whereas only Tor2 is part of TORC2. Each complex signals through a different set of effectors [9]. One of the major effectors of TORC1 is the AGC kinase Sch9 that is homologous to the mammalian kinase S6K [10,11]. Deletions of TOR1 [[12], [13], [14]] and SCH9 [15,16] genes promoted longevity [17]. However, there is evidence that these genes affected differentially lifespan and stress resistance. The deficiency in TOR1 produces less robust effects than those observed in cells lacking Sch9 [18]. The TORC1-Sch9 pathway controls many cellular processes in response to multiple stimuli including amino acids, and in doing so this via produces relevant changes in cell growth and metabolism [17,19].

Fourier Transform Infrared (FTIR) spectroscopy has been used to detect changes in biomolecules of whole cells [20,21]. This powerful tool has been applied in various specialities including biochemistry and biomedical science due to the benefits of low cost and rapidity, providing biochemical fingerprint extremely informative about the global metabolite pools [22]. FTIR technique has proven to be sensitive for the identification and for the metabolic monitoring of microorganisms and of mammalian cells [[23], [24], [25], [26]]. FTIR has been recognized as a valuable technique to determine the metabolic status of cells as it is able to analyse carbohydrates, fatty acids, proteins, nucleic acids rapidly and simultaneously with a minimum amount of sample preparation [21,25,[27], [28], [29]]. Thereby, by using its advantages, FTIR spectroscopy has been successfully applied to evaluate metabolic changes detected when yeast cells are subjected to subtle growth variations (i.e. modifying only one nutrient) or when just one gene is deleted or mutated [24,24,25,26,30,31]. This technique has been also used to detect that prolonged post-reproductive lifespan is associated with modifications of amounts of fatty acids [32].

The aim of this work was to better understand how particular genotypes and growth conditions affect cellular metabolomes and how these metabolomes impact on the ageing process. As the way the cells live determines their final fate, one goal was to establish the effect of the initial nutritional status on lifespan of cells deficient in the kinases Tor1 and Sch9 both participating in one of the key nutrient-sensitive pathway that modulate ultimately cellular physiology. Taking into account that the metabolic status certainly determines if cells will live or die, metabolic profiles of these cells were determined by FTIR spectroscopy. So, another goal was to study the changes produced on the cellular metabolome as cells age. The metabolome of cells is an excellent probe of their phenotype and, unlike the genome or even the proteome, it is a highly dynamic entity. Hence, the search of metabolic biomarkers of lifespan extension was another of our aims.