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TANK-binding kinase 1 (TBK1) is a multifunctional kinase with an essential role in mitophagy, the selective clearance of damaged mitochondria. More than 90 distinct mutations in TBK1 are linked to amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia, including missense mutations that disrupt the abilities of TBK1 to dimerize, associate with the mitophagy receptor optineurin (OPTN), autoactivate, or catalyze phosphorylation. We investigated how ALS-associated mutations in TBK1 affect Parkin-dependent mitophagy using imaging to dissect the molecular mechanisms involved in clearing damaged mitochondria. Some mutations cause severe dysregulation of the pathway, while others induce limited disruption. Mutations that abolish either TBK1 dimerization or kinase activity were insufficient to fully inhibit mitophagy, while mutations that reduced both dimerization and kinase activity were more disruptive. Ultimately, both TBK1 recruitment and OPTN phosphorylation at S177 are necessary for engulfment of damaged mitochondra by autophagosomal membranes. Surprisingly, we find that ULK1 activity contributes to the phosphorylation of OPTN in the presence of either wild-type or kinase-inactive TBK1. In primary neurons, TBK1 mutants induce mitochondrial stress under basal conditions; network stress is exacerbated with further mitochondrial insult. Our study further refines the model for TBK1 function in mitophagy, demonstrating that some ALS-linked mutations likely contribute to disease pathogenesis by inducing mitochondrial stress or inhibiting mitophagic flux. Other TBK1 mutations exhibited much less impact on mitophagy in our assays, suggesting that cell-type–specific effects, cumulative damage, or alternative TBK1-dependent pathways such as innate immunity and inflammation also factor into the development of ALS in affected individuals.

Exonic DNA sequence variants in the Tbk1 gene associate with both sporadic and familial amyotrophic lateral sclerosis (ALS). Here, we examine functional defects in 25 missense TBK1 mutations, focusing on kinase activity and protein–protein interactions. We identified kinase domain (KD) mutations that abolish kinase activity or display substrate-specific defects in specific pathways, such as innate immunity and autophagy. By contrast, mutations in the scaffold dimerization domain (SDD) of TBK1 can cause the loss of kinase activity due to structural disruption, despite an intact KD. Familial ALS mutations in ubiquitin-like domain (ULD) or SDD display defects in dimerization; however, a subset retains kinase activity. These observations indicate that TBK1 dimerization is not required for kinase activation. Rather, dimerization seems to increase protein stability and enables efficient kinase–substrate interactions. Our study revealed many aspects of TBK1 activities affected by ALS mutations, highlighting the complexity of disease pathogenicity and providing insights into TBK1 activation mechanism.

The monogenic and monoallelic expression of only one out of >1000 mouse olfactory receptor (ORs) genes requires the formation of large heterochromatic chromatin domains that sequester the OR gene clusters. Within these domains, intergenic transcriptional enhancers evade heterochromatic silencing and converge into interchromosomal hubs that assemble over the transcriptionally active OR. The significance of this nuclear organization in OR choice remains elusive. Here, we show that transcription factors Lhx2 and Ebf specify OR enhancers by binding in a functionally cooperative fashion to stereotypically spaced motifs that defy heterochromatin. Specific displacement of Lhx2 and Ebf from OR enhancers resulted in pervasive, long-range, and trans downregulation of OR transcription, whereas pre-assembly of a multi-enhancer hub increased the frequency of OR choice in cis. Our data provide genetic support for the requirement and sufficiency of interchromosomal interactions in singular OR choice and generate general regulatory principles for stochastic, mutually exclusive gene expression programs.

