Biology subjects

Rogers, J. M.

Publications and source records attributed to Rogers, J. M..

14 recordsLinked to original sources

bioRxiv2026

De novo macrocyclic peptide discovery produces potent class A GPCR ligands with diverse pharmacology

G protein-coupled receptors (GPCRs) represent one of the largest protein families of therapeutic importance, as over one third of all currently approved drugs target GPCRs. A growing proportion of GPCR drugs are peptides. However, class A GPCRs (over 80% of clinically studied targets) are underexploited as peptide targets, presumably since their only structured domain is the core, seven-helix, integral membrane fold presenting a limited solvent-exposed surface area. Screening libraries of de novo macrocyclic peptides have allowed for the targeting of otherwise intractable proteins, but their potential to generate selective ligands for class A GPCRs has not been demonstrated. Here, we used random nonstandard peptides integrated discovery (RaPID) to identify de novo macrocyclic peptide ligands targeting the chemokine receptor CXCR4. CXCR4 regulates cell migration, immune surveillance, and inflammation, making it an important therapeutic target for multiple diseases. We discovered novel, potent CXCR4-selective antagonists binding within the orthosteric pocket with nanomolar affinity. The most active peptides effectively inhibited receptor signaling, suppressed chemokine-driven cell migration, and blocked CXCR4-driven HIV entry. Our results show that even the structurally minimal class A GPCRs are viable targets for de novo cyclic peptide discovery, highlighting the potential for uncovering ligands towards other members of the GPCR superfamily.

pharmacology and toxicology↗

bioRxiv2026

A deep learning-generated hASIC1a miniprotein inhibitor confers neuroprotection in stroke

Ion channels remain notoriously recalcitrant drug targets despite their extensive pathophysiological significance. Acid-sensing ion channels (ASICs) are proton-gated cation channels that cause neuronal death when excessively activated, such as during ischaemic stroke. This acidosis-driven injury pathway remains unchallenged by existing therapeutics. Here we generate two de novo designed hASIC1a-specific miniproteins by developing a platform that integrates computational miniprotein design, high-throughput automated patch clamp screening, biophysical and structural characterisation, and in vivo validation. We identify a subtype-selective hASIC1a inhibitor, Denasin1, which achieves near-complete inhibition with nanomolar potency. Denasin1 is disordered in solution but adopts the computationally predicted helix-turn-helix fold when bound to the extracellular acidic pocket of hASIC1a. Crucially, Denasin1 reduces infarct volume by > 50% in a murine model of ischaemic stroke. We thereby establish our de novo design platform as a route to develop potent and selective ion channel modulators.

biophysics↗

bioRxiv2025

A distinct phase of cyclin B (Cdc13) nuclear export at mitotic entry in S. pombe

In eukaryotes, cell division requires coordination between the nucleus and cytoplasm. Entry into cell division is driven by cyclin-dependent kinases (CDKs), which need a cyclin binding partner for their activity. In Schizosaccharomyces pombe (fission yeast), the B-type cyclin Cdc13 is essential and sufficient for cell cycle progression and is strongly enriched in the nucleus. Here, we show that a fraction of Cdc13 is exported from the nucleus to the cytoplasm just prior to mitosis. This export could be critical to propagate CDK activity throughout the cell. Mutating three Cdc13 nuclear localization signals (NLSs) led to precocious enrichment of Cdc13 in the cytoplasm but did not accelerate mitotic entry, indicating that the export is not sufficient to trigger entry into mitosis. The export coincides with spindle pole body integration into the nuclear envelope and may be required to coordinate nuclear and cytoplasmic signaling required for this integration. The onset and stop of Cdc13 nuclear export are remarkably abrupt, underscoring that S. pombe mitotic entry consists of several switch-like transitions over the course of minutes. Our findings add another instance to the various cyclin nuclear transport events known to occur at critical cell cycle transitions throughout eukaryotes.

cell biology↗

bioRxiv2024

Proteome-scale quantification of the interactions driving condensate formation of intrinsically disordered proteins

