Biology subjects

MacMillan, D. W. C.

Publications and source records attributed to MacMillan, D. W. C..

6 recordsLinked to original sources

bioRxiv2026

μMap-uAA: Photocatalytic proximity labeling targeted with single-residue precision

Mapping protein microenvironments with residue-level precision in living cells remains challenging. We report Map-uAA, a genetically encoded proximity labeling platform that uses unnatural amino acid incorporation to install a tetrazine-quenched iridium photocatalyst at user-defined protein sites through click chemistry. Photocatalysis is activated by covalent attachment to the incorporated uAA, enabling localized catalytic labeling of proximal biomolecules. Applied to membrane receptors, Map-uAA captures domain- and time-dependent GPCR interaction networks with residue-level spatial precision.

biochemistry↗

bioRxiv2025

The molecular mechanism of uptake and cell-to-cell transmission of arginine-containing dipeptide repeat proteins.

Micro-satellite repeat expansion of the 5 GGGGCC 3 sequence in the C9orf72 gene is the most common monogenic form of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Dipeptide repeat proteins (DPRs) translated from the mutant allele can be detected in postmortem brains of afflicted individuals. The arginine containing peptides, poly-PR and poly-GR, are particularly noxious to cells. Both have been shown to undergo cell-cell transmission, but the underlying mechanisms are not understood. We found rapid internalization and nucleolar localization of bath-applied hemagglutinin (HA) tagged poly-PR with twenty repeats (HA-PR20) in cell lines and neurons. Small molecule and RNAi approaches implicated a temperature-dependent, fluid phase endocytosis mechanism in HA-PR20 uptake. We sought to identify DPR-related cell surface uptake factors using a high-resolution proximity labeling technique developed in the MacMillan group, termed {micro}Map. DPR-iridium conjugates identified candidate cell-surface proteins which were interrogated in an RNAi screen. Focusing on our strongest candidate, chondroitin sulfate proteoglycan 4 (CSPG4), we showed that cellular uptake of HA-PR20 is blocked by inhibition of glycosaminoglycan chain synthesis (using drugs or RNAi) and knockdown or ablation of CSPG4 (using RNAi or CRISPR editing). Reduction of CSPG4 protected PR20-induced neuronal toxicity. We used a dual reporter system to interrogate in vitro neuron-to-neuron transmission of PR50 and found that PR50 synthesized by one neuron readily spread to neighboring neurons. Transmission was significantly reduced when CSPG4 was knocked down. These results suggest CSPG4 is an important factor in poly-PR internalization and transmission and therefore may be a therapeutic target to slow DPR transmission and disease progression. Significance StatementA GGGGCC hexanucleotide repeat expansion in the C9orf72 gene is the most common monogenic form of ALS/FTD. This expansion leads to dipeptide repeat protein (DPR) production through non-canonical translation of repeat-containing RNA. DPRs have been shown to transmit between cells, but how this occurs is not well understood. We identified the cell surface protein chondroitin sulfate proteoglycan 4 (CSPG4) as a mediator of the uptake and intercellular spread of toxic arginine-rich DPRs. Targeting CSPG4 may provide a strategy to block DPR transmission and slow disease progression.

neuroscience↗

bioRxiv2025

Mesoscale proximity labeling to study macro changes to chromatin occupancy

Proximity labeling traditionally identifies interactomes of a single protein or RNA, though this approach limits mechanistic understanding of biomolecules functioning within complex systems. Here, we demonstrate a strategy for deciphering ligand-induced changes to global biomolecular interactions by enabling proximity labelling at the mesoscale, across an entire cellular system. By inserting nanoscale proximity labelling catalysts throughout chromatin, this system, MesoMap, provided new insights into how HDAC inhibitors regulate gene expression. Furthermore, it revealed that the orphaned drug candidate, SR-1815, regulates disease-linked Syngap1 gene expression through direct inhibition of kinases implicated in both neurological disorders and cancer. Through precise mapping of global chromatin mobility, MesoMap promotes insights into how drug-like chemical probes induce transcriptional dynamics within healthy and disease-associated cellular states.

molecular biology↗

bioRxiv2025

Temporal Microenvironment Mapping (microMap) of Intracellular Trafficking Pathways of Cell-Penetrating Peptides Across the Blood-Brain Barrier

