Shady Saad

All Publications

• Reversible amyloids of pyruvate kinase couple cell metabolism and stress granule disassembly. Nature cell biology Cereghetti, G., Wilson-Zbinden, C., Kissling, V. M., Diether, M., Arm, A., Yoo, H., Piazza, I., Saad, S., Picotti, P., Drummond, D. A., Sauer, U., Dechant, R., Peter, M. 2021

  Abstract

  Cells respond to stress by blocking translation, rewiring metabolism and forming transient messenger ribonucleoprotein assemblies called stress granules (SGs). After stress release, re-establishing homeostasis and disassembling SGs requires ATP-consuming processes. However, the molecular mechanisms whereby cells restore ATP production and disassemble SGs after stress remain poorly understood. Here we show that upon stress, the ATP-producing enzyme Cdc19 forms inactive amyloids, and that their rapid re-solubilization is essential to restore ATP production and disassemble SGs in glucose-containing media. Cdc19 re-solubilization is initiated by the glycolytic metabolite fructose-1,6-bisphosphate, which directly binds Cdc19 amyloids, allowing Hsp104 and Ssa2 chaperone recruitment and aggregate re-solubilization. Fructose-1,6-bisphosphate then promotes Cdc19 tetramerization, which boosts its activity to further enhance ATP production and SG disassembly. Together, these results describe a molecular mechanism that is critical for stress recovery and directly couples cellular metabolism with SG dynamics via the regulation of reversible Cdc19 amyloids.

  View details for DOI 10.1038/s41556-021-00760-4

  View details for PubMedID 34616026

• Protein self-assembly: A new frontier in cell signaling. Current opinion in cell biology Saad, S., Jarosz, D. F. 2021; 69: 62–69

  Abstract

  Long viewed as paradigm-shifting, but rare, prions have recently been discovered in all domains of life. Protein sequences that can drive this form of self-assembly are strikingly common in eukaryotic proteomes, where they are enriched in proteins involved in information flow and signal transduction. Although prions were thought to be a consequence of random errors in protein folding, recent studies suggest that prion formation can be a controlled process initiated by defined cellular signals. Many are present in normal biological contexts, yet are invisible to most technologies used to interrogate the proteome. Here, we review mechanisms by which protein self-assembly can create a stable record of past stimuli, altering adaptive responses, and how prion behavior is controlled by signaling processes. We touch on the diverse implications that this has for normal biological function and regulation, ranging from drug resistance in fungi to the innate immune response in humans. Finally, we discuss the potential for prion domains in transcription factors and RNA-binding proteins to orchestrate heritable gene expression changes in response to transient signals, such as during development.

  View details for DOI 10.1016/j.ceb.2020.12.013

  View details for PubMedID 33493989

• Author Correction: Reversible amyloids of pyruvate kinase couple cell metabolism and stress granule disassembly. Nature cell biology Cereghetti, G., Wilson-Zbinden, C., Kissling, V. M., Diether, M., Arm, A., Yoo, H., Piazza, I., Saad, S., Picotti, P., Drummond, D. A., Sauer, U., Dechant, R., Peter, M. 2021

  View details for DOI 10.1038/s41556-021-00799-3

  View details for PubMedID 34702981

• A hydrophobic low-complexity region regulates aggregation of the yeast pyruvate kinase Cdc19 into amyloid-like aggregates in vitro JOURNAL OF BIOLOGICAL CHEMISTRY Grignaschi, E., Cereghetti, G., Grigolato, F., Kopp, M. G., Caimi, S., Faltova, L., Saad, S., Peter, M., Arosio, P. 2018; 293 (29): 11424–32

  Abstract

  Cells form stress granules (SGs) upon stress stimuli to protect sensitive proteins and RNA from degradation. In the yeast Saccharomyces cerevisiae, specific stresses such as nutrient starvation and heat-shock trigger recruitment of the yeast pyruvate kinase Cdc19 into SGs. This RNA-binding protein was shown to form amyloid-like aggregates that are physiologically reversible and essential for cell cycle restart after stress. Cellular Cdc19 exists in an equilibrium between a homotetramer and monomer state. Here, we show that Cdc19 aggregation in vitro is governed by protein quaternary structure, and we investigate the physical-chemical basis of Cdc19's assembly properties. Equilibrium shift toward the monomer state exposes a hydrophobic low-complexity region (LCR), which is prone to induce intermolecular interactions with surrounding proteins. We further demonstrate that hydrophobic/hydrophilic interfaces can trigger Cdc19 aggregation in vitro Moreover, we performed in vitro biophysical analyses to compare Cdc19 aggregates with fibrils produced by two known dysfunctional amyloidogenic peptides. We show that the Cdc19 aggregates share several structural features with pathological amyloids formed by human insulin and the Alzheimer's disease-associated Aβ42 peptide, particularly secondary β-sheet structure, thermodynamic stability, and staining by the thioflavin T dye. However, Cdc19 aggregates could not seed aggregation. These results indicate that Cdc19 adopts an amyloid-like structure in vitro that is regulated by the exposure of a hydrophobic LCR in its monomeric form. Together, our results highlight striking structural similarities between functional and dysfunctional amyloids and reveal the crucial role of hydrophobic/hydrophilic interfaces in regulating Cdc19 aggregation.

