Binding Of Domain Research Articles

Longitudinal analysis of genetic and epigenetic changes in human pluripotent stem cells in the landscape of culture-induced abnormality.

Human embryonic stem cells (hESCs) are naturally equipped to maintain genome integrity to minimize genetic mutations during early embryo development. However, genetic aberration risks and subsequent cellular changes in hESCs during in vitro culture pose a significant threat to stem cell therapy. While a few studies have reported specific somatic mutations and copy number variations (CNVs), the molecular mechanisms underlying the acquisition of 'culture-adapted phenotypes' by hESCs are largely unknown. Therefore, we conducted comprehensive genomic, single-cell transcriptomic, and single-cell ATAC-seq analyses of an isogenic hESC model displaying definitive 'culture-adapted phenotypes'. We found that hESCs lacking TP53, in which loss-of-function mutations were identified in human pluripotent stem cells (hPSCs), presented a surge in somatic mutations. Notably, hPSCs with a copy number gain of 20q11.21 during early passage did not present 'culture-adapted phenotypes' or BCL2L1 induction. Single-cell RNA-seq and ATAC-seq analyses revealed active transcriptional regulation at the 20q11.21 locus. Furthermore, the induction of BCL2L1 and TPX2 to trigger 'culture-adapted phenotypes' was associated with epigenetic changes facilitating TEA domain (TEAD) binding. These results suggest that 20q11.21 copy number gain and additional epigenetic changes are necessary for expressing 'culture-adapted phenotypes' by activating gene transcription at this specific locus.

Cognitive Impact of Neurotropic Pathogens: Investigating Molecular Mimicry through Computational Methods

Neurotropic pathogens, notably, herpesviruses, have been associated with significant neuropsychiatric effects. As a group, these pathogens can exploit molecular mimicry mechanisms to manipulate the host central nervous system to their advantage. Here, we present a systematic computational approach that may ultimately be used to unravel protein–protein interactions and molecular mimicry processes that have not yet been solved experimentally. Toward this end, we validate this approach by replicating a set of pre-existing experimental findings that document the structural and functional similarities shared by the human cytomegalovirus-encoded UL144 glycoprotein and human tumor necrosis factor receptor superfamily member 14 (TNFRSF14). We began with a thorough exploration of the Homo sapiens protein database using the Basic Local Alignment Search Tool (BLASTx) to identify proteins sharing sequence homology with UL144. Subsequently, we used AlphaFold2 to predict the independent three-dimensional structures of UL144 and TNFRSF14. This was followed by a comprehensive structural comparison facilitated by Distance-Matrix Alignment and Foldseek. Finally, we used AlphaFold-multimer and PPIscreenML to elucidate potential protein complexes and confirm the predicted binding activities of both UL144 and TNFRSF14. We then used our in silico approach to replicate the experimental finding that revealed TNFRSF14 binding to both B- and T-lymphocyte attenuator (BTLA) and glycoprotein domain and UL144 binding to BTLA alone. This computational framework offers promise in identifying structural similarities and interactions between pathogen-encoded proteins and their host counterparts. This information will provide valuable insights into the cognitive mechanisms underlying the neuropsychiatric effects of viral infections.Graphical

Population-Based Study of Rare Coding Variants in NR5A1/SF-1.

Steroidogenic Factor 1/Nuclear Receptor Subfamily 5 Group A Member 1 (SF-1/NR5A1) is critical for the development and function of sex organs, influencing steroidogenesis and reproduction. While rare deleterious NR5A1/SF-1 variants have been identified in individuals with various differences of sex development (DSD), primary ovarian insufficiency, and infertility, their impact on the general population remains unclear. We analyzed health records and exome sequencing data from up to 420 162 individuals (227 858 women) from the UK Biobank study to assess the impact of rare (frequency < 0.1%) predicted deleterious NR5A1/SF-1 variants on age at menopause and 26 other traits. No carriers of rare protein truncating variants in NR5A1/SF-1 were identified. We found that the previously reported association of rare deleterious missense NR5A1/SF-1 variants with earlier age at menopause is driven by variants in the DNA binding domain (DBD) and ligand binding domain (LBD) (combined test: beta = -2.36 years/allele, [95% CI: 3.21, -1.51], N = 107 carriers, P = 4.6 × 10-8). Carriers also had a higher risk of adult obesity (OR = 1.061, [95% CI: 1.003, 1.104], N = 344, P = .015), particularly among women (OR = 1.095 [95% CI: 1.034, 1.163, P = 3.87 × 10-3], N = 176), but not men (OR = 1.019, [95% CI: 0.955, 1.088], P = .57, N = 168). Deleterious missense variants in the DBD and LBD likely disrupt NR5A1/SF-1 function. This study broadens the relevance of deleterious NR5A1/SF-1 variants beyond rare DSDs, suggesting the need for extended phenotyping and monitoring of affected individuals.