Peptides play critical roles in cellular functions such as signaling and immune regulation, and peptide-based biotherapeutics show great promise for treating various diseases. Among these, cell-penetrating peptides (CPPs) are particularly valuable for drug delivery due to their ability to cross cell membranes. However, the mechanisms underlying CPP-mediated transport, especially across the blood-brain barrier (BBB), remain poorly understood. Mapping intracellular CPP pathways is essential for advancing drug delivery systems, particularly for neurological disorders, as understanding how CPPs navigate the complex environment of the BBB could enable the development of more effective brain-targeted therapies. Here, we leverage a nanoscale proximity labeling technique, termed {micro}Map, to precisely probe the peptide- receptor interactions and intracellular trafficking mechanisms of photocatalyst-tagged CPPs. The unique advantage of the Map platform lies in the ability to control the timing of light exposure, which enables the collection of time-gated data, depending on when the blue light is applied to the cells. By harnessing this spatiotemporal precision, we can uncover key peptide-receptor interactions and cellular processes, setting the stage for new innovations in drug design and brain-targeted therapies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/633151v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1585958org.highwire.dtl.DTLVardef@151b288org.highwire.dtl.DTLVardef@a03efeorg.highwire.dtl.DTLVardef@87d6a4_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Tracking chromatin state changes using μMap photo-proximity labeling

Interactions between biomolecules, particularly proteins, underlie all cellular processes, and ultimately control cell fate. Perturbation of native interactions through mutation, changes in expression levels, or external stimuli leads to altered cellular physiology and can result in either disease or therapeutic effects.1,2 Mapping these interactions and determining how they respond to stimulus is the genesis of many drug development efforts, leading to new therapeutic targets and improvements in human health.1 However, in the complex environment of the nucleus it is challenging to determine protein-protein interactions due to low abundance, transient or multi-valent binding, and a lack of technologies that are able to interrogate these interactions without disrupting the protein binding surface under study.3 Chromatin remodelers, modifying enzymes, interactors, and transcription factors can all be redirected by subtle changes to the microenvironment, causing global changes in protein expression levels and subsequent physiology. Here, we describe the Chroma-Map method for the traceless incorporation of Ir-photosensitizers into the nuclear microenvironment using engineered split inteins. These Ir-catalysts can activate diazirine warheads to form reactive carbenes within a ~10 nm radius, cross-linking with proteins within the immediate microenvironment for analysis via quantitative chemoproteomics.4 We demonstrate this concept on nine different nuclear proteins with varied function and in each case, elucidating their microenvironments. Additionally, we show that this short-range proximity labeling method can reveal the critical changes in interactomes in the presence of cancer-associated mutations, as well as treatment with small-molecule inhibitors. Chroma-Map improves our fundamental understanding of nuclear protein-protein interactions, as well as the effects that small molecule therapeutics have on the local chromatin environment, and in doing so is expected to have a significant impact on the field of epigenetic drug discovery in both academia and industry.

cancer biology↗

bioRxiv2021

Small molecule photocatalysis enables drug target identification via energy transfer

The identification of cellular targets that can be exploited for therapeutic benefit, broadly known as target ID, remains a fundamental goal in drug discovery. In recent years, the application of new chemical and biological technologies that accelerate target ID has become commonplace within drug discovery programs, as a complete understanding of how molecules react in a cellular environment can lead to increased binding selectivity, improved safety profiles, and clinical efficacy. Established approaches using photoaffinity labelling (PAL) are often costly and time-consuming due to poor signal-to-noise coupled with extensive probe optimization. Such challenges are exacerbated when dealing with low abundance membrane proteins or multiple protein target engagement, typically rendering target ID unfeasible. Herein, we describe a general platform for photocatalytic small molecule target ID, which hinges upon the generation of high-energy carbene intermediates via visible light-mediated Dexter energy transfer. By decoupling the reactive warhead from the drug, catalytic signal amplification results in multiple labelling events per drug, leading to unprecedented levels of target enrichment. Through the development of cell permeable photo-catalyst conjugates, this method has enabled the quantitative target and off target identification of several drugs including (+)-JQ1, paclitaxel, and dasatinib. Moreover, this methodology has led to the target ID of two GPCRs - ADORA2A and GPR40 - a class of drug target seldom successfully uncovered in small molecule PAL campaigns.

molecular biology↗