Yusuke Sato

The AAA-ATPase p97 and its primary adaptor UFD1–NPL4 (UN) unfold ubiquitinated substrates for their proteasomal degradation. Although cryo-EM has shown that substrate processing begins with unfolding of the initiator ubiquitin and its capture by NPL4, the specific contribution of UFD1 to K48-linked ubiquitin chain engagement has not been fully defined. Here we report a 1.31 Å crystal structure of the human UFD1-UT3 bound to a K48-linked di-ubiquitin mimic, revealing that UFD1-UT3 engages the unfolded initiator ubiquitin via its Nc subdomain and the proximal ubiquitin via its Nn subdomain. AlphaFold3-guided analyses further demonstrate that accessory adaptors FAF1, FAF2, and UBXD7 enhance p97–UN activity through divergent strategies. FAF1 and FAF2 scaffold UFD1-UT3 near the NPL4-tower, whereas UBXD7 stabilizes transient interactions between UFD1 and the ubiquitin chain. Our results provide a comprehensive molecular model for the initiation of p97-mediated substrate unfolding.

Monobodies, engineered protein scaffolds derived from the fibronectin type III domain, are powerful alternatives to conventional antibodies. While the native scaffold is robust, engineering the variable loops can often compromise solubility and promote aggregation. Here, we report the crystallization and structure determination at 2.57 Å resolution of a monobody (Mb-P′) engineered to bind the synthetic small molecule HPPU [1-(4-hydroxyphenyl)-3-phenylurea] with nanomolar affinity. Although Mb-P′ exhibited severe polydispersity and heterogeneous oligomerization in solution, N-terminal fusion with maltose-binding protein (MBP) using an optimized linker successfully yielded monodisperse species and diffraction-quality crystals. The crystal structure exhibited pseudo- D ₃ symmetry in the asymmetric unit, in which the MBP moiety interacts with and partially covers the F and G β-strands of the monobody. This steric masking suggests that MBP acts as a solubility enhancer by shielding the aggregation-prone surface patches generated by loop engineering. Our results demonstrate that this fusion strategy effectively enables structural studies of aggregation-prone proteins obtained from engineered scaffolds.

Ubiquitination is a central post-translational modification that regulates processes like protein degradation and signalling. This versatility is derived from ubiquitin (Ub) chains, which are formed via eight distinct linkages. Analysing these complex functions requires the use of Ub chains with precisely defined linkage types and lengths. Although chemical synthesis allows for the preparation of these Ub chains, enzymatic reconstitution offers a more broadly accessible strategy. This review focuses on recent enzymatic advances for the in vitro synthesis of high-purity Ub chains, employing E1-E2-E3 cascades, engineered enzymes and linkage-specific deubiquitinases. Key discussion points include methods to control chain length, prevent unwanted cyclization and generate complex mixed or branched topologies. Furthermore, methods for site-specific substrate ubiquitination are summarized. These robust enzymatic systems are indispensable tools, enabling the reconstruction of complex Ub modifications in vitro and advancing structural and biochemical studies of the Ub code.

Supplementary Table 1. Clinical characteristics of patients with MET rearrangement-positive tumors in the DFCI/GRCC clinicopathologic cohort. Supplementary Table 2. Clinical characteristics of patients with MET fusion-positive tumors in the SPARTA-II clinical trial, cohort D. Supplementary Table 3. Treatment history of patients with MET fusion-positive solid tumors in the SPARTA-II clinical trial, cohort D. Supplementary Table 4. Summary of treatment emergent adverse events (TEAEs) in the SPARTAII clinical trial, cohort D. Supplementary Table 5. Details of treatment emergent adverse events (TEAEs), ordered by decreasing frequency for all grades. Supplementary Table 6. Details of treatment-related treatment emergent adverse events (TEAEs), ordered by decreasing frequency for all grades.