A "Goldilocks Zone" for Recruiting BET Proteins with Bromodomain-1-Selective Ligands.

Synthetic genome readers/regulators (SynGRs) are bifunctional molecules that are rationally designed to bind specific genomic sequences and engage cellular machinery that regulates the expression of targeted genes. The prototypical SynGR1 targets GAA trinucleotide repeats and recruits the BET family of transcriptional regulatory proteins via a flexibly tethered ligand, JQ1. This pan-BET ligand binds both tandem bromodomains of BET proteins (BD1 and BD2). Second-generation SynGRs, which substituted JQ1 with bromodomain-selective ligands, unexpectedly revealed that BD1-selective ligands failed to functionally engage BET proteins in living cells despite displaying the ability to bind BD1 in vitro. Mechanistically, recruiting a BET protein via BD1- or BD2-selective SynGRs should have resulted in indistinguishable functional outcomes. Here we report the conversion of inactive BD1-targeting SynGRs into functional gene regulators by a structure-guided redesign of the chemical linker that bridges the DNA-binding molecule to the highly selective BD1 ligand GSK778. The results point to an optimal zone for positioning the BD1-selective ligand for functional engagement of BET proteins on chromatin, consistent with the preferred binding of BD1 domains to distal acetyllysine residues on histone tails. The results not only resolve the mechanistic conundrum but also provide insight into domain-selective targeting and nuanced design of chemo probes and therapeutics.

Oxoammonium salts exert antiviral effects against coronavirus via denaturation of their spike proteins

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV2) infection has forced social changes worldwide. Development of potent antiviral agents is necessary to prevent future pandemics. Titanium oxide, a photocatalyst, is a long-acting antiviral agent; however, its effects are weakened in the dark. Therefore, new antiviral substances that can be used in the dark are needed. Two types of nitroxyl radicals, 2,2,6,6-tetramethylpiperidine N-oxyl (TEMPO) and 2-azaadamantane N-oxyl (AZADO), are commonly used as oxidation catalysts utilizing oxygen in the air as the terminal oxidant. Therefore, in this study, we aimed to evaluate the potential of these radicals as antiviral compounds with sustained activity even in the dark. We evaluated the antiviral effects of oxoammonium salts corresponding to TEMPO and AZADO (TEMPO-Oxo and AZADO-Oxo, respectively), which are the active forms of nitroxyl radicals in oxidation reactions. TEMPO-Oxo and AZADO-Oxo inhibited the binding of SARS-CoV2 spike protein receptor-binding domain (S-RBD) to angiotensin-converting enzyme 2. Notably, AZADO-Oxo exhibited a 10-fold stronger inhibitory effect than TEMPO-Oxo. TEMPO-Oxo and AZADO-Oxo also denatured S-RBD; however, effects of AZADO-Oxo were 10-fold stronger than those of TEMPO-Oxo and did not change in the dark. Some S-RBD peptides treated with AZADO-Oxo were cleaved at the N-terminal side of tyrosine residues. TEMPO-Oxo and AZADO-Oxo exhibited concentration-dependent antiviral effects against feline coronavirus. In conclusion, active forms of the nitroxyl radicals, TEMPO-Oxo and AZADO-Oxo, exerted antiviral effects by denaturing S-RBD, regardless of the presence or absence of light, suggesting their potential as novel antiviral agents.

