DNA polymerases are today used throughout scientific research, biotechnology, and medicine, in part for their ability to interact with unnatural forms of DNA created by synthetic biologists. Here especially, natural DNA polymerases often do not have the “performance specifications” needed for transformative technologies. This creates a need for science-guided rational (or semi-rational) engineering to identify variants that replicate unnatural base pairs (UBPs), unnatural backbones, tags, or other evolutionarily novel features of unnatural DNA. In this review, we provide a brief overview of the chemistry and properties of replicative DNA polymerases and their evolved variants, focusing on the Klenow fragment of Taq DNA polymerase (Klentaq). We describe comparative structural, enzymatic, and molecular dynamics studies of WT and Klentaq variants, complexed with natural or noncanonical substrates. Combining these methods provides insight into how specific amino acid substitutions distant from the active site in a Klentaq DNA polymerase variant (ZP Klentaq) contribute to its ability to replicate UBPs with improved efficiency compared with Klentaq. This approach can therefore serve to guide any future rational engineering of replicative DNA polymerases.

RecQ family helicases are highly conserved from bacteria to humans and have essential roles in maintaining genome stability. Mutations in three human RecQ helicases cause severe diseases with the main features of premature aging and cancer predisposition. Most RecQ helicases shared a conserved domain arrangement which comprises a helicase core, an RecQ C-terminal domain, and an auxiliary element helicase and RNaseD C-terminal (HRDC) domain, the functions of which are poorly understood. In this study, we systematically characterized the roles of the HRDC domain in E. coli RecQ in various DNA transactions by single-molecule FRET. We found that RecQ repetitively unwinds the 3'-partial duplex and fork DNA with a moderate processivity and periodically patrols on the ssDNA in the 5'-partial duplex by translocation. The HRDC domain significantly suppresses RecQ activities in the above transactions. In sharp contrast, the HRDC domain is essential for the deep and long-time unfolding of the G4 DNA structure by RecQ. Based on the observations that the HRDC domain dynamically switches between RecA core- and ssDNA-binding modes after RecQ association with DNA, we proposed a model to explain the modulation mechanism of the HRDC domain. Our findings not only provide new insights into the activities of RecQ on different substrates but also highlight the novel functions of the HRDC domain in DNA metabolisms.

Rhodopsin is a canonical class A photosensitive G protein–coupled receptor (GPCR), yet relatively few pharmaceutical agents targeting this visual receptor have been identified, in part due to the unique characteristics of its light-sensitive, covalently bound retinal ligands. Rhodopsin becomes activated when light isomerizes 11-cis-retinal into an agonist, all-trans-retinal (ATR), which enables the receptor to activate its G protein. We have previously demonstrated that, despite being covalently bound, ATR can display properties of equilibrium binding, yet how this is accomplished is unknown. Here, we describe a new approach for both identifying compounds that can activate and attenuate rhodopsin and testing the hypothesis that opsin binds retinal in equilibrium. Our method uses opsin-based fluorescent sensors, which directly report the formation of active receptor conformations by detecting the binding of G protein or arrestin fragments that have been fused onto the receptor's C terminus. We show that these biosensors can be used to monitor equilibrium binding of the agonist, ATR, as well as the noncovalent binding of β-ionone, an antagonist for G protein activation. Finally, we use these novel biosensors to observe ATR release from an activated, unlabeled receptor and its subsequent transfer to the sensor in real time. Taken together, these data support the retinal equilibrium binding hypothesis. The approach we describe should prove directly translatable to other GPCRs, providing a new tool for ligand discovery and mutant characterization.

Pyruvate kinase muscle isoform 2 (PKM2) is a key glycolytic enzyme and transcriptional coactivator and is critical for tumor metabolism. In cancer cells, native tetrameric PKM2 is phosphorylated or acetylated, which initiates a switch to a dimeric/monomeric form that translocates into the nucleus, causing oncogene transcription. However, it is not known how these post-translational modifications (PTMs) disrupt the oligomeric state of PKM2. We explored this question via crystallographic and biophysical analyses of PKM2 mutants containing residues that mimic phosphorylation and acetylation. We find that the PTMs elicit major structural reorganization of the fructose 1,6-bisphosphate (FBP), an allosteric activator, binding site, impacting the interaction with FBP and causing a disruption in oligomerization. To gain insight into how these modifications might cause unique outcomes in cancer cells, we examined the impact of increasing the intracellular pH (pHi) from ∼7.1 (in normal cells) to ∼7.5 (in cancer cells). Biochemical studies of WT PKM2 (wtPKM2) and the two mimetic variants demonstrated that the activity decreases as the pH is increased from 7.0 to 8.0, and wtPKM2 is optimally active and amenable to FBP-mediated allosteric regulation at pHi 7.5. However, the PTM mimetics exist as a mixture of tetramer and dimer, indicating that physiologically dimeric fraction is important and might be necessary for the modified PKM2 to translocate into the nucleus. Thus, our findings provide insight into how PTMs and pH regulate PKM2 and offer a broader understanding of its intricate allosteric regulation mechanism by phosphorylation or acetylation.