  View details for DOI 10.1074/jbc.RA117.001628

  View details for Web of Science ID 000439449700013

  View details for PubMedID 29853641

  View details for PubMedCentralID PMC6065187

• Reversible, functional amyloids: towards an understanding of their regulation in yeast and humans. Cell cycle (Georgetown, Tex.) Cereghetti, G. n., Saad, S. n., Dechant, R. n., Peter, M. n. 2018: 1–14

  Abstract

  Protein aggregates, and in particular amyloids, are generally considered to be inherently irreversible aberrant clumps, and are often associated with pathologies, such as Alzheimer's disease, Parkinson's disease, or systemic amyloidosis. However, recent evidence demonstrates that some aggregates are not only fully reversible, but also perform essential physiological functions. Despite these new findings, very little is known about how these functional protein aggregates are regulated in a physiological context. Here, we take the yeast pyruvate kinase Cdc19 as an example of a protein forming functional, reversible, solid, amyloid-like aggregates in response to stress conditions. Cdc19 aggregation is regulated via an aggregation-prone low complexity region (LCR). In favorable growth conditions, this LCR is prevented from aggregating by phosphorylation or oligomerization, while upon glucose starvation it becomes exposed and allows aggregation. We suggest that LCR phosphorylation, oligomerization or partner-binding may be general and widespread mechanisms regulating LCR-mediated reversible protein aggregation. Moreover, we show that, as predicted by computational tools, Cdc19 forms amyloid-like aggregates in vitro. Interestingly, we also observe striking similarities between Cdc19 and its mammalian counterpart, PKM2. Indeed, also PKM2 harbors a LCR and contains several peptides with high amyloidogenic propensity, which coincide with known phosphorylation sites. Thus, we speculate that the formation of reversible, amyloid-like aggregates may be a general physiological mechanism for cells to adapt to stress conditions, and that the underlying regulatory mechanisms may be conserved from yeast to humans.

  View details for PubMedID 29963943

• Reversible protein aggregation is a protective mechanism to ensure cell cycle restart after stress NATURE CELL BIOLOGY Saad, S., Cereghetti, G., Feng, Y., Picotti, P., Peter, M., Dechant, R. 2017; 19 (10): 1202-+

  Abstract

  Protein aggregation is mostly viewed as deleterious and irreversible causing several pathologies. However, reversible protein aggregation has recently emerged as a novel concept for cellular regulation. Here, we characterize stress-induced, reversible aggregation of yeast pyruvate kinase, Cdc19. Aggregation of Cdc19 is regulated by oligomerization and binding to allosteric regulators. We identify a region of low compositional complexity (LCR) within Cdc19 as necessary and sufficient for reversible aggregation. During exponential growth, shielding the LCR within tetrameric Cdc19 or phosphorylation of the LCR prevents unscheduled aggregation, while its dephosphorylation is necessary for reversible aggregation during stress. Cdc19 aggregation triggers its localization to stress granules and modulates their formation and dissolution. Reversible aggregation protects Cdc19 from stress-induced degradation, thereby allowing cell cycle restart after stress. Several other enzymes necessary for G1 progression also contain LCRs and aggregate reversibly during stress, implying that reversible aggregation represents a conserved mechanism regulating cell growth and survival.

