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Membrane contact sites (MCSs) are dynamic subcellular compartments formed between organelles that coordinate diverse aspects of cellular communication, including signaling, metabolism, and membrane organization. Tools capable of monitoring and controlling the spatially localized and dynamic properties of MCSs are needed to dissect their regulatory mechanisms and physiological roles. Recent advances in protein engineering have begun to address this need. Proximity-based reporters, chemogenetic approaches, and optogenetic systems have been developed to enable the visualization, interrogation, and manipulation of inter-organelle contacts with improved spatial and temporal precision. This minireview highlights key developments in these molecular toolkits and their representative applications in studying MCS biology. These approaches provide new insights into organelle crosstalk and may inform future therapeutic strategies targeting MCSs.

Autocatalytic activation of an initiator caspase triggers the onset of apoptosis. In dying cells, caspase-9 activation is mediated by a multimeric adaptor complex known as the Apaf-1 apoptosome. The molecular mechanism by which caspase-9 is activated by the Apaf-1 apoptosome remains largely unknown. Here we demonstrate that the previously reported 1:1 interaction between Apaf-1 caspase recruitment domain (CARD) and caspase-9 CARD is insufficient for the activation of caspase-9. Rather, formation of a multimeric CARD:CARD assembly between Apaf-1 and caspase-9, which requires three types of distinct interfaces, underlies caspase-9 activation. Importantly, an additional surface area on the multimeric CARD assembly is essential for caspase-9 activation. Together, these findings reveal mechanistic insights into the activation of caspase-9 by the Apaf-1 apoptosome and support the induced conformation model for initiator caspase activation by adaptor complexes. Significance Upstream cell death stimuli culminate in the activation of an initiator caspase, marking the onset of apoptosis. Activation of the initiator caspase, caspase-9, is mediated by the heptameric Apaf-1 apoptosome. How Apaf-1 apoptosome facilitates the autocatalytic activation of caspase-9 has remained controversial and largely enigmatic. Two contrasting but not mutually exclusive hypotheses, proximity-induced dimerization vs. induced conformation, emphasize different aspects of initiator caspase activation. This study provides compelling evidence to support the induced conformation model for caspase-9 activation. A previously unknown interface between Apaf-1 and caspase-9 was identified to play an essential role in caspase-9 activation, and formation of a multimeric complex between Apaf-1 caspase recruitment domain (CARD) and caspase-9 was shown to be indispensable for caspase-9 activation.

HighlightsNovel enzyme-induced proximity labeling technology in nanoparticles (nano-EPL).Nano-EPL enables dynamic molecular mapping of the intracellular delivery of nanoparticles in macrophages.Nano-EPL enables the elucidation of a comprehensive phagosome-centered timeline during the vesicular transportation of nanoparticles, revealing distinct organelle engagement and its differential impact on drug delivery efficiency.Achieving increasingly finely targeted drug delivery to organs, tissues, cells, and even to intracellular biomacromolecules is one of the core goals of nanomedicines. As the delivery destination is refined to cellular and subcellular targets, it is essential to explore the delivery of nanomedicines at the molecular level. However, due to the lack of technical methods, the molecular mechanism of the intracellular delivery of nanomedicines remains unclear to date. Here, we develop an enzyme-induced proximity labeling technology in nanoparticles (nano-EPL) for the real-time monitoring of proteins that interact with intracellular nanomedicines. Poly(lactic-co-glycolic acid) nanoparticles coupled with horseradish peroxidase (HRP) were fabricated as a model (HRP(+)-PNPs) to evaluate the molecular mechanism of nano delivery in macrophages. By adding the labeling probe biotin-phenol and the catalytic substrate H2O2 at different time points in cellular delivery, nano-EPL technology was validated for the real-time in situ labeling of proteins interacting with nanoparticles. Nano-EPL achieves the dynamic molecular profiling of 740 proteins to map the intracellular delivery of HRP (+)-PNPs in macrophages over time. Based on dynamic clustering analysis of these proteins, we further discovered that different organelles, including endosomes, lysosomes, the endoplasmic reticulum, and the Golgi apparatus, are involved in delivery with distinct participation timelines. More importantly, the engagement of these organelles differentially affects the drug delivery efficiency, reflecting the spatial–temporal heterogeneity of nano delivery in cells. In summary, these findings highlight a significant methodological advance toward understanding the molecular mechanisms involved in the intracellular delivery of nanomedicines.