Defining discontinuous antigenic epitopes remains a substantial challenge, as exemplified by the case of lipid transfer polyproteins, which are common pollen allergens. Hydrogen/deuterium exchange monitored by NMR can be used to map epitopes onto folded protein surfaces, but only if the complex rapidly dissociates. Modifying the standard NMR-exchange measurement to detect substoichiometric complexes overcomes this time scale limitation and provides new insights into recognition of lipid transfer polyprotein by antibodies. In the future, this new and exciting development should see broad application to a range of tight macromolecular interactions.

Myosins generate force and motion by precisely coordinating their mechanical and chemical cycles, but the nature and timing of this coordination remains controversial. We utilized a FRET approach to examine the kinetics of structural changes in the force-generating lever arm in myosin V. We directly compared the FRET results with single-molecule mechanical events examined by optical trapping. We introduced a mutation (S217A) in the conserved switch I region of the active site to examine how myosin couples structural changes in the actin- and nucleotide-binding regions with force generation. Specifically, S217A enhanced the maximum rate of lever arm priming (recovery stroke) while slowing ATP hydrolysis, demonstrating that it uncouples these two steps. We determined that the mutation dramatically slows both actin-induced rotation of the lever arm (power stroke) and phosphate release (≥10-fold), whereas our simulations suggest that the maximum rate of both steps is unchanged by the mutation. Time-resolved FRET revealed that the structure of the pre– and post–power stroke conformations and mole fractions of these conformations were not altered by the mutation. Optical trapping results demonstrated that S217A does not dramatically alter unitary displacements or slow the working stroke rate constant, consistent with the mutation disrupting an actin-induced conformational change prior to the power stroke. We propose that communication between the actin- and nucleotide-binding regions of myosin assures a proper actin-binding interface and active site have formed before producing a power stroke. Variability in this coupling is likely crucial for mediating motor-based functions such as muscle contraction and intracellular transport.

Centrioles are key eukaryotic organelles that are responsible for the formation of cilia and flagella, and for organizing the microtubule network and the mitotic spindle in animals. Centriole assembly requires oligomerization of the essential protein spindle assembly abnormal 6 (SAS-6), which forms a structural scaffold templating the organization of further organelle components. A dimerization interaction between SAS-6 N-terminal “head” domains was previously shown to be essential for protein oligomerization in vitro and for function in centriole assembly. Here, we developed a pharmacophore model allowing us to assemble a library of low-molecular-weight ligands predicted to bind the SAS-6 head domain and inhibit protein oligomerization. We demonstrate using NMR spectroscopy that a ligand from this family binds at the head domain dimerization site of algae, nematode, and human SAS-6 variants, but also that another ligand specifically recognizes human SAS-6. Atomistic molecular dynamics simulations starting from SAS-6 head domain crystallographic structures, including that of the human head domain which we now resolve, suggest that ligand specificity derives from favorable Van der Waals interactions with a hydrophobic cavity at the dimerization site.

RNA-protein interfaces control key replication events during the HIV-1 life cycle. The viral trans-activator of transcription (Tat) protein uses an archetypal arginine-rich motif (ARM) to recruit the host positive transcription elongation factor b (pTEFb) complex onto the viral trans-activation response (TAR) RNA, leading to activation of HIV transcription. Efforts to block this interaction have stimulated production of biologics designed to disrupt this essential RNA-protein interface. Here, we present four co-crystal structures of lab-evolved TAR-binding proteins (TBPs) in complex with HIV-1 TAR. Our results reveal that high-affinity binding requires a distinct sequence and spacing of arginines within a specific β2-β3 hairpin loop that arose during selection. Although loops with as many as five arginines were analyzed, only three arginines could bind simultaneously with major-groove guanines. Amino acids that promote backbone interactions within the β2-β3 loop were also observed to be important for high-affinity interactions. Based on structural and affinity analyses, we designed two cyclic peptide mimics of the TAR-binding β2-β3 loop sequences present in two high-affinity TBPs (KD values of 4.2 ± 0.3 and 3.0 ± 0.3 nm). Our efforts yielded low-molecular weight compounds that bind TAR with low micromolar affinity (KD values ranging from 3.6 to 22 μm). Significantly, one cyclic compound within this series blocked binding of the Tat-ARM peptide to TAR in solution assays, whereas its linear counterpart did not. Overall, this work provides insight into protein-mediated TAR recognition and lays the ground for the development of cyclic peptide inhibitors of a vital HIV-1 RNA-protein interaction.

