Mammalian transglutaminases, a family of Ca2+-dependent proteins, are implicated in a variety of diseases. For example, celiac disease (CeD) is an autoimmune disorder whose pathogenesis requires transglutaminase 2 (TG2) to deamidate select glutamine residues in diet-derived gluten peptides. Deamidation involves the formation of transient γ-glutamyl thioester intermediates. Recent studies have revealed that in addition to the deamidated gluten peptides themselves, their corresponding thioester intermediates are also pathogenically relevant. A mechanistic understanding of this relevance is hindered by the absence of any structure of Ca2+-bound TG2. We report the X-ray crystallographic structure of human TG2 bound to an inhibitory gluten peptidomimetic and two Ca2+ ions in sites previously designated as S1 and S3. Together with additional structure-guided experiments, this structure provides a mechanistic explanation for how S1 regulates formation of an inhibitory disulfide bond in TG2, while also establishing that S3 is essential for γ-glutamyl thioester formation. Furthermore, our crystallographic findings and associated analyses have revealed that i) two interacting residues, H305 and E363, play a critical role in resolving the thioester intermediate into an isopeptide bond (transamidation) but not in thioester hydrolysis (deamidation); and ii) residues N333 and K176 stabilize preferred TG2 substrates and inhibitors via hydrogen bonding to nonreactive backbone atoms. Overall, the intermediate-state conformer of TG2 reported here represents a superior model to previously characterized conformers for both transition states of the TG2-catalyzed reaction.

The TATA-box binding protein (TBP) is the sole transcription factor common in the initiation complexes of the three major eukaryotic RNA Polymerases (Pol I, II and III). Although TBP is central to transcription by the three RNA Pols in various species, the emergence of TBP paralogs throughout evolution has expanded the complexity in transcription initiation. Furthermore, recent studies have emerged that questioned the centrality of TBP in mammalian cells, particularly in Pol II transcription, but the role of TBP and its paralogs in Pol I transcription remains to be re-evaluated. In this report, we show that in murine embryonic stem cells TBP localizes onto Pol I promoters, whereas the TBP paralog TRF2 only weakly associates to the Spacer Promoter of rDNA, suggesting that it may not be able to replace TBP for Pol I transcription. Importantly, acute TBP depletion does not fully disrupt Pol I occupancy or activity on ribosomal RNA genes, but TBP binding in mitosis leads to efficient Pol I reactivation following cell division. These findings provide a more nuanced role for TBP in Pol I transcription in murine embryonic stem cells.

Development is regulated by coordinated changes in gene expression. Control of these changes in expression is largely governed by the binding of transcription factors to specific regulatory elements. However, the packaging of DNA into chromatin prevents the binding of many transcription factors. Pioneer factors overcome this barrier owing to unique properties that enable them to bind closed chromatin, promote accessibility and, in so doing, mediate binding of additional factors that activate gene expression. Because of these properties, pioneer factors act at the top of gene-regulatory networks and drive developmental transitions. Despite the ability to bind target motifs in closed chromatin, pioneer factors have cell type-specific chromatin occupancy and activity. Thus, developmental context clearly shapes pioneer-factor function. Here, we discuss this reciprocal interplay between pioneer factors and development: how pioneer factors control changes in cell fate and how cellular environment influences pioneer-factor binding and activity.

The binding affinity determination of protein-ligand complexes is a cornerstone of drug design. State-of-the-art techniques are limited by lengthy and expensive processes. Building upon our recently introduced novel screening method utilizing photochemically induced dynamic nuclear polarization (photo-CIDNP) NMR, we provide the methodological framework to determine binding affinities within 5-15 min using 0.1 mg of protein. The accuracy of our method is demonstrated for the affinity constants of peptides binding to a PDZ domain and fragment ligands binding to the protein PIN1. The method can also be extended to measure the affinity of nonphoto-CIDNP-polarizable ligands in competition binding experiments. Finally, we demonstrate a strong correlation between the ligand-reduced signals in photo-CIDNP-based NMR fragment screening and the well-established saturation transfer difference (STD) NMR. Thus, our methodology measures protein-ligand affinities in the micro- to millimolar range in only a few minutes and informs on the binding epitope in a single-scan experiment, opening new avenues for early stage drug discovery approaches.

