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Targeted protein degradation (TPD) has rapidly emerged as a powerful modality for drugging previously “undruggable” proteins. TPD employs small molecules like PROTACs and molecular glue degraders (MGD) to induce target protein degradation via the formation of a ternary complex with an E3 ligase. However, the rational design of these degraders is severely hindered by the difficulty of obtaining these ternary structures. Here we introduce DeepTernary, a novel end-to-end deep learning approach using an SE(3)-equivariant encoder and a query-based decoder to accurately and rapidly predict these critical structures. Trained on carefully curated TernaryDB, DeepTernary achieves state-of-the-art performance on PROTAC benchmarks without prior exposure to known PROTACs and shows notable prediction capability on the more challenging MGD benchmark with a blind docking protocol. Remarkably, the buried surface areas calculated from predicted structures correlate with experimental degradation potency metrics. Overall, DeepTernary offers a powerful tool for the development of targeted protein degraders. This work introduces DeepTernary, a deep learning method for rapid and accurate prediction of PROTAC and molecular glue-induced ternary complex structures, achieving state-of-the-art results by learning from a curated dataset, TernaryDB.

Plant cryptochromes undergo blue light-dependent phosphorylation to regulate their activity and abundance, but the protein kinases that phosphorylate plant cryptochromes have remained unclear. Here we show that photoexcited Arabidopsis cryptochrome 2 (CRY2) is phosphorylated in vivo on as many as 24 different residues, including 7 major phosphoserines. We demonstrate that four closely related Photoregulatory Protein Kinases (previously referred to as MUT9-like kinases) interact with and phosphorylate photoexcited CRY2. Analyses of the ppk123 and ppk124 triple mutants and amiR 4k artificial microRNA-expressing lines demonstrate that PPKs catalyse blue light-dependent CRY2 phosphorylation to both activate and destabilize the photoreceptor. Phenotypic analyses of these mutant lines indicate that PPKs may have additional substrates, including those involved in the phytochrome signal transduction pathway. These results reveal a mechanism underlying the co-action of cryptochromes and phytochromes to coordinate plant growth and development in response to different wavelengths of solar radiation in nature. Plant cryptochromes are regulated by blue-light dependent phosphorylation. Here the authors map the in vivo phosphorylation sites of Arabidopsis cryptochrome 2 and identify four closely related kinases that act to both activate and destabilize the receptor in response to blue light.

Cryptochromes in different evolutionary lineages act as either photoreceptors or light-independent transcription repressors. The flavin cofactor of both types of cryptochromes can be photoreduced in vitro by electron transportation via three evolutionarily conserved tryptophan residues known as the “Trp triad.” It was hypothesized that Trp triad-dependent photoreduction leads directly to photoexcitation of cryptochrome photoreceptors. We tested this hypothesis by analyzing mutations of Arabidopsis cryptochrome 1 (CRY1) altered in each of the three Trp-triad tryptophan residues (W324, W377, and W400). Surprisingly, in contrast to a previous report all photoreduction-deficient Trp-triad mutations of CRY1 remained physiologically and biochemically active in Arabidopsis plants. ATP did not enhance rapid photoreduction of the wild-type CRY1, nor did it rescue the defective photoreduction of the CRY1 ᵂ³²⁴ᴬ and CRY1 ᵂ⁴⁰⁰F mutants that are photophysiologically active in vivo. The lack of correlation between rapid flavin photoreduction or the effect of ATP on the rapid flavin photoreduction and the in vivo photophysiological activities of plant cryptochromes argues that the Trp triad-dependent photoreduction is not required for the function of cryptochromes and that further efforts are needed to elucidate the photoexcitation mechanism of cryptochrome photoreceptors. Significance The Trp triad-dependent photoreduction of the flavin chromophore has been widely accepted as the photoexcitation mechanism of cryptochrome photoreceptors. However, the experimental evidence supporting this hypothesis derived primarily from the biophysical studies in vitro, except for one genetics study of Arabidopsis cryptochrome 1 (CRY1). In contrast to the previous report, we found that all Trp-triad mutations of Arabidopsis CRY1 remained physiologically active in plants, and this result cannot be readily explained by the ATP-dependent enhancement of Trp triad-dependent photoreduction. Our results challenge the widely accepted Trp-triad hypothesis and call for further investigation of alternative electron transport mechanisms to explain cryptochrome photoexcitation.