Erythromycin-resistance methyltransferases are SAM dependent Rossmann fold methyltransferases that convert A2058 of 23S rRNA to m6 2A2058. This modification sterically blocks binding of several classes of antibiotics to 23S rRNA, resulting in a multidrug-resistant phenotype in bacteria expressing the enzyme. ErmC is an erythromycin resistance methyltransferase found in many Gram-positive pathogens, whereas ErmE is found in the soil bacterium that biosynthesizes erythromycin. Whether ErmC and ErmE, which possess only 24% sequence identity, use similar structural elements for rRNA substrate recognition and positioning is not known. To investigate this question, we used structural data from related proteins to guide site-saturation mutagenesis of key residues and characterized selected variants by antibiotic susceptibility testing, single turnover kinetics, and RNA affinity–binding assays. We demonstrate that residues in α4, α5, and the α5-α6 linker are essential for methyltransferase function, including an aromatic residue on α4 that likely forms stacking interactions with the substrate adenosine and basic residues in α5 and the α5-α6 linker that likely mediate conformational rearrangements in the protein and cognate rRNA upon interaction. The functional studies led us to a new structural model for the ErmC or ErmE-rRNA complex.

We are extremely excited to announce on behalf of the American Society for Biochemistry and Molecular Biology (ASBMB) that the Journal of Biological Chemistry (JBC), Molecular & Cellular Proteomics (MCP), and the Journal of Lipid Research (JLR) will be published as fully open-access journals beginning in January 2021. This is a landmark decision that will have huge impact for readers and authors. As many of you know, many researchers have called for journals to become open access to facilitate scientific progress, and many funding agencies across the globe are either already requiring or considering a requirement that all scientific publications based on research they support be published in open-access journals. The ASBMB journals have long supported open access, making the accepted author versions of manuscripts immediately and permanently available, allowing authors to opt in to the immediate open publication of the final version of their paper, and endorsing the goals of the larger open-access movement (1). However, we are no longer satisfied with these measures. To live up to our goals as a scientific society, we want to freely distribute the scientific advances published in JBC, MCP, and JLR as widely and quickly as possible to support the scientific community. How better can we facilitate the dissemination of new information than to make our scientific content freely open to all?For ASBMB journals and others who have contemplated or made the transition to publishing all content open access, achieving this milestone generally requires new financial mechanisms. In the case of the...

Christian Weiss
Theor. Probability and Math. Statist. 111 (), 199-209.
Abstract, references and article information

Marko Voutilainen, Lauri Viitasaari and Pauliina Ilmonen
Theor. Probability and Math. Statist. 111 (), 181-197.
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Denise Uwamariya and Xiangfeng Yang
Theor. Probability and Math. Statist. 111 (), 167-179.
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Ta Cong Son, Tran Manh Cuong, Le Quang Dung and Le Van Dung
Theor. Probability and Math. Statist. 111 (), 153-165.
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Bero Roos
Theor. Probability and Math. Statist. 111 (), 137-151.
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Olha Prykhodko and Kostiantyn Ralchenko
Theor. Probability and Math. Statist. 111 (), 123-135.
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Lutz Mattner
Theor. Probability and Math. Statist. 111 (), 45-122.
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Claudio Macci and Barbara Pacchiarotti
Theor. Probability and Math. Statist. 111 (), 21-43.
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Chris A.J. Klaassen
Theor. Probability and Math. Statist. 111 (), 9-19.
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Abdessamad Dehaj and Mohamed Guessous
Theor. Probability and Math. Statist. 111 (), 1-8.
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Roland Donninger
Bull. Amer. Math. Soc. 61 (), 659-685.
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Alin Bostan, Xavier Caruso and Julien Roques
Bull. Amer. Math. Soc. 61 (), 609-658.
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Mohammed Abouzaid and Nathaniel Bottman
Bull. Amer. Math. Soc. 61 (), 525-608.
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The two branches of the Kennedy pathways (CDP-choline and CDP-ethanolamine) are the predominant pathways responsible for the synthesis of the most abundant phospholipids, phosphatidylcholine and phosphatidylethanolamine, respectively, in mammalian membranes. Recently, hereditary diseases associated with single gene mutations in the Kennedy pathways have been identified. Interestingly, genetic diseases within the same pathway vary greatly, ranging from muscular dystrophy to spastic paraplegia to a childhood blinding disorder to bone deformations. Indeed, different point mutations in the same gene (PCYT1; CCTα) result in at least three distinct diseases. In this review, we will summarize and review the genetic diseases associated with mutations in genes of the Kennedy pathway for phospholipid synthesis. These single-gene disorders provide insight, indeed direct genotype-phenotype relationships, into the biological functions of specific enzymes of the Kennedy pathway. We discuss potential mechanisms of how mutations within the same pathway can cause disparate disease.