Intrinsically disordered protein regions facilitate cellular organization through phase separation into biomolecular condensates. However, the molecular interactions driving this process remain poorly understood because of experimental limitations. Here, we advance experimental throughput by several orders of magnitude by developing Condensate Partitioning by mRNA-Display (CPmD). The method allows analysis of partitioning of hundred thousand peptides derived from the disordered proteome into reconstituted condensates. Our results demonstrate that the amino acid content, rather than specific sequence, primarily determines partitioning behavior. Importantly, quantification of the partitioning energies of peptides allows us to decipher the molecular grammar of the relevant interactions, allowing accurate prediction of the formation of condensates of diverse full-length disordered protein regions. The results reveal how physicochemical properties of disordered regions encode biological functions through formation of biomolecular condensates.

biophysics↗

bioRxiv2024

Notch induces transcription by stimulating release of paused RNA Pol II without increasing chromatin accessibility

Notch proteins undergo ligand-induced proteolysis to release a nuclear effector that influences a wide range of cellular processes by regulating transcription. Despite years of study, however, how Notch induces the transcription of its target genes remains unclear. Here, we comprehensively examined the response to human Notch1 across a time course of activation using high-resolution genomic assays of chromatin accessibility and nascent RNA production. Our data reveal that Notch induces target gene transcription primarily by releasing paused RNA polymerase II (RNAPII). Moreover, in contrast to prevailing models suggesting that Notch acts by promoting chromatin accessibility, we found that open chromatin was established at Notch-responsive regulatory elements prior to Notch signal induction, through SWI/SNF-mediated remodeling. Together, these studies show that the nuclear response to Notch signaling is dictated by the pre-existing chromatin state and RNAPII distribution at the time of signal activation.

genomics↗

bioRxiv2023

Temporal Dynamics and Stoichiometry in Notch Signaling - from Notch Synaptic Complex Formation to NICD Nuclear Entry

Mammalian Notch signaling occurs when binding of Delta or Jagged to Notch stimulates proteolytic release of the Notch intracellular domain (NICD), which enters the nucleus to regulate target gene expression. To determine the temporal dynamics of events associated with Notch signaling under native conditions, we fluorescently tagged Notch and Delta at their endogenous genomic loci and visualized them upon pairing of receiver (Notch) and sender (Delta) cells as a function of time after cell contact. At contact sites, Notch and Delta immediately accumulated at 1:1 stoichiometry in synapses, which resolved by 15-20 min after contact. Synapse formation preceded entrance of the Notch extracellular domain into the sender cell and accumulation of NICD in the nucleus of the receiver cell, which approached a maximum after [~]45 min and was prevented by chemical and genetic inhibitors of signaling. These findings directly link Notch-Delta synapse dynamics to NICD production with unprecedented spatiotemporal precision.

cell biology↗

bioRxiv2023

DNA binding analysis of rare variants in homeodomains reveals novel homeodomain specificity-determining residues

Homeodomains (HDs) are the second largest class of DNA binding domains (DBDs) among eukaryotic sequence-specific transcription factors (TFs) and play important roles in regulating development, body patterning, and cellular differentiation. Here, we analyzed 92 human HD mutants, including disease-associated variants and variants of unknown significance (VUSs), for their effects on DNA binding activity. Many of the variants altered DNA binding affinity and/or specificity. Biochemical analysis and structural modeling identified 14 novel specificity-determining positions, 5 of which do not contact DNA. The same missense substitution at analogous positions within different HDs often exhibited different effects on DNA binding. Variant effect prediction tools perform moderately well in distinguishing variants with altered binding affinity, but poorly in identifying those with altered specificity. Our results highlight the need for biochemical assays of TF coding variants and prioritize dozens of variants for further investigations into their pathogenicity and development of clinical diagnostics and precision therapies.

genetics↗

bioRxiv2023

Fluorogenic Substrates and Cyclic Peptide Inhibitors of the Oligonucleotide Activated SIRT7