Proximity-inducing modalities that co-opt cellular pathways offer new opportunities to regulate oncogenic drivers. Inspired by the success of proximity-based chimeras in both intracellular and extracellular target space, here we describe the development of LY sosome M embrane TA rgeting C himera s (LYMTACs) as a small molecule-based platform that functions intracellularly to modulate the membrane proteome. Conceptually, LYMTACs are heterobifunctional small molecules that co-opt short-lived lysosomal membrane proteins (LMPs) as effectors to deliver targets for lysosomal degradation. We demonstrate that a promiscuous kinase inhibitor-based LYMTAC selectively targets membrane proteins for lysosomal degradation via RNF152, a short-lived LMP. We extend this concept by showing that oncogenic KRAS G12D signaling can be potently inhibited by LYMTACs. Mechanistically, LYMTACs display multi-pharmacology and exert their activity through both target relocalization into the lysosome and degradation. We further generalize LYMTACs across various LMPs and thus offer a platform to access challenging membrane proteins through targeted protein relocalization and degradation. LYMTACs are heterobifunctional small molecules that take advantage of lysosomal membrane proteins to degrade membrane targets via relocalization and lysosomal degradation, offering a modular approach to drug otherwise intractable membrane proteins.

Cryptochromes are photolyase-like blue light receptors that are conserved in plants and animals. Although the light-dependent catalytic mechanism of photolyase is well studied, the photochemical mechanism of cryptochromes remains largely unknown. Lack of an appropriate protein expression system to obtain photochemically active cryptochrome holoproteins is a technical obstacle for the study of plant cryptochromes. We report here an easy-to-use method to express and study Arabidopsis cryptochrome in HEK293T cells. Our results indicate that Arabidopsis cryptochromes expressed in HEK293T are photochemically active. We envision a broad use of this method in the functional investigation of plant proteins, especially in the large-scale analyses of photochemical activities of cryptochromes such as blue light-dependent protein-protein interactions.

The epigenetic mechanisms that maintain differentiated cell states remain incompletely understood. Here we employed histone mutants to uncover a crucial role for H3K36 methylation in the maintenance of cell identities across diverse developmental contexts. Focusing on the experimental induction of pluripotency, we show that H3K36M-mediated depletion of H3K36 methylation endows fibroblasts with a plastic state poised to acquire pluripotency in nearly all cells. At a cellular level, H3K36M facilitates epithelial plasticity by rendering fibroblasts insensitive to TGFβ signals. At a molecular level, H3K36M enables the decommissioning of mesenchymal enhancers and the parallel activation of epithelial/stem cell enhancers. This enhancer rewiring is Tet dependent and redirects Sox2 from promiscuous somatic to pluripotency targets. Our findings reveal a previously unappreciated dual role for H3K36 methylation in the maintenance of cell identity by integrating a crucial developmental pathway into sustained expression of cell-type-specific programmes, and by opposing the expression of alternative lineage programmes through enhancer methylation. Hoetker et al. show that H3K36 methylation exerts a dual role in cell identity maintenance: it integrates TGFβ signals at mesenchymal targets to keep them active and prevents the activation of alternative lineage programmes via enhancer methylation.

Ectopically expressing the transcription factors (TFs) Oct4, Sox2, Klf4, and c-Myc (OSKM) leads to the reprogramming of somatic cells to induced pluripotent stem cells (iPSCs). iPSC reprogramming takes several weeks and yields pluripotent cells only at low frequencies indicating that the reprogramming factors need to overcome barriers established in somatic cells to preserve cell identity. Yet, the mechanisms driving the successful decommissioning of the starting somatic program and the activation of the target pluripotency program are currently unclear. A recent study from the Plath lab has begun to determine how OSKM induce the remodeling of enhancers and induce the transition from somatic to pluripotency enhancers during the reprogramming of mouse embryonic fibroblasts (MEFs) to iPSCs. The key finding was that the collaborative binding of OSK is essential for the step-wise selection and activation of pluripotency enhancers (PEs) throughout reprogramming. Consequently, the target sites of OSKM gradually change during reprogramming to mediate the step-wise induction of pluripotency enhancers. Intriguingly, the reprogramming factors also act on MEF enhancers (MEs). Based on ChIP-seq results, it was suggested that OSK redirect somatic (endogenously expressed) TFs away from MEs to new sites opened by the reprogramming factors. Concomitantly, the active enhancer mark H3K27ac is decreasing at MEs, suggesting that the movement of somatic TFs is critical for the inactivation of MEs. The key questions in the field now are to understand how OSKM, at a mechanistic level, induce (i) the redistribution of somatic TFs and (ii) the decommissioning of MEs in the early stage of reprogramming. In my graduate work, I am addressing these questions by taking advantage of co- mentorship in the Plath and Wohlschlegel labs. In Chapter 2, I hypothesize that both protein-protein interactions (PPIs) of the reprogramming factors with somatic TFs and the newly opened sites containing somatic TFs’ binding motifs are critical for the redistribution of somatic TFs binding and the following somatic enhancer decommissioning. Consequently, I am combining functional experiments with mass spectrometry (MS) methodologies to i) define co-binding between OSK and somatic TFs, ii) to distinguish mechanisms of somatic TFs redistribution through direct PPIs, cooperative binding, and open-sites free binding and iii) identify the mechanism of how active histone mark is removed from MEs. However, since TFs are often of low abundance in cells, MS approaches with a large dynamic range are required for the identification and quantification. Therefore, another aspect of my graduate work is to develop and optimize cutting-edge bottom-up proteomics methodologies for the assessment of low abundance proteins. In Chapter 3, to achieve the better identification and quantification of lowly abundant proteins in complex protein mixtures, I developed a bead-based off-line peptide fractionation method termed: CMMB (Carboxylate Modified Magnetic Bead) -based isopropanol gradient peptide fractionation or ‘CIF’. CIF provides an effective but low material loss alternative to other fractionation methods. In Chapter 4, by combining optimized proteomics and cell biology approaches, we uncovered an understudied mechanism of nuclear proteome regulation: activity- dependent proteasome-mediated degradation. We found that the tumor suppressor protein PDCD4 undergoes rapid stimulus-induced degradation in the nucleus of neurons. We demonstrate that degradation of PDCD4 is required for normal activity-dependent transcription and that PDCD4 target genes include those encoding proteins critical for synapse formation, remodeling, and transmission. In Chapter 5, for improving the quantification of proteins of interest, targeted proteomics assays are often used to pursue more accurate quantitation and better sensitivity. The recently launched High-field asymmetric waveform ion mobility spectrometry (FAIMS) device enables the possibility of improving conventional targeted proteomics assay data quality, while such improvement relies heavily on tuning the parameters of the FAIMS settings, in my thesis work, I investigated the molecular determinants underlying peptide separation by FAIMS and demonstrate that the machine learning model can be used to predict optimized FAIMS settings for peptides which significantly improves targeted proteomics workflows.