Recent studies have suggested that innate immune responses exhibit characteristics associated with memory linked to modulations in both vertebrates and invertebrates. However, the diverse evolutionary paths taken, particularly within the invertebrate taxa, should lead to similarly diverse innate immunity memory processes. Our understanding of innate immune memory in invertebrates primarily comes from studies of the fruit fly Drosophila melanogaster, the generality of which is unclear. Caenorhabditis elegans typically inhabits soil harboring a variety of fatal microbial pathogens; for this invertebrate, the innate immune system and aversive behavior are the major defensive strategies against microbial infection. However, their characteristics of immunological memory remains infantile. Here we discovered an immunological memory that promoted avoidance and suppressed innate immunity during reinfection with bacteria, which we revealed to be specific to the previously exposed pathogens. During this trade-off switch of avoidance and innate immunity, the chemosensory neurons AWB and ADF modulated production of serotonin and dopamine, which in turn decreased expression of the innate immunity-associated genes and led to enhanced avoidance via the downstream insulin-like pathway. Therefore, our current study profiles the immune memories during C. elegans reinfected by pathogenic bacteria and further reveals that the chemosensory neurons, the neurotransmitter(s), and their associated molecular signaling pathways are responsible for a trade-off switch between the two immunological memories.

Animals can sense the presence of microbes in their tissues and mobilize their own defenses by recognizing and responding to conserved microbial structures (often called microbe-associated molecular patterns (MAMPs)). Successful host defenses may kill the invaders, yet the host animal may fail to restore homeostasis if the stimulatory microbial structures are not silenced. Although mice have many mechanisms for limiting their responses to lipopolysaccharide (LPS), a major Gram-negative bacterial MAMP, a highly conserved host lipase is required to extinguish LPS sensing in tissues and restore homeostasis. We review recent progress in understanding how this enzyme, acyloxyacyl hydrolase (AOAH), transforms LPS from stimulus to inhibitor, reduces tissue injury and death from infection, prevents prolonged post-infection immunosuppression, and keeps stimulatory LPS from entering the bloodstream. We also discuss how AOAH may increase sensitivity to pulmonary allergens. Better appreciation of how host enzymes modify LPS and other MAMPs may help prevent tissue injury and hasten recovery from infection.

Knockout mouse models have been extensively used to study the antiviral activity of IFIT (interferon-induced protein with tetratricopeptide repeats). Human IFIT1 binds to cap0 (m7GpppN) RNA, which lacks methylation on the first and second cap-proximal nucleotides (cap1, m7GpppNm, and cap2, m7GpppNmNm, respectively). These modifications are signatures of “self” in higher eukaryotes, whereas unmodified cap0-RNA is recognized as foreign and, therefore, potentially harmful to the host cell. IFIT1 inhibits translation at the initiation stage by competing with the cap-binding initiation factor complex, eIF4F, restricting infection by certain viruses that possess “nonself” cap0-mRNAs. However, in mice and other rodents, the IFIT1 orthologue has been lost, and the closely related Ifit1b has been duplicated twice, yielding three paralogues: Ifit1, Ifit1b, and Ifit1c. Although murine Ifit1 is similar to human IFIT1 in its cap0-RNA–binding selectivity, the roles of Ifit1b and Ifit1c are unknown. Here, we found that Ifit1b preferentially binds to cap1-RNA, whereas binding is much weaker to cap0- and cap2-RNA. In murine cells, we show that Ifit1b can modulate host translation and restrict WT mouse coronavirus infection. We found that Ifit1c acts as a stimulatory cofactor for both Ifit1 and Ifit1b, promoting their translation inhibition. In this way, Ifit1c acts in an analogous fashion to human IFIT3, which is a cofactor to human IFIT1. This work clarifies similarities and differences between the human and murine IFIT families to facilitate better design and interpretation of mouse models of human infection and sheds light on the evolutionary plasticity of the IFIT family.

