Biology subjects

Iso, K.

Publications and source records attributed to Iso, K..

4 recordsLinked to original sources

bioRxiv2025

Structural insights into YheS-mediated release of SecM-arrested ribosome.

ATPase-binding cassette subfamily F (ABCF) proteins interact with the ribosome to resolve translation defects near the peptidyl-transferase center, induced by ribosome-targeting antibiotics or "hard-to-translate" nascent peptides. In Escherichia coli, four ABCF proteins resolve the translation of distinct problematic sequences, but their molecular mechanisms remain unclear. Here, we present a 2.8-[A] cryo-EM structure of the SecM-stalled ribosome in complex with an ATPase-deficient mutant of YheS, an ABCF protein that releases ribosomes stalled during the translation of SecM, a representative ribosome-arresting peptide. YheS is bound to the E-site and relocates the P-site tRNA (P-tRNA) by displacing its CCA-end. Notably, the density of the SecM nascent chain mostly disappeared upon YheS binding. Molecular dynamics simulations support that the P-tRNA relocation generates a pulling force that disrupts key intratunnel interactions essential for arrest. This mechanism is functionally analogous to, yet mechanistically distinct from, the translocon-mediated external pulling force for SecM release. TeaserThe release mechanism of the SecM-arrested ribosome by the ABCF protein YheS was elucidated by structure-based analyses.

biochemistry↗

bioRxiv2025

Dual E3 ligase recruitment by monovalent degraders enables redundant and tuneable degradation of SMARCA2/4

Proteolysis-Targeting Chimeras (PROTACs) and Molecular Glue Degraders (MGDs) canonically target proteins for degradation by recruiting them to a single E3 ligase complex. While heterotrivalent PROTACs that can co-opt multiple E3 ligase complexes have been described, to our knowledge all MGDs reported to date are dependent on a single E3. Here, using orthogonal genetic screening, biophysical and structural analyses, we show that a monovalent MGD can covalently recruit CUL4DCAF16 and CRL1FBXO22 in a parallel and redundant manner to degrade SMARCA2/4. Deep mutational scanning identifies a single cysteine (Cys173) in DCAF16 essential for degrader activity, and intact protein MS confirms covalent adduct at this site. The cryo-EM structure of the DCAF16:SMARCA2:degrader ternary complex reveals a unique binding mode and a distinct interface of neo-interactions, providing insights into degrader specificity. We demonstrate that E3 ligase dependency can be tuned both chemically and genetically. Minimal alterations to the compounds "degradation tail" switches ligase preference from DCAF16 to FBXO22, while a single L59W mutation on DCAF16 is sufficient to drive DCAF16 engagement for otherwise FBXO22-dependent compounds. These results establish a molecular and structural framework for the design of tuneable dual glue degraders that could mitigate challenges from resistance mechanisms in degrader therapies.

biochemistry↗

bioRxiv2023

An intramolecular bivalent degrader glues an intrinsic BRD4-DCAF16 interaction

Targeted protein degradation is a pharmacological modality based on the induced proximity of an E3 ubiquitin ligase and a target protein to promote target ubiquitination and proteasomal degradation. This has been achieved either via bifunctional compounds (PROTACs) composed of two separate warheads that individually bind the target and E3 ligase, or via molecular glues that monovalently bind either the ligase or the target1-4. Using orthogonal genetic screening, biophysical characterization, and structural reconstitution, we investigate the mode of action of bifunctional BRD2/4 degraders (IBG1-4) and find that - instead of connecting target and ligase in trans as PROTACs do - they simultaneously engage two adjacent domains of the target protein in cis. This conformational change glues BRD4 to the E3 ligases DCAF11 or DCAF16, leveraging intrinsic target-ligase affinities which, albeit pre-existing, do not translate to BRD4 degradation in absence of compound. Structural insights into the ternary BRD4:IBG1:DCAF16 complex guided the rational design of improved degraders of low picomolar potency. We thus introduce a new modality in targeted protein degradation, termed intramolecular bivalent glues (IBGs), which work by bridging protein domains to enhance surface complementarity with E3 ligases for productive ubiquitination and degradation.

biochemistry↗

bioRxiv2022

Paradoxical tumor suppressive role of the musculoaponeurotic fibrosarcoma gene in colorectal cancer

Somatic cell reprogramming using the microRNAs miR200c, miR-302s, and miR-369s leads to increased expression of cyclin-dependent kinase inhibitors in human colorectal cancer (CRC) cells and suppressed tumor growth. Here, we investigated whether these microRNAs inhibit colorectal tumorigenesis in CPC;Apc mice, which are prone to colon and rectal polyps. Repeated administration of microRNAs inhibited polyp formation. Microarray analysis indicated that c-MAF, which reportedly shows oncogene-like behavior in multiple myeloma and T-cell lymphoma, decreased in tumor samples but increased in microRNA-treated normal mucosa. Immunohistochemistry identified downregulation of c-MAF as an early tumorigenesis event in CRC, with low c-MAF expression associated with poor prognosis. Of note, c-MAF expression and p53 protein levels were inversely correlated in CRC samples. c-MAF knockout led to enhanced tumor formation in azoxymethane/dextran sodium sulfate-treated mice, with activation of cancer-promoting genes. c-MAF may play a tumor-suppressive role in CRC development.

molecular biology↗