Ataxia Telangiectasia (A-T) is a genetic disease characterized by cerebellar ataxia and immunodeficiency. A-T is linked to the loss of ATM protein function, a serine/threonine kinase central in DNA damage response. ATM modulates also the activity of E3-ubiquitin-ligases, affecting the stability of target proteins. Therefore, ATM deficiency may severely impinge on the cellular proteome composition resulting in defective signaling pathways. In fact, there are increasing evidence that this protein may have an important role in the control of target proteins of the ubiquitin system. Stagni and colleagues have recently shown that ATM modulates the proteasome dependent down-regulation of c-FLIP [17,18]
. In the present study, we have pursued a comprehensive proteomic investigation to evaluate the biological effects of ATM expression on the control of protein quality and stability. To this aim, protein expression profiling were also assessed in the presence of the proteasome inhibitor MG132 to highlight those proteins whose expression is modulated by ATM most likely through the ubiquitin–proteasome system and whose half-life is particularly short and their ATM dependent modulation levels over the whole proteome would be partially masked in a direct investigation. By label-free nLC-MSE
approach, a total of 53 and 62 differentially expressed proteins were identified in the two analyzed comparison (L6ATM vs
L6ATM and MG132 treated L6ATM vs
MG132 treated L6 cell lines, respectively). Twelve proteins are regulated in the same way in both experimental dataset and we can speculate that their expression is influenced by the presence/absence of ATM but this event occurs independently of the ubiquitin–proteasome system involvement. Remarkably one of them, Plastin 3 (PLS3), differentially regulated in both dataset of the shotgun proteomic experiments (overepressed in L6ATM cell line in presence or absence of MG132, , A, B), is already known as phosphorylated upon DNA damage, probably by ATM or ATR 
, and its levels are decreased in Spinal Muscular Atrophy (SMA, a neurological disease) mouse model 
We analyzed by western blot other three proteins whose levels were influenced by ATM expression and MG132 treatment: STAT1, Lamin B1 and Matrin 3 (, E, F) to confirm the regulation observed through proteomic analysis in both L6 treated cell lines. Signal Transducer and Activator of Transcription 1 (STAT1) has been previously identified as a potential substrate of ATM in nuclear extracts from irradiated (10 Gy) HeLa cells 
enforcing the idea that this member of the STAT protein family could be a direct target of ATM. In our study STAT1 is down-regulated after proteasome blockage in L6 ATM compared to L6, an evidence that could be eventually explained by proteasome dependent degradation of STAT1 in ATM proficient cells. In response to cytokines and growth factors, STAT family members are phosphorylated by the receptor associated kinases, and then form dimers that translocate to the cell nucleus where they act as transcription activators of a variety of genes, which is thought to be important for cell viability in response to different cell stimuli and pathogens 
. There are some evidences in literature which shine a light on the interplay between ATM and STAT1 in the response to the DNA damage, that strengthen our findings [46,47]
Moreover, we observed a decrease of Lamin B1(LMNB1) in L6 ATM treated cells; recently Barascu and colleagues demonstrated an upregulation of Lamin B1 in A-T cells extract. The authors stressed the point that LMNB1 overexpression is sufficient to induce nuclear shape alterations and senescence in wild-type cells. A-T patients suffer from premature ageing and this observation led to the hypothesis that Lamin B1 dysregulation could account for senescence in A-T cells 
. The authors related LMNB1 accumulation to A-T associated DDR defects, oxidative stress and nuclear shape alterations. Finally, by a systematic analysis of human protein complexes to identify chromosome segregation proteins, ATM and LMNB1 were found as bait-prey interactors from affinity-purification - mass spectrometry experiments (www.mitocheck.org
; this experimental evidence adds an interesting discussion point for the possible direct interaction between ATM and LMNB1 occurring in the nuclear compartment while the highly ordered processes of chromosome segregation and cell division is ongoing. Chromosome alignment, movement and segregation during cell division involve interactions between the kinetochore and the mitotic spindle through microtubule depolymerization/assembly 
. Notably, we revealed differential expression of tubulins and Heat Shock Proteins (HSPs) in both proteomics dataset. Although the expression of some cytoskeleton proteins and HSPs could be related to their abundance and therefore to their more easily accessible identification by mass spectrometry experiments, in our opinion the selective presence of centrosome components, like tubulins, and Hsp 70 and 90 in our cell models is tied to their function in cell cycle control, cell death and aggresome promoting formation as described in several literature papers and already observed in our previous work 
. Mediators of stress response (i.e., Checkpoint kinase 2 and its upstream regulator ATM) indeed regulate centrosome inactivation checkpoint and use stress inducted centrosome fragmentation or amplification for eliminating damaged cells 
. The role of HSPs in cell cycle control and in signal transduction networks has been indeed described and assigned both to Hsp 90 
and Hsp 70. In particular Hsp 70, as binding partners of hSNM1B/Apollo, a protein with stimulating effect on ATM substrate phosphorylation in response to DNA-damage, results indirectly implicated in the maintenance of genome stability 
. Recent studies have also disclosed the involvement of Hsp 70 in the preservation of cytoarchitecture. Zhang X. and colleagues have indeed described the “unexpected role” of Hsp 70 in promoting aggresome formation through the interaction with the cochaperone ubiquitin ligase Carboxyl terminal of Hsp70/Hsp90 interacting protein (CHIP) 
. In conclusion the participation of tubulins and HSPs in cell surveillance mechanisms qualified them as additional implementing checkpoints recruited and activated by stress stimuli, thereby explaining in part their differentially expressed levels in ATM absence in our proteomic observations.