Reliable, specific polyclonal and monoclonal antibodies are important tools in research and medicine. However, the discovery of antibodies against their targets in their native forms is difficult. Here, we present a novel method for discovery of antibodies against membrane proteins in their native configuration in mammalian cells. The method involves the co-expression of an antibody library in a population of mammalian cells that express the target polypeptide within a natural membrane environment on the cell surface. Cells that secrete a single-chain fragment variable (scFv) that binds to the target membrane protein thereby become self-labeled, enabling enrichment and isolation by magnetic sorting and FRET-based flow sorting. Library sizes of up to 109 variants can be screened, thus allowing campaigns of naïve scFv libraries to be selected against membrane protein antigens in a Chinese hamster ovary cell system. We validate this method by screening a synthetic naïve human scFv library against Chinese hamster ovary cells expressing the oncogenic target epithelial cell adhesion molecule and identify a panel of three novel binders to this membrane protein, one with a dissociation constant (KD) as low as 0.8 nm. We further demonstrate that the identified antibodies have utility for killing epithelial cell adhesion molecule–positive cells when used as a targeting domain on chimeric antigen receptor T cells. Thus, we provide a new tool for identifying novel antibodies that act against membrane proteins, which could catalyze the discovery of new candidates for antibody-based therapies.

TNF is a highly pro-inflammatory cytokine that contributes not only to the regulation of immune responses but also to the development of severe inflammatory diseases. TNF is synthesized as a transmembrane protein, which is further matured via proteolytic cleavage by metalloproteases such as ADAM17, a process known as shedding. At present, TNF is mainly detected by measuring the precursor or the mature cytokine of bulk cell populations by techniques such as ELISA or immunoblotting. However, these methods do not provide information on the exact timing and extent of TNF cleavage at single-cell resolution and they do not allow the live visualization of shedding events. Here, we generated C-tag TNF as a genetically encoded reporter to study TNF shedding at the single-cell level. The functionality of the C-tag TNF reporter is based on the exposure of a cryptic epitope on the C terminus of the transmembrane portion of pro-TNF on cleavage. In both denatured and nondenatured samples, this epitope can be detected by a nanobody in a highly sensitive and specific manner only upon TNF shedding. As such, C-tag TNF can successfully be used for the detection of TNF cleavage in flow cytometry and live-cell imaging applications. We furthermore demonstrate its applicability in a forward genetic screen geared toward the identification of genetic regulators of TNF maturation. In summary, the C-tag TNF reporter can be employed to gain novel insights into the complex regulation of ADAM-dependent TNF shedding.

Programmed cell death protein 1 (PD-1) is a critical inhibitory receptor that limits excessive T cell responses. Cancer cells have evolved to evade these immunoregulatory mechanisms by upregulating PD-1 ligands and preventing T cell–mediated anti-tumor responses. Consequently, therapeutic blockade of PD-1 enhances T cell–mediated anti-tumor immunity, but many patients do not respond and a significant proportion develop inflammatory toxicities. To improve anti-cancer therapy, it is critical to reveal the mechanisms by which PD-1 regulates T cell responses. We performed global quantitative phosphoproteomic interrogation of PD-1 signaling in T cells. By complementing our analysis with functional validation assays, we show that PD-1 targets tyrosine phosphosites that mediate proximal T cell receptor signaling, cytoskeletal organization, and immune synapse formation. PD-1 ligation also led to differential phosphorylation of serine and threonine sites within proteins regulating T cell activation, gene expression, and protein translation. In silico predictions revealed that kinase/substrate relationships engaged downstream of PD-1 ligation. These insights uncover the phosphoproteomic landscape of PD-1–triggered pathways and reveal novel PD-1 substrates that modulate diverse T cell functions and may serve as future therapeutic targets. These data are a useful resource in the design of future PD-1–targeting therapeutic approaches.