Myosin-5a facilitates stress granule formation by interacting with G3BP1

Stress granules (SGs) are non-membranous organelles composed of mRNA and proteins that assemble in the cytosol when the cell is under stress. Although the composition of mammalian SGs is both cell-type and stress-dependent, they consistently contain core components, such as Ras GTPase activating protein SH3 domain binding protein 1 (G3BP1). Upon stress, living cells rapidly assemble micrometric SGs, sometimes within a few minutes, suggesting that SG components may be actively transported by the microtubule and/or actin cytoskeleton. Indeed, SG assembly has been shown to depend on the microtubule cytoskeleton and the associated motor proteins. However, the role of the actin cytoskeleton and associated myosin motor proteins remains controversial. Here, we identified G3BP1 as a novel binding protein of unconventional myosin-5a (Myo5a). G3BP1 uses its C-terminal RNA-binding domain to interact with the middle portion of Myo5a tail domain (Myo5a-MTD). Suppressing Myo5a function in mammalian cells, either by overexpressing Myo5a-MTD, eliminating Myo5a gene expression, or treatment with myosin-5 inhibitor, inhibits the arsenite-induced formation of both small and large SGs. This is different from the effect of microtubule disruption, which abolishes the formation of large SGs but enhances the formation of small SGs under stress conditions. We therefore propose that, under stress conditions, Myo5a facilitates the formation of SGs at an earlier stage than the microtubule-dependent process.

Pin1 WW Domain Ligand Library Synthesized with an Easy Solid-Phase Phosphorylating Reagent.

Cell cycle regulatory enzyme Pin1 both catalyzes pSer/Thr-cis/trans-Pro isomerization and binds the same motif separately in its WW domain. To better understand the function of Pin1, a way to separate these activities is needed. An unnatural peptide library, R1CO-pSer-Pro-NHR2, was designed to identify ligands specific for the Pin1 WW domain. A new solid-phase phosphorylating reagent (SPPR) containing a phosphoramidite functional group was synthesized in one step from Wang resin. The SPPR was used in the preparation of the library by parallel synthesis. The final 315-member library was screened with our WW-domain-specific, enzyme-linked enzyme-binding assay (ELEBA). Four of the best hits were resynthesized, and the competitive dissociation constants were measured by ELEBA. NMR chemical-shift perturbations (CSP) of ligands with 15N-labeled Pin1 were used to measure Kd for the best four ligands directly, demonstrating that they were specific Pin1 WW domain ligands. Models of the ligands bound to the Pin1 WW domain were used to visualize the mode of binding in the WW domain.

Phosphorylation at the D56 residue of MtrA in Mycobacterium tuberculosis enhances its DNA binding affinity by modulating inter-domain interaction

The response regulator, MtrA, plays a major role in adaptation to the host environment, cell division, replication, and dormancy activation of Mycobacterium tuberculosis (Mtb). The phosphorylation of the response regulator MtrA alters the downstream activity, typically involving changes in DNA binding activity. However, there is a substantial knowledge gap in understanding the phosphorylation-mediated structural changes in MtrA. Additionally, the active conformation of the protein has yet to be determined. Therefore, in this study, we have investigated the phosphorylation-induced conformational changes of MtrA using all-atom molecular dynamics simulations under various phosphorylation conditions. The results from this study demonstrate that the phosphorylation at D56 (pD56-MtrA) increases the compactness of the MtrA protein by stabilizing the inter-domain interaction between the regulatory domain and DNA binding domain. Notably, the higher occupancy H-bond (over 95 %) between Arg200-Asn100 in case of the pD56-MtrA condition, which is otherwise absent in the non-phosphorylated (uMtrA) condition, suggests the importance of this interaction in the active conformation of the protein. The dynamic cross-correlation analysis reveals that phosphorylation (especially pD56-MtrA) reduces the anti-correlated motions and increases correlated motions between different domains. Moreover, the higher DNA binding affinity of pD56-MtrA compared to uMtrA supported by molecular docking and MD simulation followed by MMPBSA analysis suggests that pD56-MtrA is the possible active conformation of the MtrA protein. Overall, this investigation elucidates the key structural changes in MtrA under different phosphorylated conditions, which might help in designing novel therapeutics against tuberculosis.