  View details for DOI 10.1038/ncb3600

  View details for Web of Science ID 000412013400013

  View details for PubMedID 28846094

• Therapy-relevant aberrant expression of MRP3 and BCRP mRNA in TCC-/SCC-bladder cancer tissue of untreated patients ONCOLOGY REPORTS Rady, M., Mostageer, M., Rohde, J., Zaghloul, A., Knuechel-Clarke, R., Saad, S., Attia, D., Mahran, L., Spahn-Langguth, H. 2017; 38 (1): 551–60

  Abstract

  Multidrug resistance (MDR) is a critical factor, which results in suboptimal outcomes in cancer chemotherapy. One principal mechanism of MDR is the increased expression of ATP-binding cassette (ABC) transporters. Of these, multidrug resistance-associated protein 3 (MRP3) and breast cancer resistance protein (BCRP) confer MDR when overexpressed in cancer cell lines. We measured the mRNA expression of MRP3 and BCRP in primary untreated bladder cancer specimens using reverse transcription-quantitative PCR (RT-qPCR) in comparison to normal bladder tissue. The MRP3 and BCRP expression in the two major histotypes of bladder cancer; transitional cell carcinoma (TCC; urothelial type of bladder cancer) and squamous cell carcinoma (SCC; 'Schistosoma-induced' bladder cancer) were compared. Furthermore, the association between MRP3 and BCRP expression and tumor grade and stage were investigated. MRP3 mRNA expression in bladder cancer specimens was increased ~13-fold on average compared to normal bladder tissue (n=36, P<0.0001). BCRP mRNA expression was decreased in bladder cancer specimens to ~1/5 on average, compared to normal bladder tissue (n=38, P<0.0001). TCC showed significantly increased MRP3 mRNA expression compared to SCC of the bladder (P<0.0001). BCRP mRNA expression was similar in TCC and SCC of the bladder (P=0.1072). The increased MRP3 mRNA expression was not related to bladder tumor grade (P=0.3465) but was, however, significantly higher in superficial than in invasive bladder tumors (P=0.0173). The decreased expression of BCRP was not related to bladder tumor grade (P=0.1808) or stage (P=0.8016). The current data show that bladder cancer is associated with perturbed expression of MRP3 and BCRP. Representing drug resistance factors, determining the expression of these transporters in native tumors may be predictive of the outcome of chemotherapy based-treatment of bladder cancer.

  View details for DOI 10.3892/or.2017.5695

  View details for Web of Science ID 000404089500063

  View details for PubMedID 28586062

• Non-targeted metabolomic approach reveals two distinct types of metabolic responses to telomerase dysfunction in S. cerevisiae METABOLOMICS Buettner, F., Jay, K., Wischnewski, H., Stadelmann, T., Saad, S., Jefimovs, K., Mansurova, M., Gerez, J., Azzalin, C. M., Dechant, R., Ibanez, A. J. 2017; 13 (5)

  View details for DOI 10.1007/s11306-017-1195-x

  View details for Web of Science ID 000399681400008

• Cytosolic pH Regulates Cell Growth through Distinct GTPases, Arf1 and Gtr1, to Promote Ras/PKA and TORC1 Activity MOLECULAR CELL Dechant, R., Saad, S., Ibanez, A. J., Peter, M. 2014; 55 (3): 409–21

  Abstract

  Regulation of cell growth by nutrients is governed by highly conserved signaling pathways, yet mechanisms of nutrient sensing are still poorly understood. In yeast, glucose activates both the Ras/PKA pathway and TORC1, which coordinately regulate growth through enhancing translation and ribosome biogenesis and suppressing autophagy. Here, we show that cytosolic pH acts as a cellular signal to activate Ras and TORC1 in response to glucose availability. We demonstrate that cytosolic pH is sensitive to the quality and quantity of the available carbon source (C-source). Interestingly, Ras/PKA and TORC1 are both activated through the vacuolar ATPase (V-ATPase), which was previously identified as a sensor for cytosolic pH in vivo. V-ATPase interacts with two distinct GTPases, Arf1 and Gtr1, which are required for Ras and TORC1 activation, respectively. Together, these data provide a molecular mechanism for how cytosolic pH links C-source availability to the activity of signaling networks promoting cell growth.

  View details for DOI 10.1016/j.molcel.2014.06.002

  View details for Web of Science ID 000340646600008

  View details for PubMedID 25002144

• In Scarcity and Abundance: Metabolic Signals Regulating Cell Growth PHYSIOLOGY Saad, S., Peter, M., Dechant, R. 2013; 28 (5): 298–309

  Abstract

  Although nutrient availability is a major driver of cell growth, and continuous adaptation to nutrient supply is critical for the development and survival of all organisms, the molecular mechanisms of nutrient sensing are only beginning to emerge. Here, we highlight recent advances in the field of nutrient sensing and discuss arising principles governing how metabolism might regulate growth-promoting pathways. In addition, we discuss signaling functions of metabolic enzymes not directly related to their metabolic activity.

  View details for DOI 10.1152/physiol.00005.2013

  View details for Web of Science ID 000324094400006

  View details for PubMedID 23997189

  View details for PubMedCentralID PMC3768095