Carnosine (β-alanyl-l-histidine) and anserine (β-alanyl-3-methyl-l-histidine) are abundant peptides in the nervous system and skeletal muscle of many vertebrates. Many in vitro and in vivo studies demonstrated that exogenously added carnosine can improve muscle contraction, has antioxidant activity, and can quench various reactive aldehydes. Some of these functions likely contribute to the proposed anti-aging activity of carnosine. However, the physiological role of carnosine and related histidine-containing dipeptides (HCDs) is not clear. In this study, we generated a mouse line deficient in carnosine synthase (Carns1). HCDs were undetectable in the primary olfactory system and skeletal muscle of Carns1-deficient mice. Skeletal muscle contraction in these mice, however, was unaltered, and there was no evidence for reduced pH-buffering capacity in the skeletal muscle. Olfactory tests did not reveal any deterioration in 8-month-old mice lacking carnosine. In contrast, aging (18–24-month-old) Carns1-deficient mice exhibited olfactory sensitivity impairments that correlated with an age-dependent reduction in the number of olfactory receptor neurons. Whereas we found no evidence for elevated levels of lipoxidation and glycation end products in the primary olfactory system, protein carbonylation was increased in the olfactory bulb of aged Carns1-deficient mice. Taken together, these results suggest that carnosine in the olfactory system is not essential for information processing in the olfactory signaling pathway but does have a role in the long-term protection of olfactory receptor neurons, possibly through its antioxidant activity.

Distinct cell types emerge from embryonic stem cells through a precise and coordinated execution of gene expression programs during lineage commitment. This is established by the action of lineage specific transcription factors along with chromatin complexes. Numerous studies have focused on epigenetic factors that affect embryonic stem cells (ESC) self-renewal and pluripotency. However, the contribution of chromatin to lineage decisions at the exit from pluripotency has not been as extensively studied. Using a pooled epigenetic shRNA screen strategy, we identified chromatin-related factors critical for differentiation toward mesodermal and endodermal lineages. Here we reveal a critical role for the chromatin protein, ARID4B. Arid4b-deficient mESCs are similar to WT mESCs in the expression of pluripotency factors and their self-renewal. However, ARID4B loss results in defects in up-regulation of the meso/endodermal gene expression program. It was previously shown that Arid4b resides in a complex with SIN3A and HDACS 1 and 2. We identified a physical and functional interaction of ARID4B with HDAC1 rather than HDAC2, suggesting functionally distinct Sin3a subcomplexes might regulate cell fate decisions Finally, we observed that ARID4B deficiency leads to increased H3K27me3 and a reduced H3K27Ac level in key developmental gene loci, whereas a subset of genomic regions gain H3K27Ac marks. Our results demonstrate that epigenetic control through ARID4B plays a key role in the execution of lineage-specific gene expression programs at pluripotency exit.

Thoracic great vessels such as the aorta and subclavian arteries are formed through dynamic remodeling of embryonic pharyngeal arch arteries (PAAs). Previous work has shown that loss of a basic helix-loop-helix transcription factor Hey1 in mice causes abnormal fourth PAA development and lethal great vessel anomalies resembling congenital malformations in humans. However, how Hey1 mediates vascular formation remains unclear. In this study, we revealed that Hey1 in vascular endothelial cells, but not in smooth muscle cells, played essential roles for PAA development and great vessel morphogenesis in mouse embryos. Tek-Cre–mediated Hey1 deletion in endothelial cells affected endothelial tube formation and smooth muscle differentiation in embryonic fourth PAAs and resulted in interruption of the aortic arch and other great vessel malformations. Cell specificity and signal responsiveness of Hey1 expression were controlled through multiple cis-regulatory regions. We found two distal genomic regions that had enhancer activity in endothelial cells and in the pharyngeal epithelium and somites, respectively. The novel endothelial enhancer was conserved across species and was specific to large-caliber arteries. Its transcriptional activity was regulated by Notch signaling in vitro and in vivo, but not by ALK1 signaling and other transcription factors implicated in endothelial cell specificity. The distal endothelial enhancer was not essential for basal Hey1 expression in mouse embryos but may likely serve for Notch-dependent transcriptional control in endothelial cells together with the proximal regulatory region. These findings help in understanding the significance and regulation of endothelial Hey1 as a mediator of multiple signaling pathways in embryonic vascular formation.