Among the proteins whose levels were influenced by ATM expression and MG132 treatment and were analyzed by western blot, Matrin 3 (MATR3) has already been identified as cross-reacting protein to phospho-specific antibodies against known ATM/ATR substrates 
. Moreover, Matrin 3 has been already described in literature as involved in early stage of DSB response 
. In fact, treatment with the radiomimetic agent neocarzinostatin and MATR3 depletion led to abnormal accumulation of cells at the S-phase of the cell cycle. We observed an up-regulated protein expression in L6ATM treated cells by nLC-MSE approach. On the other hand, we could not confirm these data through western blot analysis, thus we could not completely rely on the proteomic evidence.
Therefore, as first conclusion we can argue that our experimental data pointed out some stimulating proteins whose expression changes depending on ATM in presence of proteasome inhibition and could be considered potential ATM activity substrate through the Ub–P system: the transcription activator STAT1 and Lamin B1.
The second interesting point of discussion concerns the significant overrepresentation of proteins involved in glycolysis/gluconeogenesis pathway and carbohydrate metabolism molecular function supporting the idea that there is an evident switch of the metabolism, and in particular of the carbohydrate process, in absence of the ATM expression. Our observations showed how expression of ATM in L6 cells drives higher expression of glycolytic enzymes (PGK1, PGAM1 and PKM2), lower intermediate glycolytic metabolites and higher pyruvate production probably by a stimulation of the cellular rate of glycolysis. The higher lactate amounts may depend consequently both on higher levels of its precursor (pyruvate) and on its function as NADH depleting compound in order to avoid the blockage of glycolysis due to the GAPDH enzymatic step which is operated in near equilibrium condition. These findings are related with the emerging role of ATM as central regulator of cellular metabolism in response to oxidative stress, linking genome stability, cell cycle and carbon catabolism [57–60]
. ATM is largely nuclear, acting as modulator of the cellular response to genotoxic stress and indeed our observed up-regulation of hnRNPH in ATM cells could possibly be related to its function in maintaining the genome integrity. In fact, hnRNPH has been described as part of a rescue mechanism of p53 mRNA 3′-end processing regulation in DNA-damaged cells 
. Moreover, there are increasing evidences that ATM deficiency is not only cause of damage response lack of function; ATM localizes predominantly in the cytoplasm in neuronal and neuron-like cells [62,63]
and cytoplasmatic ATM activity is involved in insulin signaling pathways [30,32]
). Cosentino et al. 
demonstrated the link between ATM and the pentose phosphate pathway (PPP) by inducing Glucose-6-phosphate dehydrogenase (G6PD) activity. G6PD is the limiting enzyme of the PPP metabolic pathway which in turn is responsible for the production of the essential antioxidant NADPH cofactor and nucleotide synthesis required to promote DSB repair. Acting as a sensor of reactive oxygen species (ROS), ATM could possibly shift the carbohydrate metabolism from glycolysis to the oxidative PPP under stress condition like DSBs. Shifting the energy source glucose-6–phosphate from glycolysis to PPP, the energy stored in carbohydrate backbones molecules will be shifted toward NADPH production and nucleotide synthesis instead of ATP and NADH produced by glycolysis. In our study we hypothesize a shift of the glycolytic pathway itself in ATM activity absence which may be due to an impairment in the functional link between glycolysis and mitochondrial metabolism. In a recent published paper 
, Mongiardi et al. demonstrated that ATM defective cells have an impaired mitochondrial activity, a reduced response to hypoxia in terms of HIF-1α stabilization and transcription of Hypoxia-responsive genes, including PGK1 and MIF. Accordingly, we identified these two gene products as down-regulated in L6 cells respect to L6ATM. The proposed explanation relays on a blunted response to hypoxia and intracellular concentration of ROS in response to hypoxia which in turn is due to an impaired sensing of oxygen variation. On the other end, in our study, the observed up-regulation of GLRX1 in ATM deficient cells could possibly be related to an adaptive response to mitigate the challenge of redox unbalance in ATM absence, a continuous stress state leading to genomic instability, accumulation of unrepaired DNA, constant activation of the DNA repair mechanisms and impaired mitochondrial activity. The transcription factor NF- κB, which has a pivotal role in cell survival and proliferation, is subject to regulation by redox changes; this regulation relies in part on the oxidative inactivation by means of S-glutathionylation of the Inhibitory κB kinase (IKK) β-subunit of the IKK signalosome; overexpression of GLRX1 catalyzes deglutathionylation of IKKβ and enhances NF-κB activation 
. This evidence, our observation of GLRX1 up-regulation in ATM absence and the ATM dependent NEMO ubiquitylation and NF-κB activation 
could possibly open a new route to an interesting vision on the linkage between ATM, NF-κB, genotoxic and oxidative stress, and cellular metabolism.
The present study provides initial evidences toward a new scenario of ATM function in cellular homeostasis; we are aware of the necessity to go deep inside this issue to complete the schema of signaling pathways beyond the differences in the metabolism response correlated to the loss of function of ATM. Nevertheless, all the described evidences begin to explain the intricate scenario beyond the A-T syndrome which could be hardly understood as consequence only of the DNA damage response lack of function.