Biology subjects

Lemak, A.

Publications and source records attributed to Lemak, A..

4 recordsLinked to original sources

bioRxiv2021

The PNUTS-PAD domain recruits MYC to the PNUTS:PP1 phosphatase complex via the oncogenic MYC-MB0 region

Despite MYC dysregulation in most human cancers, strategies to target this potent oncogenic driver remains an urgent unmet need. Recent evidence shows the PP1 phosphatase and its regulatory subunit PNUTS control MYC phosphorylation and stability, however the molecular basis remains unclear. Here we demonstrate that MYC interacts directly with PNUTS through the MYC homology Box 0 (MB0), a highly conserved region recently shown to be important for MYC oncogenic activity. MB0 interacts with PNUTS residues 1-148, a functional unit here termed, PNUTS amino-terminal domain (PAD). Using NMR spectroscopy we determined the solution structure of PAD, and characterised its interaction with MYC. Point mutations of residues at the MYC-PNUTS interface significantly weaken their interaction both in vitro and in vivo. These data demonstrate the MB0 binding pocket of the PAD represents an attractive site for pharmacological disruption of the MYC-PNUTS interaction. In BriefSolving the structure of MYC-PNUTS direct interaction reveals how the intrinsically disordered MYC-Box0 (MB0) region anchors into a binding pocket in the N-terminal PAD domain of PNUTS. These data provide insight into the molecular mechanism of how the PNUTS:PP1 phosphatase complex regulates MYC phosphorylation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/470928v1_ufig1.gif" ALT="Figure 1"> View larger version (86K): org.highwire.dtl.DTLVardef@179b89corg.highwire.dtl.DTLVardef@47338eorg.highwire.dtl.DTLVardef@89b6f4org.highwire.dtl.DTLVardef@14a80bd_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIA region critical for MYC oncogenesis, MYC-Box0 (MB0), directly interacts with PNUTS C_LIO_LIPNUTS amino-terminal domain (PAD) is a structural domain that interacts with MYC MB0 C_LIO_LIMutation of single residues at the interaction interface disrupts MYC-PNUTS binding in cells C_LIO_LIMYC-PNUTS binding releases MYC intramolecular interactions to enable PP1substrate access C_LI

cancer biology↗

bioRxiv2021

HAP40 orchestrates huntingtin structure for differential interaction with polyglutamine expanded exon 1

Huntingtons disease results from expansion of a glutamine-coding CAG tract in the huntingtin (HTT) gene, producing an aberrantly functioning form of HTT. Both wildtype and disease-state HTT form a hetero-dimer with HAP40 of unknown functional relevance. We demonstrate in vivo that HTT and HAP40 cellular abundance are coupled. Integrating data from a 2.6 [A] cryo-electron microscopy structure, cross-linking mass spectrometry, small-angle X-ray scattering, and modeling, we provide a near-atomic-level view of HTT, its molecular interaction surfaces and compacted domain architecture, orchestrated by HAP40. Native mass-spectrometry reveals a remarkably stable hetero-dimer, potentially explaining the cellular inter-dependence of HTT and HAP40. The polyglutamine tract containing N-terminal exon 1 region of HTT is dynamic, but shows greater conformational variety in the mutant than wildtype exon 1. By providing novel insight into the structural consequences of HTT polyglutamine expansion, our data provide a foundation for future functional and drug discovery studies targeting Huntingtons disease.

biochemistry↗

bioRxiv2018

Design and characterisation of mutant and wild-type huntingtin proteins produced from a toolkit of scalable eukaryotic expression systems

The gene mutated in Huntingtons disease (HD) patients encodes the 348 kDa huntingtin (HTT) protein. The pathogenic HD CAG-expansion mutation causes a polyglutamine (polyQ) tract at the N-terminus of the HTT protein to expand above a critical threshold of ~35 glutamine residues. The effect of HD mutations on HTT is not well understood, in part due to difficulties in carrying out biochemical, biophysical and structural studies of this large protein. To facilitate such studies, we have generated expression constructs for the scalable production of HTT in multiple eukaryotic expression systems. Our set of HTT expression clones comprises both N and C-terminally FLAG-tagged HTT constructs with polyQ lengths representative of the general population, HD patients, juvenile HD patients as well as the more extreme polyQ expansions used in some HD tissue and animal models. These reagents yield milligram quantities of pure recombinant HTT protein, including many of the previously mapped posttranslational modifications. We have characterised both apo and HTT-HAP40 complex samples produced using this HD resource, demonstrating that this toolkit can be used to generate physiologically meaningful complexes of HTT. We demonstrate how these resources can produce sufficient material for protein-intensive experiments such as small angle X-ray scattering (SAXS), providing biochemical insight into HTT protein structure. The work outlined in this manuscript and the tools generated, lay a foundation for further biochemical and structural work on the HTT protein and its functional interactions with other biomolecules.

biochemistry↗

bioRxiv2018

MLL1 minimal catalytic complex is a dynamic conformational ensemble susceptible to pharmacological allosteric disruption

Histone H3K4 methylation is an epigenetic mark associated with actively transcribed genes. This modification is catalyzed by the mixed lineage leukaemia (MLL) family of histone methyltransferases including MLL1, MLL2, MLL3, MLL4, SET1A and SET1B. Catalytic activity of MLL proteins is dependent on interactions with additional conserved proteins but the structural basis for subunit assembly and the mechanism of regulation is not well understood. We used a hybrid methods approach to study the assembly and biochemical function of the minimally active MLL1 complex (MLL1, WDR5 and RbBP5). A combination of small angle X-ray scattering (SAXS), cross-linking mass spectrometry (XL-MS), NMR spectroscopy, and computational modeling were used to generate a dynamic ensemble model in which subunits are assembled via multiple weak interaction sites. We identified a new interaction site between the MLL1 SET domain and the WD40 repeat domain of RbBP5, and demonstrate the susceptibility of the catalytic function of the complex to disruption of individual interaction sites.

biophysics↗