Biology subjects

Örd, M.

Publications and source records attributed to Örd, M..

7 recordsLinked to original sources

bioRxiv2026

HCF1 orchestrates O-GlcNAcylation and affinity-dependent transcription through extended molecular determinants and register-shifted binding

We recently identified Host Cell Factor 1 (HCF1), a transcriptional co-regulator discovered more than thirty years ago, as a cancer dependency. To further understand its molecular functions and expand its known interactome, here, we screened a proteome-wide library of candidate HCF1 binding peptides and identified previously uncharacterized canonical and non-canonical HCF1-binding partners. Through deep mutational scanning (DMS) screening, we uncovered an extended set of molecular determinants of binding and show how mutations outside its previously established interacting residues impact binding affinity. Next, we uncover non-canonical HCF1 binders with an extended register-shifted two-amino acid sequence between their "anchor" histidine and tyrosine amino-acid residues, which we show critically contributes, in an affinity-dependent manner, to the downstream transcriptional activity of IRF1. Our data also shows that HCF1 promotes O-GlcNAcylation of the majority of its transcriptional binders. Overall, our results significantly expand the number and diversity of HCF1 binders and propose an enhanced mechanistic understanding of how HCF1 orchestrates transcription and O-GlcNAcylation.

molecular biology↗

bioRxiv2026

Uncovering cancer dependencies in peptide-interacting protein pockets

Cancer cells often become dependent on specific molecular functions. As many proteins perform multiple functions mediated by different pockets and interfaces, we hypothesized that we could identify distinct cancer dependencies and therapeutic vulnerabilities by disrupting peptide-binding pockets. To test this hypothesis, we screened a proteome-wide library of 7152 genetically encoded peptides across nine cancer cell lines. We identify common and selective dependencies on peptide-binding pockets and find that gene knockout and peptide-mediated inhibition of pockets often drive divergent phenotypes. For the common-essential gene HCF1, we identify a therapeutic window by using inhibitory peptides with varying affinity. Moreover, peptides targeting TLE1-4 reveal a dependency hidden in genetic screens by homolog redundancy. We also uncover that peptides inhibiting cyclin D drive specific suppression of leukemia cell proliferation and demonstrate that these peptides improve the potency of CDK4/6 inhibitors. Overall, our screening platform facilitates data-driven prioritization of molecular pockets for subsequent therapeutic translation.

cancer biology↗

bioRxiv2024

A quantitative intracellular peptide binding assay reveals recognition determinants and context dependence of short linear motifs

Transient protein-protein interactions play key roles in controlling dynamic cellular responses. Many examples involve globular protein domains that bind to peptide sequences known as Short Linear Motifs (SLiMs), which are enriched in intrinsically disordered regions of proteins. Here we describe a novel functional assay for measuring SLiM binding, called Systematic Intracellular Motif Binding Analysis (SIMBA). In this method, binding of a foreign globular domain to its cognate SLiM peptide allows yeast cells to proliferate by blocking a growth arrest signal. A high-throughput application of the SIMBA method involving competitive growth and deep sequencing provides rapid quantification of the relative binding strength for thousands of SLiM sequence variants, and a comprehensive interrogation of SLiM sequence features that control their recognition and potency. We show that multiple distinct classes of SLiM-binding domains can be analyzed by this method, and that the relative binding strength of peptides in vivo correlates with their biochemical affinities measured in vitro. Deep mutational scanning provides high-resolution definitions of motif recognition determinants and reveals how sequence variations at non-core positions can modulate binding strength. Furthermore, mutational scanning of multiple parent peptides that bind human tankyrase ARC or YAP WW domains identifies distinct binding modes and uncovers context effects in which the preferred residues at one position depend on residues elsewhere. The findings establish SIMBA as a fast and incisive approach for interrogating SLiM recognition via massively parallel quantification of protein-peptide binding strength in vivo.

biochemistry↗

bioRxiv2021

Differentially accessible Cdc4 phospho-degrons regulate Ctf19CCAN kinetochore subunit stability in mitosis

Kinetochores are multi-subunit protein assemblies that link chromosomes to microtubules of the mitotic and meiotic spindle. How effective, yet strictly centromere-dependent kinetochore assembly is coupled to cell cycle progression is incompletely understood. Here, by combining comprehensive phosphorylation analysis of native Ctf19CCAN subunits with biochemical and functional assays in the model system budding yeast, we demonstrate that Cdk1 phosphorylation activates phospho-degrons on the essential subunit Ame1CENP-U which are recognized by the E3 ubiquitin ligase complex SCF-Cdc4. Gradual phosphorylation of degron motifs culminates in M-Phase and targets the protein for degradation. Binding of the Mtw1 complex shields the proximal phospho-degron, protecting kinetochore-bound Ame1 from the degradation machinery. Artificially increasing degron strength partially suppresses the temperature-sensitivity of a cdc4 mutant, while overexpression of Ame1-Okp1 is toxic to cells, demonstrating the physiological importance of this mechanism. We propose that phospho-regulated clearance of excess CCAN subunits protects against ectopic kinetochore assembly and contributes to mitotic checkpoint silencing. Our results suggest a novel strategy for how phospho-degrons can be used to regulate the assembly of multi-subunit complexes.

