The N-end rule finds a physiological function: The N-end–rule pathway for protein degradation is a canonical degradation pathway discovered in the 1980s. In recent years, studies have focused on finding novel variant pathways of N-end recognition. The “classical” pathway is blocked by N-terminal acetylation of the substrate. However, in yeast, N-terminal acetylation need not block degradation, because a second pathway can act on acetylated N-termini. But is this alternate pathway a major player in the physiology of mammals? Park et al. now confirm the existence of the alternate pathway in mammalian cells. Most notably, patient-derived point mutations thought to confer hypertension in humans affect susceptibility to this pathway for the encoded protein substrate, Rgs2. Science , this issue p. 1249

Another N-end rule to add: Proteins that emerge from a ribosome bear the N-terminal methionine (Met) residue. In bacteria, Met is formylated before translation starts, whereas in eukaryotes, most nascent proteins seemed to start with unmodified Met. Working in yeast, Kim et al. found that the N-terminal formylation of eukaryotic proteins is detectable even under normal conditions and is greatly increased upon specific stresses, which cause some Fmt1 formyltransferase to be retained in the cytoplasm. The retention of this normally mitochondrial protein was found to require the Gcn2 kinase. In addition, the Psh1 ubiquitin ligase was shown to target N-terminally formylated eukaryotic proteins for proteasome-dependent degradation by the so-called fMet/N-end rule pathway. Science , this issue p. eaat0174

Kang-Eun Lee, Ji-Eun Heo, Jeong-Mok Kim, and Cheol-Sang Hwang. Mol. Cells 2016;39:169-78. https://doi.org/10.14348/molcells.2016.2329

All organisms begin protein synthesis with methionine (Met). The resulting initiator Met of nascent proteins is irreversibly processed by Met aminopeptidases (MetAPs). N-terminal (Nt) Met excision (NME) is an evolutionarily conserved and essential process operating on up to two-thirds of proteins. However, the universal function of NME remains largely unknown. MetAPs have a well-known processing preference for Nt-Met with Ala, Ser, Gly, Thr, Cys, Pro, or Val at position 2, but using CHX-chase assays to assess protein degradation in yeast cells, as well as protein-binding and RT-qPCR assays, we demonstrate here that NME also occurs on nascent proteins bearing Met–Asn or Met–Gln at their N termini. We found that the NME at these termini exposes the tertiary destabilizing Nt residues (Asn or Gln) of the Arg/N-end rule pathway, which degrades proteins according to the composition of their Nt residues. We also identified a yeast DNA repair protein, MQ-Rad16, bearing a Met–Gln N terminus, as well as a human tropomyosin–receptor kinase–fused gene (TFG) protein, MN-TFG, bearing a Met–Asn N terminus as physiological, MetAP-processed Arg/N-end rule substrates. Furthermore, we show that the loss of the components of the Arg/N-end rule pathway substantially suppresses the growth defects of naa20Δ yeast cells lacking the catalytic subunit of NatB Nt acetylase at 37 °C. Collectively, the results of our study reveal that NME is a key upstream step for the creation of the Arg/N-end rule substrates bearing tertiary destabilizing residues in vivo.

Extensive progress in understanding the molecular mechanisms of cancer growth and proliferation has led to the remarkable development of drugs that target cancer-driving molecules. Most target molecules are proteins such as kinases and kinase-associated receptors, which have enzymatic activities needed for the signaling cascades of cells. The small molecule inhibitors for these target molecules greatly improved therapeutic efficacy and lowered the systemic toxicity in cancer therapies. However, long-term and high-dosage treatment of small inhibitors for cancer has produced other obstacles, such as resistance to inhibitors. Among recent approaches to overcoming drug resistance to cancers, targeted protein degradation (TPD) such as proteolysis-targeting chimera (PROTAC) technology adopts a distinct mechanism of action by which a target protein is destroyed through the cellular proteolytic system, such as the ubiquitin–proteasome system or autophagy. Here, we review the currently developed PROTACs as the representative TPD molecules for cancer therapy and the N-degrons of the N-degron pathways as the potential TPD ligands.

BldD is a central regulator of the developmental process in Streptomyces coelicolor. The 1.8 Å resolution structure of the DNA‐binding domain of BldD (BldDN) reveals that BldDN forms a compact globular domain composed of four helices (α1–α4) containing a helix‐turn‐helix motif (α2–α3) resembling that of the DNA‐binding domain of lambda repressor. The BldDN/DNA complex model led us to design a series of mutants, which revealed the important role of α3 and the ‘turn’ region between α2 and α3 for DNA recognition. Based on the fact that BldD occupies two operator sites of bldN and whiG and shows significant disparity in the affinity toward the two operator sites when they are disconnected, we propose a model of cooperative binding, which means that the binding of one BldD dimer to the high affinity site facilitates that of the second BldD dimer to the low affinity site. In addition, structural and mutational investigation reveals that the Tyr62Cys mutation, found in the first‐identified bldD mutant, can destabilize BldD structure by disrupting the hydrophobic core.