Supplementary Figure 1. Identification of MET rearrangement cases in the DFCI and GRCC cohorts. Supplementary Figure 2. Genomic features of MET rearrangements. Supplementary Figure 3. Oncoprint of likely oncogenic MET rearrangements and of unknown significance with select co-occurring genomic alterations that are generally recognized as common standalone mitogenic drivers. Supplementary Figure 4. Treatment and response to vebreltinib as per independent review committee (IRC) according to RECIST 1.1. Supplementary Figure 5. Clinical presentation of salivary gland tumor with ETV6::MET and in vitro modelling. Supplementary Figure 6. In vitro data of HLA-DRB1::MET Ba/F3 cells. Supplementary Figure 7. Sensitivity pattern of HLA-DRB1::MET WT and D1228V to MET TKIs. Supplementary Figure 8. Molecular dynamics simulation graphs. Supplementary Figure 9. Co-immunoprecipitation assay by HA.

Oncogenic translocations involving the MET gene have been reported in several cancer types, but detailed clinicogenomic characterization of these cancers is not well defined. In addition, prospective clinical trials evaluating the antitumor activity of MET inhibitors in MET rearrangement–positive cancers are limited. In this study, in a pan-cancer analysis of >46,000 solid tumors with comprehensive genomic profiling, we identified oncogenic MET rearrangements in ∼0.04% of cancers. Preliminary analysis from a phase II clinical trial of the type I MET tyrosine kinase inhibitor (TKI) vebreltinib in MET fusion–positive solid tumors demonstrated an objective response rate of 50% and disease control rate of 79%, with antitumor activity seen in diverse cancer types, including lung adenocarcinoma and intrahepatic cholangiocarcinoma, among others. Similar to MET exon 14–altered lung cancer, secondary mutations in the kinase domain can confer resistance to MET TKIs in MET fusion–positive cancers. Overall, these data categorize MET rearrangements as actionable targets in solid tumors. Significance: MET rearrangement–positive cancers are not well-characterized, and optimal treatment strategies are yet to be defined. Through comprehensive genomic analysis, preclinical modeling, and preliminary results of a phase II clinical trial, we demonstrate that MET fusions are a unique molecular subtype of cancers targetable with vebreltinib, a TKI in development.

Protein aggregates are degraded by both the autophagy–lysosomal and the ubiquitin–proteasome pathways. Macroautophagy and microautophagy, two forms of the autophagy–lysosomal pathway, are widely conserved across eukaryotes. While macroautophagy has been extensively studied in the context of degradation of protein aggregates, microautophagy remains less explored. Here, we identify the UBAP1-containing ESCRT-I complex and PTPN23 as new regulators for degradation of aggregated proteins through an unbiased genome-wide CRISPR knockout screen, using a cell line expressing tau repeat domain (tauRD) aggregates. ESCRT-I recognizes ubiquitylated tauRD via the UEV domain of TSG101. The accessory protein PTPN23, instead of ESCRT-II, bridges ESCRT-I and ESCRT-III to complete the endosomal microautophagy of ubiquitylated tauRD aggregates. Our results uncover the molecular mechanism underlying the degradation of tau aggregates by endosomal microautophagy.

The human enzyme p97 regulates various cellular pathways by unfolding hundreds of protein substrates in an ATP-dependent manner, making it an essential component of protein homeostasis and an impactful pharmacological target. The hexameric complex undergoes substantial conformational changes throughout its catalytic cycle. Here we elucidate the molecular motions that occur at the active site in the temporal window immediately before and after ATP hydrolysis by merging cryo-EM, NMR spectroscopy and molecular dynamics simulations. p97 populates a metastable reaction intermediate, the ADP·P_i state, which is poised between hydrolysis and product release. Detailed snapshots reveal that the active site is finely tuned to trap and eventually discharge the cleaved phosphate. Signalling pathways originating at the active site coordinate the action of the hexamer subunits and couple hydrolysis with allosteric conformational changes. Our multidisciplinary approach enables a glimpse into the sophisticated spatial and temporal orchestration of ATP handling by a prototype AAA+ protein.