Clostridium difficile is an anaerobic and spore-forming bacterium responsible for 15–25% of postantibiotic diarrhea and 95% of pseudomembranous colitis. Peptidoglycan is a crucial element of the bacterial cell wall that is exposed to the host, making it an important target for the innate immune system. The C. difficile peptidoglycan is largely N-deacetylated on its glucosamine (93% of muropeptides) through the activity of enzymes known as N-deacetylases, and this N-deacetylation modulates host–pathogen interactions, such as resistance to the bacteriolytic activity of lysozyme, virulence, and host innate immune responses. C. difficile genome analysis showed that 12 genes potentially encode N-deacetylases; however, which of these N-deacetylases are involved in peptidoglycan N-deacetylation remains unknown. Here, we report the enzymes responsible for peptidoglycan N-deacetylation and their respective regulation. Through peptidoglycan analysis of several mutants, we found that the N-deacetylases PdaV and PgdA act in synergy. Together they are responsible for the high level of peptidoglycan N-deacetylation in C. difficile and the consequent resistance to lysozyme. We also characterized a third enzyme, PgdB, as a glucosamine N-deacetylase. However, its impact on N-deacetylation and lysozyme resistance is limited, and its physiological role remains to be dissected. Finally, given the influence of peptidoglycan N-deacetylation on host defense against pathogens, we investigated the virulence and colonization ability of the mutants. Unlike what has been shown in other pathogenic bacteria, a lack of N-deacetylation in C. difficile is not linked to a decrease in virulence.

Stromal interaction molecule 1 (STIM1) plays a pivotal role in store-operated Ca2+ entry (SOCE), an essential mechanism in cellular calcium signaling and in maintaining cellular calcium balance. Because O-GlcNAcylation plays pivotal roles in various cellular function, we examined the effect of fluctuation in STIM1 O-GlcNAcylation on SOCE activity. We found that both increase and decrease in STIM1 O-GlcNAcylation impaired SOCE activity. To determine the molecular basis, we established STIM1-knockout HEK293 (STIM1-KO-HEK) cells using the CRISPR/Cas9 system and transfected STIM1 WT (STIM1-KO-WT-HEK), S621A (STIM1-KO-S621A-HEK), or T626A (STIM1-KO-T626A-HEK) cells. Using these cells, we examined the possible O-GlcNAcylation sites of STIM1 to determine whether the sites were O-GlcNAcylated. Co-immunoprecipitation analysis revealed that Ser621 and Thr626 were O-GlcNAcylated and that Thr626 was O-GlcNAcylated in the steady state but Ser621 was not. The SOCE activity in STIM1-KO-S621A-HEK and STIM1-KO-T626A-HEK cells was lower than that in STIM1-KO-WT-HEK cells because of reduced phosphorylation at Ser621. Treatment with the O-GlcNAcase inhibitor Thiamet G or O-GlcNAc transferase (OGT) transfection, which increases O-GlcNAcylation, reduced SOCE activity, whereas treatment with the OGT inhibitor ST045849 or siOGT transfection, which decreases O-GlcNAcylation, also reduced SOCE activity. Decrease in SOCE activity due to increase and decrease in O-GlcNAcylation was attributable to reduced phosphorylation at Ser621. These data suggest that both decrease in O-GlcNAcylation at Thr626 and increase in O-GlcNAcylation at Ser621 in STIM1 lead to impairment of SOCE activity through decrease in Ser621 phosphorylation. Targeting STIM1 O-GlcNAcylation could provide a promising treatment option for the related diseases, such as neurodegenerative diseases.

Myelination plays an important role in cognitive development and in demyelinating diseases like multiple sclerosis (MS), where failure of remyelination promotes permanent neuro-axonal damage. Modification of cell surface receptors with branched N-glycans coordinates cell growth and differentiation by controlling glycoprotein clustering, signaling, and endocytosis. GlcNAc is a rate-limiting metabolite for N-glycan branching. Here we report that GlcNAc and N-glycan branching trigger oligodendrogenesis from precursor cells by inhibiting platelet-derived growth factor receptor-α cell endocytosis. Supplying oral GlcNAc to lactating mice drives primary myelination in newborn pups via secretion in breast milk, whereas genetically blocking N-glycan branching markedly inhibits primary myelination. In adult mice with toxin (cuprizone)-induced demyelination, oral GlcNAc prevents neuro-axonal damage by driving myelin repair. In MS patients, endogenous serum GlcNAc levels inversely correlated with imaging measures of demyelination and microstructural damage. Our data identify N-glycan branching and GlcNAc as critical regulators of primary myelination and myelin repair and suggest that oral GlcNAc may be neuroprotective in demyelinating diseases like MS.