Chimeric Antigen Receptor T-cell (CAR-T) immunotherapies are dependent upon designed transmembrane proteins to bind target antigens and stimulate an immune response. The success or failure of these CARs is only partially predictable, yet recent work has highlighted the importance of antigen binding scFvs driving distinct oligomerization states with varied CAR-T efficacy. Here, we sought to determine the extracellular structure of the anti-CD19 CAR 47G4-CD828Z. Unexpectedly, the resolved crystal structure revealed an IgVL homodimer bound along an inverted VL|VL interface. We found that the VL-VH linker, designed to be cleavage resistant, was cleaved, and the VH and CAR hinge domains were absent from the crystal structure lattice. Molecular Dynamics simulations revealed that the inverted VL|VL interface was more stable than the canonical VL|VL configuration. Our work substantiates the need to interrogate the scFv structure and CAR oligomerization state for optimal CAR-T design.

In the future, we may be faced with the need to provide treatment for an emergent biological threat against which existing vaccines and drugs have limited efficacy or availability. To prepare for this eventuality, our objective was to use a metabolic network-based approach to rapidly identify potential drug targets and prospectively screen and validate novel small-molecule antimicrobials. Our target organism was the fully virulent Francisella tularensis subspecies tularensis Schu S4 strain, a highly infectious intracellular pathogen that is the causative agent of tularemia and is classified as a category A biological agent by the Centers for Disease Control and Prevention. We proceeded with a staggered computational and experimental workflow that used a strain-specific metabolic network model, homology modeling and X-ray crystallography of protein targets, and ligand- and structure-based drug design. Selected compounds were subsequently filtered based on physiological-based pharmacokinetic modeling, and we selected a final set of 40 compounds for experimental validation of antimicrobial activity. We began screening these compounds in whole bacterial cell-based assays in biosafety level 3 facilities in the 20th week of the study and completed the screens within 12 weeks. Six compounds showed significant growth inhibition of F. tularensis, and we determined their respective minimum inhibitory concentrations and mammalian cell cytotoxicities. The most promising compound had a low molecular weight, was non-toxic, and abolished bacterial growth at 13 µM, with putative activity against pantetheine-phosphate adenylyltransferase, an enzyme involved in the biosynthesis of coenzyme A, encoded by gene coaD. The novel antimicrobial compounds identified in this study serve as starting points for lead optimization, animal testing, and drug development against tularemia. Our integrated in silico/in vitro approach had an overall 15% success rate in terms of active versus tested compounds over an elapsed time period of 32 weeks, from pathogen strain identification to selection and validation of novel antimicrobial compounds.

Significance Mutations in the catalytic subunit of protein kinase A (PKA) have been found in tumors associated with the kidney disorder Cushing’s syndrome and with the rare liver cancer fibrolamellar hepatocellular carcinoma (FL-HCC). Crystal structures and biochemical characterizations of the relevant PKA mutants clarify the molecular basis for disease caused by these mutations. We find contrasting underlying mechanisms for increased PKA signaling in these cancers. In Cushing’s syndrome, the L205R PKA mutation abolishes regulatory-subunit binding, whereas in FL-HCC, the recurring DnaJ–PKA fusion that results from a chromosomal deletion exhibits wild-type characteristics, but is overproduced by a more active promoter. Our findings provide a structural basis for designing selective drugs that may lead to effective treatments for these diseases. The extensively studied cAMP-dependent protein kinase A (PKA) is involved in the regulation of critical cell processes, including metabolism, gene expression, and cell proliferation; consequentially, mis-regulation of PKA signaling is implicated in tumorigenesis. Recent genomic studies have identified recurrent mutations in the catalytic subunit of PKA in tumors associated with Cushing’s syndrome, a kidney disorder leading to excessive cortisol production, and also in tumors associated with fibrolamellar hepatocellular carcinoma (FL-HCC), a rare liver cancer. Expression of a L205R point mutant and a DnaJ–PKA fusion protein were found to be linked to Cushing's syndrome and FL-HCC, respectively. Here we reveal contrasting mechanisms for increased PKA signaling at the molecular level through structural determination and biochemical characterization of the aberrant enzymes. In the Cushing’s syndrome disorder, we find that the L205R mutation abolishes regulatory-subunit binding, leading to constitutive, cAMP-independent signaling. In FL-HCC, the DnaJ–PKA chimera remains under regulatory subunit control; however, its overexpression from the DnaJ promoter leads to enhanced cAMP-dependent signaling. Our findings provide a structural understanding of the two distinct disease mechanisms and they offer a basis for designing effective drugs for their treatment.