Targeted protein degradation (TPD) is a novel strategy for developing therapeutics against pathogens. Prior to causing malaria, Plasmodium parasites replicate within hepatocytes as liver stages, surrounded by a parasitophorous vacuole membrane (PVM). We hypothesized that TPD can be employed to trigger host-driven degradation of essential liver stage PVM proteins and lead to parasite death. To explore this, we took advantage of the proteolysis-targeting-chimera HaloPROTAC3, a molecule that recruits the host von Hippel-Lindau (VHL) E3 ligase to the HaloTag (HT). Parasites expressing HT fused to the host cytosol-exposed domain of the PVM protein UIS4 (UIS4-HT) were generated in Plasmodium berghei and Plasmodium cynomolgi , but only P. berghei UIS4-HT enabled productive liver stage infection experiments in vitro. Although HaloPROTAC3 triggered the degradation of HT proteins in host cells, it had no impact on the survival of P. berghei UIS4-HT liver stages. Furthermore, HaloPROTAC3 bound to P. berghei UIS4-HT but did not recruit VHL or trigger ubiquitination of the PVM. Overall, although this study did not establish whether host-driven TPD can degrade Plasmodium PVM proteins, it highlights the challenges of developing TPD approaches against novel targets and offers insights for advancing this therapeutic strategy against pathogens.

Chromatin regulation is a critical aspect of nuclear function. Recent advances have provided detailed information about dynamic three-dimensional organization of chromatin and its regulatory factors. Mechanisms crucial for normal nuclear function and epigenetic control include compartmentalization of biochemical reactions by liquid-phase separated condensates and signal-dependent regulation of protein stability. Synthetic control of these phenomena by small molecules provides deep insight into essential activities such as histone modification, BAF (SWI/SNF) and PBAF remodeling, Polycomb repression, enhancer looping by cohesin and CTCF, as well as many other processes that contribute to transcription. As a result, a complete understanding of the spatiotemporal mechanisms that underlie chromatin regulation increasingly requires the use of fast-acting drugs and chemical probes. Here, we provide a comprehensive review of next-generation chemical biology tools to interrogate the chromatin regulatory landscape, including selective PROTAC E3 ubiquitin ligase degraders, degrons, fluorescent ligands, dimerizers, inhibitors, and other drugs. These small molecules provide important insights into the mechanisms that govern gene regulation, DNA repair, development, and diseases like cancer.

The slow delayed rectifier potassium current, I Ks , conducted through pore-forming Q1 and auxiliary E1 ion channel complexes is important for human cardiac action potential repolarization. During exercise or fright, I Ks is up-regulated by protein kinase A (PKA)-mediated Q1 phosphorylation to maintain heart rhythm and optimum cardiac performance. Sympathetic up-regulation of I Ks requires recruitment of PKA holoenzyme (two regulatory – RI or RII – and two catalytic Cα subunits) to Q1 C-terminus by an A kinase anchoring protein (AKAP9). Mutations in Q1 or AKAP9 that abolish their functional interaction result in long QT syndrome type 1 and 11, respectively, which increases the risk of sudden cardiac death during exercise. Here, we investigated the utility of a targeted protein phosphorylation (TPP) approach to reconstitute PKA regulation of I Ks in the absence of AKAP9. Targeted recruitment of endogenous Cα to E1-YFP using a GFP/YFP nanobody (nano) fused to RIIα enabled acute cAMP-mediated enhancement of I Ks , reconstituting physiological regulation of the channel complex. By contrast, nano-mediated tethering of RIIα or Cα to Q1-YFP constitutively inhibited I Ks by retaining the channel intracellularly in the endoplasmic reticulum and Golgi. Proteomic analysis revealed that distinct phosphorylation sites are modified by Cα targeted to Q1-YFP compared to free Cα. Thus, functional outcomes of synthetically recruited PKA on I Ks regulation is critically dependent on the site of recruitment within the channel complex. The results reveal insights into divergent regulation of I Ks by phosphorylation across different spatial and time scales, and suggest a TPP approach to develop new drugs to prevent exercise-induced sudden cardiac death.