Nuclear Receptor Interdomain Communication is Mediated by the Hinge with Ligand Specificity

Nuclear receptors are ligand-induced transcription factors that bind directly to target genes and regulate their expression. Ligand binding initiates conformational changes that propagate to other domains, allosterically regulating their activity. The nature of this interdomain communication in nuclear receptors is poorly understood, largely owing to the difficulty of experimentally characterizing full-length structures. We have applied computational modeling approaches to describe and study the structure of the full-length farnesoid X receptor (FXR), approximated by the DNA binding domain (DBD) and ligand binding domain (LBD) connected by the flexible hinge region. Using extended molecular dynamics simulations (>10 microseconds) and enhanced sampling simulations, we provide evidence that ligands selectively induce domain rearrangement, leading to interdomain contact. We use protein–protein interaction assays to provide experimental evidence of these interactions, identifying a critical role of the hinge in mediating interdomain contact. Our results illuminate previously unknown aspects of interdomain communication in FXR and provide a framework to enable characterization of other full-length nuclear receptors.

The impact of the carbohydrate-binding module on how a lytic polysaccharide monooxygenase modifies cellulose fibers

BackgroundIn recent years, lytic polysaccharide monooxygenases (LPMOs) that oxidatively cleave cellulose have gained increasing attention in cellulose fiber modification. LPMOs are relatively small copper-dependent redox enzymes that occur as single domain proteins but may also contain an appended carbohydrate-binding module (CBM). Previous studies have indicated that the CBM “immobilizes” the LPMO on the substrate and thus leads to more localized oxidation of the fiber surface. Still, our understanding of how LPMOs and their CBMs modify cellulose fibers remains limited.ResultsHere, we studied the impact of the CBM on the fiber-modifying properties of NcAA9C, a two-domain family AA9 LPMO from Neurospora crassa, using both biochemical methods as well as newly developed multistep fiber dissolution methods that allow mapping LPMO action across the fiber, from the fiber surface to the fiber core. The presence of the CBM in NcAA9C improved binding towards amorphous (PASC), natural (Cell I), and alkali-treated (Cell II) cellulose, and the CBM was essential for significant binding of the non-reduced LPMO to Cell I and Cell II. Substrate binding of the catalytic domain was promoted by reduction, allowing the truncated CBM-free NcAA9C to degrade Cell I and Cell II, albeit less efficiently and with more autocatalytic enzyme degradation compared to the full-length enzyme. The sequential dissolution analyses showed that cuts by the CBM-free enzyme are more evenly spread through the fiber compared to the CBM-containing full-length enzyme and showed that the truncated enzyme can penetrate deeper into the fiber, thus giving relatively more oxidation and cleavage in the fiber core.ConclusionsThese results demonstrate the capability of LPMOs to modify cellulose fibers from surface to core and reveal how variation in enzyme modularity can be used to generate varying cellulose-based materials. While the implications of these findings for LPMO-based cellulose fiber engineering remain to be explored, it is clear that the presence of a CBM is an important determinant of the three-dimensional distribution of oxidation sites in the fiber.

NMR Dynamic View of the Stabilization of the WW4 Domain by Neutral NaCl and Kosmotropic Na2SO4 and NaH2PO4.