cell biology↗

bioRxiv2020

Regulation of trehalase activity by multi-site phosphorylation and 14-3-3 interaction

Protein phosphorylation enables a rapid adjustment of cellular activities to diverse intracellular and environmental stimuli. Many phosphoproteins are targeted on more than one site, which allows the integration of multiple signals and the implementation of complex responses. However, the hierarchy and interplay between multiple phospho-sites are often unknown. Here, we study multi-site phosphorylation using the yeast trehalase Nth1 and its activator, the 14-3-3 protein Bmh1, as a model. Nth1 is known to be phosphorylated by the metabolic kinase PKA on four serine residues and by the cell cycle kinase CDK on one residue. However, how these five phospho-sites adjust Nth1 activity remains unclear. Using a novel reporter construct, we investigated the contribution of the individual sites for the regulation of the trehalase and its 14-3-3 interactor. In contrast to the constitutively phosphorylated S20 and S83, the weaker sites S21 and S60 are only phosphorylated by increased PKA activity. For binding Bmh1, S83 functions as the high-affinity "gatekeeper" site, but successful binding of the Bmh1 dimer and thus Nth1 activation requires S60 as a secondary site. Under nutrient-poor conditions with low PKA activity, S60 is not efficiently phosphorylated and the cell cycle dependent phosphorylation of S66 by Cdk1 contributes to Nth1 activity, likely by providing an alternative Bmh1 binding site. Additionally, the PKA sites S20 and S21 modulate the dephosphorylation of Nth1 on downstream Bmh1 sites. In summary, our results expand our molecular understanding of Nth1 regulation and provide a new aspect of the interaction of 14-3-3 proteins with their targets.

biochemistry↗

bioRxiv2020

Biochemical evidence of furin specificity and potential for phospho-regulation at Spike protein S1/S2 cleavage site in SARS-CoV2 but not in SARS-CoV1 or MERS-CoV

The Spike protein of the novel coronavirus SARS-CoV2 contains an insertion 680SPRRAR{downarrow}SV687 forming a cleavage motif RxxR for furin-like enzymes at the boundary of S1/S2 subunits. Cleavage at S1/S2 is important for efficient viral entry into target cells. The insertion is absent in other CoV-s of the same clade, including SARS-CoV1 that caused the 2003 outbreak. However, an analogous insertion was present in the Spike protein of the more distant Middle East Respiratory Syndrome coronavirus MERS-CoV. We show that a crucial third arginine at the left middle position, comprising a motif RRxR is required for furin recognition in vitro, while the general motif RxxR in common with MERS-CoV is not sufficient for cleavage. Further, we describe a surprising finding that the two serines at the edges of the insert SPRRAR{downarrow}SV can be efficiently phosphorylated by proline-directed and basophilic protein kinases. Both phosphorylations switch off furins ability to cleave the site. Although phosphoregulation of secreted proteins is still poorly understood, further studies, supported by a recent report of ten in vivo phosphorylated sites in the Spike protein of SARS-CoV2, could potentially uncover important novel regulatory mechanisms for SARS-CoV2.

molecular biology↗

bioRxiv2020

Comprehensive analysis of fungal G1 cyclin docking motif sequences that control CDK regulatory potency in vivo

Cyclin-dependent kinases (CDKs) control the ordered series of events during eukaryotic cell division. The stage at which individual CDK substrates are phosphorylated can be dictated by cyclin-specific docking motifs. In budding yeast, substrates with Leu/Pro-rich (LP) docking motifs are recognized by Cln1/2 cyclins in late G1 phase, yet the key sequence features of these motifs and the conservation of this mechanism were unknown. Here we comprehensively analyzed LP motif requirements in vivo by combining a competitive growth assay with mutational scanning and deep sequencing. We quantified the impact of all single-residue replacements in five different LP motifs, using six distinct G1 cyclins from diverse fungi including medical and agricultural pathogens. The results reveal the basis for variations in potency among wild-type motifs, and allow derivation of a quantitative matrix that predicts the potency of other candidate motifs. In one protein, Whi5, we found overlapping LP and phosphorylation motifs with partly redundant effects. In another protein, the CDK inhibitor Sic1, we found that its LP motif is inherently weak due to unfavorable residues at key positions, and this imposes a beneficial delay in its phosphorylation and degradation. The overall results provide a general method for surveying viable docking motif sequences and quantifying their potency in vivo, and they reveal how variations in LP motif potency can tune the strength and timing of CDK regulation.

molecular biology↗