Tissue-specific antigens can serve as targets for adoptive T cell transfer-based cancer immunotherapy. Recognition of tumor by T cells is mediated by interaction between peptide–major histocompatibility complexes (pMHCs) and T cell receptors (TCRs). Revealing the identity of peptides bound to MHC is critical in discovering cognate TCRs and predicting potential toxicity. We performed multimodal immunopeptidomic analyses for human prostatic acid phosphatase (PAP), a well-recognized tissue antigen. Three physical methods, including mild acid elution, coimmunoprecipitation, and secreted MHC precipitation, were used to capture a thorough signature of PAP on HLA-A*02:01. Eleven PAP peptides that are potentially A*02:01-restricted were identified, including five predicted strong binders by NetMHCpan 4.0. Peripheral blood mononuclear cells (PBMCs) from more than 20 healthy donors were screened with the PAP peptides. Seven cognate TCRs were isolated which can recognize three distinct epitopes when expressed in PBMCs. One TCR shows reactivity toward cell lines expressing both full-length PAP and HLA-A*02:01. Our results show that a combined multimodal immunopeptidomic approach is productive in revealing target peptides and defining the cloned TCR sequences reactive with prostatic acid phosphatase epitopes.

Cryptochromes in different evolutionary lineages act as either photoreceptors or light-independent transcription repressors. The flavin cofactor of both types of cryptochromes can be photoreduced in vitro by electron transportation via three evolutionarily conserved tryptophan residues known as the “Trp triad.” It was hypothesized that Trp triad-dependent photoreduction leads directly to photoexcitation of cryptochrome photoreceptors. We tested this hypothesis by analyzing mutations ofArabidopsiscryptochrome 1 (CRY1) altered in each of the three Trp-triad tryptophan residues (W324, W377, and W400). Surprisingly, in contrast to a previous report all photoreduction-deficient Trp-triad mutations of CRY1 remained physiologically and biochemically active inArabidopsisplants. ATP did not enhance rapid photoreduction of the wild-type CRY1, nor did it rescue the defective photoreduction of the CRY1W324Aand CRY1W400Fmutants that are photophysiologically active in vivo. The lack of correlation between rapid flavin photoreduction or the effect of ATP on the rapid flavin photoreduction and the in vivo photophysiological activities of plant cryptochromes argues that the Trp triad-dependent photoreduction is not required for the function of cryptochromes and that further efforts are needed to elucidate the photoexcitation mechanism of cryptochrome photoreceptors.

Due to the special hydrographic and physiographic conditions in Taiwan, flooding is likely to occur in the middle and lower reaches of a plain whenever serious rainstorm events occurred. Note worthily, the loss of lives and property caused by flooding are always most considerable in a metropolitan area, and the densely distributed buildings would, not only increase the impervious area, but also decrease the water storage area. Furthermore, a large number of intensive buildings have changed the original land flow conditions, resulting in a beam shrinking flow and the additional form drag phenomenon, which makes the flooding phenomenon more serious. The main purpose of this research is to find the correlation between building coverage and the Manning’s coefficient n through a water flume model experiment. To probe into this issue, the Manning’s roughness adjustment is further divided into a part caused by the surface impedance and a part caused by the building impedance. Thus, building coverage can be added to the general computing grid to reflect the flooding situation with buildings. The two-dimensional inundation model, based on this research, was applied to Taichung City for an actual case simulation. The simulation result of Typhoon Kalmaegi showed that the presented model can obtain a more accurate flooding situation in urban area by considering the blockage effects of buildings and adjusting the surface roughness.