The Hofmeister series categorizes ions based on their effects on protein stability, yet the microscopic mechanism remains a mystery. In this series, NaCl is neutral, Na2SO4 and Na2HPO4 are kosmotropic, while GdmCl and NaSCN are chaotropic. This study employs CD and NMR to investigate the effects of NaCl, Na2SO4, and Na2HPO4 on the conformation, stability, binding, and backbone dynamics (ps-ns and µs-ms time scales) of the WW4 domain with a high stability and accessible side chains at concentrations ≤ 200 mM. The results indicated that none of the three salts altered the conformation of WW4 or showed significant binding to the four aliphatic hydrophobic side chains. NaCl had no effect on its thermal stability, while Na2SO4 and Na2HPO4 enhanced the stability by ~5 °C. Interestingly, NaCl only weakly interacted with the Arg27 amide proton, whereas Na2SO4 bound to Arg27 and Phe31 amide protons with Kd of 32.7 and 41.6 mM, respectively. Na2HPO4, however, bound in a non-saturable manner to Trp9, His24, and Asn36 amide protons. While the three salts had negligible effects on ps-ns backbone dynamics, NaCl and Na2SO4 displayed no effect while Na2HPO4 significantly increased the µs-ms backbone dynamics. These findings, combined with our recent results with GdmCl and NaSCN, suggest a microscopic mechanism for the Hofmeister series. Additionally, the data revealed a lack of simple correlation between thermodynamic stability and backbone dynamics, most likely due to enthalpy-entropy compensation. Our study rationalizes the selection of chloride and phosphate as the primary anions in extracellular and intracellular spaces, as well as polyphosphate as a primitive chaperone in certain single-cell organisms.

Ionizable networks mediate pH-dependent allostery in SH2 signaling proteins.

Transient intracellular pH dynamics 1 regulate mammalian proliferation 2,3 , migration 4 , and differentiation 5 . However, for many pH-dependent cell processes, the molecular mediators are unknown 6 . Prior work identified histidine residues as molecular switches in pH-sensitive proteins, but how other ionizable residues contribute to pH-dependent protein allostery is understudied. Here, we develop an in silico computational pipeline to identify putative pH-sensitive proteins and their molecular mechanisms. We first apply this pipeline to SHP2, a known pH-sensitive signaling protein with an uncharacterized molecular mechanism. We show wild-type SHP2 phosphatase activity is pH-sensitive in vitro and in cells, and mutation of identified H116 and E252 to non-titratable alanine residues abolishes pH-sensitive function. We also show that c-Src is a previously unrecognized pH-dependent kinase, and mutation of the identified ionizable network again abolishes pH-sensitive activity. Constant pH molecular dynamics simulations support a conserved allosteric mechanism of pH-dependent binding of inhibitory SH2 domains to the functional catalytic domains of SHP2 and c-Src. We apply our computational pipeline across SH2 domain-containing signaling proteins and identify evolutionarily conserved putative pH-sensing networks. Our results reveal that pH is an allosteric regulator of SH2 domain-containing signaling proteins providing insight into normal pH-dependent cell biology and diseases where pHi is dysregulated, such as cancer.

Exploring the Ligand Binding and Conformational Dynamics of the Substrate-Binding Domain 1 of the ABC Transporter GlnPQ.

The adenosine triphosphate (ATP)-binding cassette (ABC) importer GlnPQ from Lactococcus lactis has two sequential covalently linked substrate-binding domains (SBDs), which capture the substrates and deliver them to the translocon. The two SBDs differ in their ligand specificities, binding affinities and the distance to the transmembrane domain; interestingly, both SBDs can bind their ligands simultaneously without affecting each other. In this work, we studied the binding of ligands to both SBDs using X-ray crystallography and molecular dynamics simulations. We report three high-resolution structures of SBD1, namely, the wild-type SBD1 with bound asparagine or arginine, and E184D SBD1 with glutamine bound. Molecular dynamics (MD) simulations provide a detailed insight into the dynamics associated with open-closed transitions of the SBDs.

Evolution of Einkorn wheat centromeres is driven by the mutualistic interplay of two LTR retrotransposons