The sirtuins are NAD+-dependent lysine deacylases, comprising seven isoforms (SIRT1-7) in humans, which are involved in the regulation of a plethora of biology, including gene expression and metabolism. The sirtuins share a common hydrolytic mechanism but display preferences for different {varepsilon}-N-acyllysine substrates. SIRT7 deacetylates targets in nuclei and nucleoli but remains one of the lesser studied of the seven isoforms; in part, because of a lack of chemical tools to specifically probe SIRT7 activity. Here we expressed SIRT7 and, using small-angle X-ray scattering, reveal SIRT7 to be a monomeric enzyme with low degree of globular flexibility in solution. We developed a fluorogenic assay for investigation of the substrate preferences of SIRT7 and to evaluate compounds that modulate its activity. We report several mechanism-based SIRT7 inhibitors as well as de novo cyclic peptide inhibitors selected from mRNA-display library screening that exhibit selectivity for SIRT7 over other sirtuin isoforms and stabilize SIRT7 in cells.

biochemistry↗

bioRxiv2022

A Spatiotemporal Notch Interaction Map from Membrane to Nucleus

Notch signaling relies on ligand-induced proteolysis to liberate a nuclear effector that drives cell fate decisions. The location and timing of individual steps required for proteolysis and movement of Notch from membrane to nucleus, however, remain unclear. Here, we use proximity labeling with quantitative multiplexed mass spectrometry to monitor the microenvironment of endogenous Notch2 after ligand stimulation in the presence of a gamma secretase inhibitor and then as a function of time after inhibitor removal. Our studies show that gamma secretase cleavage of Notch2 occurs in an intracellular compartment and that formation of nuclear complexes and recruitment of chromatin-modifying enzymes occurs within 45 minutes of inhibitor washout. This work provides a spatiotemporal map of unprecedented detail tracking the itinerary of Notch from membrane to nucleus after activation and identifies molecular events in signal transmission that are potential targets for modulating Notch signaling activity.

molecular biology↗

bioRxiv2021

Targeted degradation of the oncogenic phosphatase SHP2

SHP2 is a protein tyrosine phosphatase that plays a critical role in the full activation of the Ras/MAPK pathway upon stimulation of receptor tyrosine kinases (RTKs), which are frequently amplified or mutationally activated in human cancer. In addition, activating mutations in SHP2 result in developmental disorders and hematologic malignancies. Several allosteric inhibitors have been developed for SHP2 and are currently in clinical trials. Here, we report the development and evaluation of a SHP2 PROTAC created by conjugating RMC-4550 with pomalidomide using a PEG linker. This molecule is highly selective for SHP2, induces degradation of SHP2 in leukemic cells at sub-micromolar concentration, inhibits MAPK signaling, and suppresses cancer cell growth. SHP2 PROTACs serve as an alternative strategy for targeting ERK-dependent cancers and are useful tools alongside allosteric inhibitors for dissecting the mechanisms by which SHP2 exerts its oncogenic activity.

biochemistry↗

bioRxiv2021

Mitotic checkpoint gene expression is tuned by coding sequences

The mitotic checkpoint (also called spindle assembly checkpoint, SAC) is a signaling pathway that safeguards proper chromosome segregation. Proper functioning of the SAC depends on adequate protein concentrations and appropriate stoichiometries between SAC proteins. Yet very little is known about SAC gene expression. Here, we show in fission yeast (S. pombe) that a combination of short mRNA half-lives and long protein half-lives supports stable SAC protein levels. For the SAC genes mad2+ and mad3+, their short mRNA half-lives are supported by a high frequency of non-optimal codons. In contrast, mad1+ mRNA has a short half-life despite a low frequency of non-optimal codons and despite the lack of known destabilizing motifs. Hence, different SAC genes employ different strategies of expression. We further show that Mad1 homodimers form co-translationally, which may necessitate a certain codon usage pattern. Taken together, we propose that the codon usage of SAC genes is fine-tuned for proper SAC function. Our work shines light on gene expression features that promote spindle assembly checkpoint function and suggests that synonymous mutations may weaken the checkpoint.

