Patterns of resistance to first-line osimertinib are not well-established and have primarily been evaluated using plasma assays which cannot detect histologic transformation and have differential sensitivity for copy number changes and chromosomal rearrangements. Experimental Design:To characterize mechanisms of resistance to osimertinib, patients with metastatic EGFR-mutant lung cancers who received osimertinib at Memorial Sloan Kettering and had next-generation sequencing performed on tumor tissue before osimertinib initiation and after progression were identified.Results: Among 62 patients who met eligibility critieria, histologic transformation, primarily squamous transformation, was identified in 15% of first-line osimertinib cases and 14% of laterline cases. Nineteen percent (5/27) of patients treated with first-line osimertinib had off-target genetic resistance (2 MET amplification, 1 KRAS mutation, 1 RET fusion, and 1 BRAF fusion) whereas 4% (1/27) had an acquired EGFR mutation (EGFR G724S). Patients with squamous transformation exhibited considerable genomic complexity; acquired PIK3CA mutation, chromosome 3q amplification and FGF amplification were all seen. Patients with transformation had shorter time on osimertinib and shorter survival compared to patients with on-target resistance. Initial EGFR sensitizing mutation, time on osimertinib treatment and line of therapy also influenced resistance mechanism that emerged. The compound mutation EGFR S768 + V769L and the mutation MET H1094Y were identified and validated as resistance mechanisms with potential treatment options. Conclusion:Histologic transformation and other off-target molecular alterations are frequent early emerging resistance mechanisms to osimertinib and are associated with poor clinical outcomes.Research.

Rad23 is a DNA repair protein that promotes the assembly of the nucleotide excision repair complex. Rad23 can interact with the 26S proteasome through an N-terminal ubiquitin-like domain, and inhibits the assembly of substrate-linked multiubiquitin (multi-Ub) chains in vitro and in vivo. Significantly, Rad23 can bind a proteolytic substrate that is conjugated to a few ubiquitin (Ub) moieties. We report here that two ubiquitin-associated (UBA) domains in Rad23 form non-covalent interactions with Ub. A mutant that lacked either UBA sequence was capable of blocking the assembly of substratelinked multi-Ub chains, although a mutant that lacked both UBA domains was significantly impaired. These studies suggest that the interaction with Ub is required for Rad23 activity, and that other UBA-containing proteins may have a similar function.

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SummaryCopper is essential for cell metabolism as a cofactor of key metabolic enzymes. The biosynthetic incorporation of copper into secreted and plasma membrane-bound proteins requires activity of the copper-transporting ATPases (Cu-ATPases) ATP7A and ATP7B. The Cu-ATPases also export excess copper from the cell and thus critically contribute to the homeostatic control of copper. The trafficking of Cu-ATPases from the trans-Golgi network to endocytic vesicles in response to various signals allows for the balance between the biosynthetic and copper exporting functions of these transporters. Although significant progress has been made towards understanding the biochemical characteristics of human Cu-ATPase, the mechanisms that control their function and intracellular localization remain poorly understood. In this review, we summarize current information on structural features and functional properties of ATP7A and ATP7B. We also describe sequence motifs unique for each Cu-ATPase and speculate about their role in regulating ATP7A and ATP7B activity and trafficking.

Prokaryotic subtilisins and eukaryotic proprotein convertases (PCs) are two homologous protease subfamilies that belong to the larger ubiquitous super-family called subtilases. Members of the subtilase super-family are produced as zymogens wherein their propeptide domains function as dedicated intramolecular chaperones (IMCs) that facilitate correct folding and regulate precise activation of their cognate catalytic domains. The molecular and cellular determinants that modulate IMC-dependent folding and activation of PCs are poorly understood. In this chapter we review what we have learned from the folding and activation of prokaryotic subtilisin, discuss how this has molded our understanding of furin maturation, and foray into the concept of pH sensors, which may represent a paradigm that PCs (and possibly other IMC-dependent eukaryotic proteins) follow for regulating their biological functions using the pH gradient in the secretory pathway.

The folding and activation of furin occur through two pH-and compartment-specific autoproteolytic steps. In the endoplasmic reticulum (ER), profurin folds under the guidance of its prodomain and undergoes an autoproteolytic excision at the consensus furin site Arg-Thr-Lys-Arg 107 2 generating an enzymatically masked furin-propeptide complex competent for transport to late secretory compartments. In the mildly acidic environment of the trans-Golgi network/endosomal system, the bound propeptide is cleaved at the internal site 69 HRGVTKR 75 2, unmasking active furin capable of cleaving substrates in trans. Here, by using cellular, biochemical, and modeling studies, we demonstrate that the conserved His 69 is a pH sensor that regulates the compartment-specific cleavages of the propeptide. In the ER, unprotonated His 69 stabilizes a solvent-accessible hydrophobic pocket necessary for autoproteolytic excision at Arg 107 . Profurin molecules unable to form the hydrophobic pocket, and hence, the furin-propeptide complex, are restricted to the ER by a PACS-2-and COPI-dependent mechanism. Once exposed to the acidic pH of the late secretory pathway, protonated His 69 disrupts the hydrophobic pocket, resulting in exposure and cleavage of the internal cleavage site at Arg 75 to unmask the enzyme. Together, our data explain the pH-regulated activation of furin and how this His-dependent regulatory mechanism is a model for other proteins.The pH gradient formed by the various membranous compartments comprising the secretory and endocytic pathways has essential and manifold roles ranging from the regulation of protein traffic to the control of protein conformation and enzyme activities, including the processing of prohormones and proproteins by proprotein convertases (PCs). 2 The PCs, a family of calcium-dependent serine endoproteases, cleave proproteins and prohormones at doublets or clusters of basic amino acids thus generating mature bioactive proteins as well as hormonal processing intermediates that require additional post-translational modifications to gain full bioactivity (1, 2). The proteolytic maturation of prohormones by the neuroendocrine-specific PC1/3 and PC2 requires the acidic pH of maturing secretory granules (3-5), whereas the proteolytic activation of proprotein substrates by the ubiquitously expressed PC furin in the trans-Golgi network (TGN)/endosomal system is compartment-and hence pH-specific (2, 6). This compartmentspecific processing by furin is mediated in part by the pH-dependent changes in the conformation of furin substrates as well as the utilization of pH-sensitive furin cleavage sites. Furin efficiently cleaves a number of proprotein substrates at the consensus site -Arg-X-Lys/Arg-Arg-2 (2). Kinetic studies show that the P1 and P4 Arg residues are required for the efficient processing of furin substrates, whereas the P2 Arg has a modulatory role (7,8). However, at acidic pH the absence of a P2 or P4 Arg can be compensated for by the presence of positively charged residues at P6 or possibly adjacen...