BackgroundCentromere function is highly conserved across eukaryotes, but the underlying centromeric DNA sequences vary dramatically between species. Centromeres often contain a high proportion of repetitive DNA, such as tandem repeats and/or transposable elements (TEs). Einkorn wheat centromeres lack tandem repeat arrays and are instead composed mostly of the two long terminal repeat (LTR) retrotransposon families RLG_Cereba and RLG_Quinta which specifically insert in centromeres. However, it is poorly understood how these two TE families relate to each other and if and how they contribute to centromere function and evolution.ResultsBased on conservation of diagnostic motifs (LTRs, integrase and primer binding site and polypurine-tract), we propose that RLG_Cereba and RLG_Quinta are a pair of autonomous and non-autonomous partners, in which the autonomous RLG_Cereba contributes all the proteins required for transposition, while the non-autonomous RLG_Quinta contributes GAG protein. Phylogenetic analysis of predicted GAG proteins showed that the RLG_Cereba lineage was present for at least 100 million years in monocotyledon plants. In contrast, RLG_Quinta evolved from RLG_Cereba between 28 and 35 million years ago in the common ancestor of oat and wheat. Interestingly, the integrase of RLG_Cereba is fused to a so-called CR-domain, which is hypothesized to guide the integrase to the functional centromere. Indeed, ChIP-seq data and TE population analysis show only the youngest subfamilies of RLG_Cereba and RLG_Quinta are found in the active centromeres. Importantly, the LTRs of RLG_Quinta and RLG_Cereba are strongly associated with the presence of the centromere-specific CENH3 histone variant. We hypothesize that the LTRs of RLG_Cereba and RLG_Quinta contribute to wheat centromere integrity by phasing and/or placing CENH3 nucleosomes, thus favoring their persistence in the competitive centromere-niche.ConclusionOur data show that RLG_Cereba cross-mobilizes the non-autonomous RLG_Quinta retrotransposons. New copies of both families are specifically integrated into functional centromeres presumably through direct binding of the integrase CR domain to CENH3 histone variants. The LTRs of newly inserted RLG_Cereba and RLG_Quinta elements, in turn, recruit and/or phase new CENH3 deposition. This mutualistic interplay between the two TE families and the plant host dynamically maintains wheat centromeres.

Chaperone BiP controls ER stress sensor Ire1 through interactions with its oligomers.

The complex multistep activation cascade of Ire1 involves changes in the Ire1 conformation and oligomeric state. Ire1 activation enhances ER folding capacity, in part by overexpressing the ER Hsp70 molecular chaperone BiP; in turn, BiP provides tight negative control of Ire1 activation. This study demonstrates that BiP regulates Ire1 activation through a direct interaction with Ire1 oligomers. Particularly, we demonstrated that the binding of Ire1 luminal domain (LD) to unfolded protein substrates not only trigger conformational changes in Ire1-LD that favour the formation of Ire1-LD oligomers but also exposes BiP binding motifs, enabling the molecular chaperone BiP to directly bind to Ire1-LD in an ATP-dependent manner. These transient interactions between BiP and two short motifs in the disordered region of Ire1-LD are reminiscent of interactions between clathrin and another Hsp70, cytoplasmic Hsc70. BiP binding to substrate-bound Ire1-LD oligomers enables unfolded protein substrates and BiP to synergistically and dynamically control Ire1-LD oligomerisation, helping to return Ire1 to its deactivated state when an ER stress response is no longer required.

Structure-Activity Relationship of Oxacyclo- and Triazolo-Containing Respiratory Syncytial Virus Polymerase Inhibitors.

Despite the availability of medicines preventing respiratory syncytial virus (RSV) infection, post-exposure treatment options are needed for addressing patient's needs. RSV non-nucleoside polymerase inhibitors (NNI) have emerged as a promising asset for which our group previously disclosed JNJ-8003 with potent in vitro antiviral activity and pronounced in vivo efficacy. In this work, a structural-guided design to modify the linker vector of JNJ-8003 resulted in the identification of 2-oxacyclo pyridine-containing derivatives whose various ring closing strategies are described. In addition, bioisosteric replacement of an amide bond with triazole retained potency, and cryo-electron microscopy (cryo-EM) confirmed binding in the capping domain. Subsequent NMR conformational analysis suggested a correlation between the potency and conformations. Our efforts have fulfilled the aim of identifying linker modifications with maintained biological activity while enriching structural diversity and allowing modulations of other parameters.