Acetylcholinesterase (AChE) is a critical enzyme that regulates neurotransmission by degrading the neurotransmitter acetylcholine in synapses of the nervous system. It is an important target for both therapeutic drugs that treat Alzheimer’s disease and organophosphate (OP) chemical warfare agents that cripple the nervous system and cause death through paralysis. We are exploring a strategy to design compounds that bind tightly at or near a peripheral or P - site near the mouth of the AChE active site gorge and exclude OPs from the active site while interfering minimally with the passage of acetylcholine. However, to target the AChE P-site, much more information must be gathered about the structure-activity relationships of ligands that bind specifically to the P-site. Here, we review our recent reports on two uncharged, natural product inhibitors of AChE, dihydrotanshinone I and territrem B, that have relatively high affinities for the enzyme. We describe an inhibitor competition assay and comment on the structures of these inhibitors in complex with recombinant human acetylcholinesterase as determined by X-ray crystallography. Our results reveal that dihydrotanshinone I binding is specific to only the P-site, while territrem B binding spans the P-site and extends into the acylation or A - site at the base of the gorge.

Contact with the air-water interface can bias the orientation of macromolecules during cryo-EM sample preparation, leading to uneven sample distribution, preferred orientation, and damage to the molecules of interest. To prevent this, we describe a method to encapsulate target proteins within highly hydrophilic, structurally homogeneous, and stable protein shells, which we refer to as \"nanocrates\" for this purpose. Here, we describe packaging, data acquisition, and reconstruction of three proof-of-principle examples, each illuminating a different aspect of the method: apoferritin (ApoF, demonstrating high-resolution), thyroglobulin (Tg, solving a known preferred orientation problem), and 7,8-dihydroneopterin aldolase (DHNA, a structure previously uncharacterized by cryo-EM).

In the future, we may be faced with the need to provide treatment for an emergent biological threat against which existing vaccines and drugs have limited efficacy or availability. To prepare for this eventuality, our objective was to use a metabolic network-based approach to rapidly identify potential drug targets and prospectively screen and validate novel small-molecule antimicrobials. Our target organism was the fully virulent Francisella tularensis subspecies tularensis Schu S4 strain, a highly infectious intracellular pathogen that is the causative agent of tularemia and is classified as a category A biological agent by the Centers for Disease Control and Prevention. We proceeded with a staggered computational and experimental workflow that used a strain-specific metabolic network model, homology modeling and X-ray crystallography of protein targets, and ligand- and structure-based drug design. Selected compounds were subsequently filtered based on physiological-based pharmacokinetic modeling, and we selected a final set of 40 compounds for experimental validation of antimicrobial activity. We began screening these compounds in whole bacterial cell-based assays in biosafety level 3 facilities in the 20th week of the study and completed the screens within 12 weeks. Six compounds showed significant growth inhibition of F. tularensis, and we determined their respective minimum inhibitory concentrations and mammalian cell cytotoxicities. The most promising compound had a low molecular weight, was non-toxic, and abolished bacterial growth at 13 mu M, with putative activity against pantetheine-phosphate adenylyltransferase, an enzyme involved in the biosynthesis of coenzyme A, encoded by gene coaD. The novel antimicrobial compounds identified in this study serve as starting points for lead optimization, animal testing, and drug development against tularemia. Our integrated in silico/in vitro approach had an overall 15% success rate in terms of active versus tested compounds over an elapsed time period of 32 weeks, from pathogen strain identification to selection and validation of novel antimicrobial compounds.