Prediction of Quality-control Degradation Signals in Yeast Proteins.

Effective proteome homeostasis is key to cellular and organismal survival, and cells therefore contain efficient quality control systems to monitor and remove potentially toxic misfolded proteins. Such general protein quality control to a large extent relies on the efficient and robust delivery of misfolded or unfolded proteins to the ubiquitin-proteasome system. This is achieved via recognition of so-called degradation motifs-degrons-that are assumed to become exposed as a result of protein misfolding. Despite their importance, the nature and sequence properties of quality-control degrons remain elusive. Here, we have used data from a yeast-based screen of 23,600 17-residue peptides to build a predictor of quality-control degrons. The resulting model, QCDPred (Quality Control Degron Prediction), achieves good accuracy using only the sequence composition of the peptides as input. Our analysis reveals that strong degrons are enriched in hydrophobic amino acids and depleted in negatively charged amino acids, in line with the expectation that they are buried in natively folded proteins. We applied QCDPred to the yeast proteome, enabling us to analyse more widely the potential effects of degrons. As an example, we show a correlation between cellular abundance and degron potential in disordered regions of proteins. Together with recent results on membrane proteins, our work suggest that the recognition of exposed hydrophobic residues is a key and generic mechanism for proteome homeostasis. QCDPred is freely available as open source code and via a web interface.

S6K1 phosphorylates Cdk1 and MSH6 to regulate DNA repair.

The mTORC1 substrate, S6 Kinase 1 (S6K1), is involved in the regulation of cell growth, ribosome biogenesis, glucose homeostasis, and adipogenesis. Accumulating evidence has suggested a role for mTORC1 signaling in the DNA damage response. This is mostly based on the findings that mTORC1 inhibitors sensitized cells to DNA damage. However, a direct role of the mTORC1-S6K1 signaling pathway in DNA repair and the mechanism by which this signaling pathway regulates DNA repair is unknown. In this study, we discovered a novel role for S6K1 in regulating DNA repair through the coordinated regulation of the cell cycle, homologous recombination (HR) DNA repair (HRR) and mismatch DNA repair (MMR) mechanisms. Here, we show that S6K1 orchestrates DNA repair by phosphorylation of Cdk1 at serine 39, causing G2/M cell cycle arrest enabling homologous recombination and by phosphorylation of MSH6 at serine 309, enhancing MMR. Moreover, breast cancer cells harboring RPS6KB1 gene amplification show increased resistance to several DNA damaging agents and S6K1 expression is associated with poor survival of breast cancer patients treated with chemotherapy. Our findings reveal an unexpected function of S6K1 in the DNA repair pathway, serving as a tumorigenic barrier by safeguarding genomic stability.

Damage to the DNA in our cells can cause harmful changes that, if unchecked, can lead to the development of cancer. To help prevent this, cellular mechanisms are in place to repair defects in the DNA. A particular process, known as the mTORC1-S6K1 pathway is suspected to be important for repair because when this pathway is blocked, cells become more sensitive to DNA damage. It is still unknown how the various proteins involved in the mTORC1-S6K1 pathway contribute to repairing DNA. One of these proteins, S6K1, is an enzyme involved in coordinating cell growth and survival. The tumor cells in some forms of breast cancer produce more of this protein than normal, suggesting that S6K1 benefits these cells' survival. However, it is unclear exactly how the enzyme does this. Amar-Schwartz, Ben-Hur, Jbara et al. studied the role of S6K1 using genetically manipulated mouse cells and human cancer cells. These experiments showed that the protein interacts with two other proteins involved in DNA repair and activates them, regulating two different repair mechanisms and protecting cells against damage. These results might explain why some breast cancer tumors are resistant to radiotherapy and chemotherapy treatments, which aim to kill tumor cells by damaging their DNA. If this is the case, these findings could help clinicians choose more effective treatment options for people with cancers that produce additional S6K1. In the future, drugs that block the activity of the enzyme could make cancer cells more susceptible to chemotherapy.

Trans-binding of UFM1 to UBA5 stimulates UBA5 homodimerization and ATP binding.

All ubiquitin-like proteins (UBLs) undergo an activation process before their conjugation to target proteins. Although the steps required for the activation of UBLs are conserved and common to all UBLs, we have previously shown that the activation of the UBL, ubiquitin fold modifier 1 (UFM1) by the E1, Ufm1 modifier-activating enzyme 5 (UBA5) is executed in a trans-binding mechanism, not observed in any other E1. In this study, we explored the necessity of that mechanism for UFM1 activation and found that it is needed not only for UFM1 binding to UBA5 but also for stabilizing the UBA5 homodimer. Although UBA5 functions as a dimer, in solution it behaves as a weak dimer. Dimerization of UBA5 is required for ATP binding; therefore, stabilization of the dimer by UFM1 enhances ATP binding. Our results make a connection between the binding of UFM1 to UBA5 and the latter's affinity to ATP, so we propose a novel mechanism for the regulation of ATP's binding to E1.-Mashahreh, B., Hassouna, F., Soudah, N., Cohen-Kfir, E., Strulovich, R., Haitin, Y., Wiener, R. Trans-binding of UFM1 to UBA5 stimulates UBA5 homodimerization and ATP binding.

Novel insights into the interaction of UBA5 with UFM1 via a UFM1-interacting sequence.

The modification of proteins by ubiquitin-fold modifier 1 (UFM1) is implicated in many human diseases. Prior to conjugation, UFM1 undergoes activation by its cognate activating enzyme, UBA5. UBA5 is a non-canonical E1 activating enzyme that possesses an adenylation domain but lacks a distinct cysteine domain. Binding of UBA5 to UFM1 is mediated via an amino acid sequence, known as the UFM1-interacting sequence (UIS), located outside the adenylation domain that is required for UFM1 activation. However, the precise boundaries of the UIS are yet not clear and are still under debate. Here we revisit the interaction of UFM1 with UBA5 by determining the crystal structure of UFM1 fused to 13 amino acids of human UBA5. Using binding and activity assays, we found that His 336 of UBA5, previously not reported to be part of the UIS, occupies a negatively charged pocket on UFM1's surface. This His is involved in UFM1 binding and if mutated perturbs activation of UFM1. Surprisingly, we also found that the interaction between two UFM1 molecules mimics how the UIS binds UFM1. Specifically, UFM1 His 70 resembles UBA5 His336 and enters a negatively charged pocked on the other UFM1 molecule. Our results refine our understanding of UFM1-UBA5 binding.