The fork protection complex promotes parental histone recycling and epigenetic memory

The inheritance of parental histones across the replication fork is thought to mediate epigenetic memory. Here, we reveal that fission yeast Mrc1 (CLASPIN in humans) binds H3-H4 tetramers and operates as a central coordinator of symmetric parental histone inheritance. Mrc1 mutants in a key connector domain disrupted segregation of parental histones to the lagging strand comparable to Mcm2 histone-binding mutants. Both mutants showed clonal and asymmetric loss of H3K9me-mediated gene silencing. AlphaFold predicted co-chaperoning of H3-H4 tetramers by Mrc1 and Mcm2, with the Mrc1 connector domain bridging histone and Mcm2 binding. Biochemical and functional analysis validated this model and revealed a duality in Mrc1 function: disabling histone binding in the connector domain disrupted lagging-strand recycling while another histone-binding mutation impaired leading strand recycling. We propose that Mrc1 toggles histones between the lagging and leading strand recycling pathways, in part by intra-replisome co-chaperoning, to ensure epigenetic transmission to both daughter cells.

Expression and molecular insights of lima1 in cholangiocarcinoma

ABSTRACT Lim Domain and Actin Binding protein1 (lima1) influence cancer cell function. Thus far, functional role of lima1 in cholangiocarcinoma remains unknown. We used public databases, in vitro experiments, and multi-omics analysis to investigate the Lima1 in cholangiocarcinoma. Our results showed that lima1 expression is significantly upregulated and high levels of lima1 are significantly associated with vascular invasion in cholangiocarcinoma. Furthermore, lima1 knocking out inhibits the RBE cell invasion. Multi-omics data suggest that lima1 affect a broad spectrum of cancer related pathways, promoting tumor progression and metastatic ability in cholangiocarcinoma. This study provides insights into molecular associations of lima1 with tumorigenesist and establishes a preliminary picture of the correlation network in cholangiocarcinoma.

BCL-2 and BOK regulate apoptosis by interaction of their C-terminal transmembrane domains

The Bcl-2 family controls apoptosis by direct interactions of pro- and anti-apoptotic proteins. The principle mechanism is binding of the BH3 domain of pro-apoptotic proteins to the hydrophobic groove of anti-apoptotic siblings, which is therapeutically exploited by approved BH3-mimetic anti-cancer drugs. Evidence suggests that also the transmembrane domain (TMD) of Bcl-2 proteins can mediate Bcl-2 interactions. We developed a highly-specific split luciferase assay enabling the analysis of TMD interactions of pore-forming apoptosis effectors BAX, BAK, and BOK with anti-apoptotic Bcl-2 proteins in living cells. We confirm homotypic interaction of the BAX-TMD, but also newly identify interaction of the TMD of anti-apoptotic BCL-2 with the TMD of BOK, a peculiar pro-apoptotic Bcl-2 protein. BOK-TMD and BCL-2-TMD interact at the endoplasmic reticulum. Molecular dynamics simulations confirm dynamic BOK-TMD and BCL-2-TMD dimers and stable heterotetramers. Mutation of BCL-2-TMD at predicted key residues abolishes interaction with BOK-TMD. Also, inhibition of BOK-induced apoptosis by BCL-2 depends specifically on their TMDs. Thus, TMDs of Bcl-2 proteins are a relevant interaction interface for apoptosis regulation and provide a novel potential drug target.

A ligand discovery toolbox for the WWE domain family of human E3 ligases

The WWE domain is a relatively under-researched domain found in twelve human proteins and characterized by a conserved tryptophan-tryptophan-glutamate (WWE) sequence motif. Six of these WWE domain-containing proteins also contain domains with E3 ubiquitin ligase activity. The general recognition of poly-ADP-ribosylated substrates by WWE domains suggests a potential avenue for development of Proteolysis-Targeting Chimeras (PROTACs). Here, we present novel crystal structures of the HUWE1, TRIP12, and DTX1 WWE domains in complex with PAR building blocks and their analogs, thus enabling a comprehensive analysis of the PAR binding site structural diversity. Furthermore, we introduce a versatile toolbox of biophysical and biochemical assays for the discovery and characterization of novel WWE domain binders, including fluorescence polarization-based PAR binding and displacement assays, 15N-NMR-based binding affinity assays and 19F-NMR-based competition assays. Through these assays, we have characterized the binding of monomeric iso-ADP-ribose (iso-ADPr) and its nucleotide analogs with the aforementioned WWE proteins. Finally, we have utilized the assay toolbox to screen a small molecule fragment library leading to the successful discovery of novel ligands targeting the HUWE1 WWE domain.