cell biology↗

bioRxiv2020

IER5, a DNA-damage response gene, is required for Notch-mediated induction of squamous cell differentiation

Notch signaling regulates normal squamous cell proliferation and differentiation and is frequently disrupted in squamous cell carcinomas, in which Notch is a key tumor suppressive pathway. To identify the direct targets of Notch that produce these phenotypes, we introduced a conditional Notch transgene into squamous cell carcinoma cell lines, which respond to Notch activation in 2D culture and in organoid cultures by undergoing differentiation. RNA-seq and ChIP-seq analyses show that in squamous cells Notch activates a context-specific program of gene expression that depends on lineage-specific regulatory elements, most of which lie in long- range enhancers. Among the direct Notch target genes are multiple DNA damage response genes, including IER5, which is regulated by Notch through several enhancer elements. We show that IER5 is required for Notch-induced differentiation in squamous carcinoma cells and in TERT-immortalized keratinocytes. Its function is epistatic to PPP2R2A, which encodes the B55 subunit of PP2A, and IER5 interacts with B55 in cells and in purified systems. These results show that Notch and DNA-damage response pathways converge in squamous cells and that some components of these pathways promote differentiation, which may serve to eliminate DNA-damaged cells from the proliferative pool in squamous epithelia. Crosstalk involving Notch and PP2A may enable Notch signaling to be tuned and integrated with other pathways that regulate squamous differentiation. Our work also suggests that squamous cell carcinomas remain responsive to Notch signaling, providing a rationale for reactivation of Notch as a therapeutic strategy in these cancers. ImpactOur findings highlight context-specific crosstalk between Notch, DNA damage response genes, and PP2A, and provide a roadmap for understanding how Notch induces the differentiation of squamous cells.

cancer biology↗

bioRxiv2020

Varying co-factor requirements for MAML1-dependent transcription at different Notch-responsive target genes

Mastermind proteins are required for transcription of Notch target genes, yet the molecular basis for mastermind function remains incompletely understood. Previous work has shown that Notch can induce transcriptional responses by binding to promoters, but more often by binding to enhancers, with HES4 and DTX1 as representative mammalian examples of promoter and enhancer responsiveness, respectively. Here, we show that mastermind dependence of the Notch response at these loci is differentially encoded in Jurkat T-ALL cells. Knockout of Mastermind-like1 (MAML1) eliminates Notch responsive activation of both these genes, and reduced target gene expression is accompanied by a decrease in H3K27 acetylation, consistent with the importance of MAML1 for p300 activity. Add-back of defined MAML1 constructs in knockout cells identifies residues 151-350 of MAML1 as essential for expression of either Notch-responsive gene. Fusion of the Notch-binding region of MAML1 to the HAT domain of p300 rescues expression of HES4 but not DTX1, suggesting that an additional activity of MAML1 is needed for gene induction at a distance. Together, these studies establish the functional importance of the MAML1151-350 region for Notch-dependent transcriptional induction, and reveal differential requirements for MAML1-dependent recruitment activities at different Notch responsive loci, highlighting the molecular complexity of NTC-stimulated transcription.

molecular biology↗

bioRxiv2019

Lineage-specific control of convergent cell identity by a Forkhead repressor

During convergent differentiation, multiple developmental lineages produce a highly similar or identical cell type. However, few molecular players that drive convergent differentiation are known. Here, we show that the C. elegans Forkhead transcription factor UNC-130 is required in only one of three convergent lineages that produce the same glial cell type. UNC-130 acts transiently as a repressor in progenitors and newly-born terminal cells to allow the proper specification of cells related by lineage rather than by cell type or function. Specification defects correlate with UNC-130:DNA binding, and UNC-130 can be functionally replaced by its human homolog, the neural crest lineage determinant FoxD3. We propose that, in contrast to terminal selectors that activate cell-type specific transcriptional programs in terminally differentiating cells, UNC-130 acts early and specifically in one convergent lineage to produce a cell type that also arises from molecularly distinct progenitors in other lineages.

developmental biology↗