Precise measurement of the binding activity changes of therapeutic antibodies is important to determine the potential critical quality attributes (CQAs) in developability assessment at the early stage of antibody development. Here, we report a surface plasmon resonance (SPR)-based relative binding activity method, which incorporates both binding affinity and binding response and allows us to determine relative binding activity of antibodies with high accuracy and precision. We applied the SPR-based relative binding activity method in multiple forced degradation studies of antibody developability assessment. The current developability assessment strategy provided comprehensive, precise characterization of antibody binding activity in the stability studies, enabling us to perform correlation analysis and establish the structure-function relationship between relative binding activity and quality attributes. The impact of a given quality attribute on binding activity could be confidently determined without isolating antibody variants. We identified several potential CQAs, including Asp isomerization, Asn deamidation, and fragmentation. Some potential CQAs affected binding affinity of antibody and resulted in a reduction of binding activity. Certain potential CQAs impaired antibody binding to antigen and led to a loss of binding activity. A few potential CQAs could influence both binding affinity and binding response and cause a substantial decrease in antibody binding activity. Specifically, we identified low abundance Asn33 deamidation in the light chain complementarity-determining region as a potential CQA, in which all the stressed antibody samples showed Asn33 deamidation abundances ranging from 4.2% to 27.5% and a mild binding affinity change from 1.76 nM to 2.16 nM.

BACKGROUND: We have previously identified an unsuspected role for GJB3 showing that the deficiency of this connexin protein induces aneuploidy in human and murine cells and accelerates cell transformation as well as tumor formation in xenograft models. The molecular mechanisms by which loss of GJB3 leads to aneuploidy and cancer initiation and progression remain unsolved.
METHODS: GJB3 expression levels were determined by RT-qPCR and Western blot. The consequences of GJB3 knockdown on genome instability were assessed by metaphase chromosome counting, multinucleation of cells, by micronuclei formation and by the determination of spindle orientation. Interactions of GJB3 with α-tubulin and F-actin was analyzed by immunoprecipitation and immunocytochemistry. Consequences of GJB3 deficiency on microtubule and actin dynamics were measured by live cell imaging and fluorescence recovery after photobleaching experiments, respectively. Immunohistochemistry was used to determine GJB3 levels on human and murine bladder cancer tissue sections. Bladder cancer in mice was chemically induced by BBN-treatment.
RESULTS: We find that GJB3 is highly expressed in the ureter and bladder epithelium, but it is downregulated in invasive bladder cancer cell lines and during tumor progression in both human and mouse bladder cancer. Downregulation of GJB3 expression leads to aneuploidy and genomic instability in karyotypically stable urothelial cells and experimental modulation of GJB3 levels alters the migration and invasive capacity of bladder cancer cell lines. Importantly, GJB3 interacts both with α-tubulin and F-actin. The impairment of these interactions alters the dynamics of these cytoskeletal components and leads to defective spindle orientation.
CONCLUSIONS: We conclude that deregulated microtubule and actin dynamics have an impact on proper chromosome separation and tumor cell invasion and migration. Consequently, these observations indicate a possible role for GJB3 in the onset and spreading of bladder cancer and demonstrate a molecular link between enhanced aneuploidy and invasive capacity cancer cells during tumor cell dissemination.