Chemogenetic tools are genetically encoded systems regulated by user‐defined chemicals. Their ability to temporally modulate protein functions in specific cell populations has facilitated in‐depth understanding of dynamic biological systems. Many chemogenetic domains have been developed for regulating a wide range of biological processes, ranging from cellular events to animal behaviors. These tools share some common mechanisms, including proximity regulation, conformational change and allosteric control, as well as steric hinderance control. Here in this review, we aim to provide an overview of different chemogenetic tool designs that utilize the above three common mechanisms to control cellular events. Chemogenetic tools have been widely applied to interrogate dynamic biological processes. They are genetically encoded systems that are controlled by user‐defined chemicals. In this review, chemogenetic tool designs for various biological applications are introduced and discussed.

Multifunctional ligand-binding domains have revolutionized cell biology by enabling the precise visualization and manipulation of fused proteins of interest through small molecule probes. Employing these domains is labor-intensive and low-throughput, limiting studies to only small subsets of proteins at a time. However, advancements in high-throughput technologies like pooled protein tagging have enabled the tagging of proteins across the entire proteome with ligand-binding domains. This allows for the generation of complex cell libraries where each cell expresses a different protein fused to a generic, ligandable handle. These libraries unlock opportunities to explore the proteome with a versatile toolbox of small molecule probes designed to interact with the fused tags, allowing researchers to visualize protein localization, induce protein misfolding, manipulate protein-protein interactions, modulate protein stability, and more in a scalable, systematic manner. This review explores recent studies employing multifunctional domains at proteome scale, delving into how the associated chemical probes have been employed to enable insights into endogenous protein function, cellular processes, and disease mechanisms. Additional multifunctional ligand-binding domains are discussed that can be used in pooled protein tagging, as well as their potential strengths and weaknesses. We also discuss potential applications in drug discovery such as high-throughput screening for therapeutic targets with insights for bifunctional ligand optimization. By integrating pooled protein tagging with ligand-binding domains and chemical probes, we highlight how the fusion of chemical biology and functional genomics is paving the way for innovative research avenues and transformative advances in cell biology and pharmacology.

The development of proteolysis targeting chimeras (PROTACs), which induce the degradation of target proteins by bringing them into proximity with cellular E3 ubiquitin ligases, has revolutionized drug development. While the human genome encodes more than 600 different E3 ligases, current PROTACs use only a handful of them, drastically limiting their full potential. Furthermore, many PROTAC development campaigns fail because the selected E3 ligase candidates are unable to induce degradation of the particular target of interest. As more and more ligands for novel E3 ligases are discovered, the chemical effort to identify the best E3 ligase for a given target is exploding. Therefore, a genetic system to identify degradation-causing E3 ligases and suitable target/E3 ligase pairs is urgently needed. Here, we used the well-established dimerization of the FKBP12 protein and FRB domain by rapamycin to bring the target protein WDR5 into proximity with candidate E3 ligases. Strikingly, this rapamycin-induced proximity assay (RiPA) revealed that VHL, but not Cereblon, is able to induce WDR5 degradation - a finding previously made by PROTACs, demonstrating its predictive power. By optimizing the steric arrangement of all components and fusing the target protein with a minimal luciferase, RiPA can identify the ideal E3 for any target protein of interest in living cells, significantly reducing and focusing the chemical effort in the early stages of PROTAC development.

Systemic toxicity is a major challenge in the development of therapeutics. Consequently, cell-type-specific targeting is needed to improve on-target efficacy while reducing off-target toxicity. Here, we describe a cell-targeting system we have termed BRAID ( BR idged A ctivation by I ntra/intermolecular D ivision) whereby an active molecule is divided into two inactive or less active parts that are subsequently brought together via a so-called ‘bridging receptor’ on the target cell. This concept was validated using the WNT/β-catenin signaling system, demonstrating that a multivalent WNT agonist molecule divided into two inactive components assembled from different epitopes via the hepatocyte receptor βKlotho induces signaling specifically on hepatocytes. These data provide proof of concept for this cell-specific targeting strategy, and in principle, this may also allow activation of multiple signaling pathways where desirable. This approach has broad application potential for other receptor systems.