Chemical probes are an indispensable tool for translating biological discoveries into new therapies, though are increasingly difficult to identify since novel therapeutic targets are often hard-to-drug proteins. We introduce FRASE-based hit-finding robot (FRASE-bot), to expedite drug discovery for unconventional therapeutic targets. FRASE-bot mines available 3D structures of ligand-protein complexes to create a database of FRAgments in Structural Environments (FRASE). The FRASE database can be screened to identify structural environments similar to those in the target protein and seed the target structure with relevant ligand fragments. A neural network model is used to retain fragments with the highest likelihood of being native binders. The seeded fragments then inform ultra-large-scale virtual screening of commercially available compounds. We apply FRASE-bot to identify ligands for Calcium and Integrin Binding protein 1 (CIB1), a promising drug target implicated in triple negative breast cancer. FRASE-based virtual screening identifies a small-molecule CIB1 ligand (with binding confirmed in a TR-FRET assay) showing specific cell-killing activity in CIB1-dependent cancer cells, but not in CIB1-depletion-insensitive cells.

Vitamin C plays important roles as a cofactor in many enzymatic reactions and as an antioxidant against oxidative stress. As some mammals including humans cannot synthesize vitamin C de novo from glucose, its uptake from dietary sources is essential, and is mediated by the sodium-dependent vitamin C transporter 1 (SVCT1). Despite its physiological significance in maintaining vitamin C homeostasis, the structural basis of the substrate transport mechanism remained unclear. Here, we report the cryo-EM structures of human SVCT1 in different states at 2.5-3.5 Å resolutions. The binding manner of vitamin C together with two sodium ions reveals the counter ion-dependent substrate recognition mechanism. Furthermore, comparisons of the inward-open and occluded structures support a transport mechanism combining elevator and distinct rotational motions. Our results demonstrate the molecular mechanism of vitamin C transport with its underlying conformational cycle, potentially leading to future industrial and medical applications.

Damage to renal tubular epithelial cells (RTECs) signaled the onset and progression of sepsis-associated acute kidney injury (SA-AKI). Recent research on mitochondria has revealed that mitophagy plays a crucial physiological role in alleviating injury to RTECs and it is suppressed progressively by the inflammation response in SA-AKI. However, the mechanism by which inflammation influences mitophagy remains poorly understood. We examined how macrophage migration inhibitory factor (MIF), a pro-inflammatory protein, influences the PINK1-Parkin pathway of mitophagy by studying protein-protein interactions when MIF was inhibited or overexpressed. Surprisingly, elevated levels of MIF were found to directly bind to PINK1, disrupting its interaction with Parkin. This interference hindered the recruitment of Parkin to mitochondria and impeded the initiation of mitophagy. Furthermore, this outcome led to significant apoptosis of RTECs, which could, however, be reversed by an MIF inhibitor ISO-1 and/or a new mitophagy activator T0467. These findings highlight the detrimental impact of MIF on renal damage through its disruption of the interaction between PINK1 and Parkin, and the therapeutic potential of ISO-1 and T0467 in mitigating SA-AKI. This study offers a fresh perspective on treating SA-AKI by targeting MIF and mitophagy.

Mutations in the microtubule-associated motor protein KIF1A lead to severe neurological conditions known as KIF1A-associated neurological disorders (KAND). Despite insights into its molecular mechanism, high-resolution structures of KIF1A-microtubule complexes remain undefined. Here, we present 2.7-3.5 Å resolution structures of dimeric microtubule-bound KIF1A, including the pathogenic P305L mutant, across various nucleotide states. Our structures reveal that KIF1A binds microtubules in one- and two-heads-bound configurations, with both heads exhibiting distinct conformations with tight inter-head connection. Notably, KIF1A\'s class-specific loop 12 (K-loop) forms electrostatic interactions with the C-terminal tails of both α- and β-tubulin. The P305L mutation does not disrupt these interactions but alters loop-12\'s conformation, impairing strong microtubule-binding. Structure-function analysis reveals the K-loop and head-head coordination as major determinants of KIF1A\'s superprocessive motility. Our findings advance the understanding of KIF1A\'s molecular mechanism and provide a basis for developing structure-guided therapeutics against KAND.