Infiltration of peripheral immune cells after blood-brain barrier dysfunction causes severe inflammation after a stroke. Although the endothelial glycocalyx, a network of membrane-bound glycoproteins and proteoglycans that covers the lumen of endothelial cells, functions as a barrier to circulating cells, the relationship between stroke severity and glycocalyx dysfunction remains unclear. In this study, glycosaminoglycans, a component of the endothelial glycocalyx, were studied in the context of ischemic stroke using a photochemically induced thrombosis mouse model. Decreased levels of heparan sulfate and chondroitin sulfate and increased activity of hyaluronidase 1 and heparanase (HPSE) were observed in ischemic brain tissues. HPSE expression in cerebral vessels increased after stroke onset and infarct volume greatly decreased after co-administration of N-acetylcysteine + glycosaminoglycan oligosaccharides as compared with N-acetylcysteine administration alone. These results suggest that the endothelial glycocalyx was injured after the onset of stroke. Interestingly, scission activity of proHPSE produced by immortalized endothelial cells and HEK293 cells transfected with hHPSE1 cDNA were activated by acrolein (ACR) exposure. We identified the ACR-modified amino acid residues of proHPSE using nano LC–MS/MS, suggesting that ACR modification of Lys139 (6-kDa linker), Lys107, and Lys161, located in the immediate vicinity of the 6-kDa linker, at least in part is attributed to the activation of proHPSE. Because proHPSE, but not HPSE, localizes outside cells by binding with heparan sulfate proteoglycans, ACR-modified proHPSE represents a promising target to protect the endothelial glycocalyx.

VAR2CSA is the placental-malaria–specific member of the antigenically variant Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family. It is expressed on the surface of Plasmodium falciparum-infected host red blood cells and binds to specific chondroitin-4-sulfate chains of the placental proteoglycan receptor. The functional ∼310 kDa ectodomain of VAR2CSA is a multidomain protein that requires a minimum 12-mer chondroitin-4-sulfate molecule for specific, high affinity receptor binding. However, it is not known how the individual domains are organized and interact to create the receptor-binding surface, limiting efforts to exploit its potential as an effective vaccine or drug target. Using small angle X-ray scattering and single particle reconstruction from negative-stained electron micrographs of the ectodomain and multidomain constructs, we have determined the structural architecture of VAR2CSA. The relative locations of the domains creates two distinct pores that can each accommodate the 12-mer of chondroitin-4-sulfate, suggesting a model for receptor binding. This model has important implications for understanding cytoadherence of infected red blood cells and potentially provides a starting point for developing novel strategies to prevent and/or treat placental malaria.

α-Linked galactose is a common carbohydrate motif in nature that is processed by a variety of glycoside hydrolases from different families. Terminal Galα1–3Gal motifs are found as a defining feature of different blood group and tissue antigens, as well as the building block of the marine algal galactan λ-carrageenan. The blood group B antigen and linear α-Gal epitope can be processed by glycoside hydrolases in family GH110, whereas the presence of genes encoding GH110 enzymes in polysaccharide utilization loci from marine bacteria suggests a role in processing λ-carrageenan. However, the structure–function relationships underpinning the α-1,3-galactosidase activity within family GH110 remain unknown. Here we focus on a GH110 enzyme (PdGH110B) from the carrageenolytic marine bacterium Pseudoalteromonas distincta U2A. We showed that the enzyme was active on Galα1–3Gal but not the blood group B antigen. X-ray crystal structures in complex with galactose and unhydrolyzed Galα1–3Gal revealed the parallel β-helix fold of the enzyme and the structural basis of its inverting catalytic mechanism. Moreover, an examination of the active site reveals likely adaptations that allow accommodation of fucose in blood group B active GH110 enzymes or, in the case of PdGH110, accommodation of the sulfate groups found on λ-carrageenan. Overall, this work provides insight into the first member of a predominantly marine clade of GH110 enzymes while also illuminating the structural basis of α-1,3-galactoside processing by the family as a whole.

RNase J enzymes are metallohydrolases that are involved in RNA maturation and RNA recycling, govern gene expression in bacteria, and catalyze both exonuclease and endonuclease activity. The catalytic activity of RNase J is regulated by multiple mechanisms which include oligomerization, conformational changes to aid substrate recognition, and the metal cofactor at the active site. However, little is known of how RNase J paralogs differ in expression and activity. Here we describe structural and biochemical features of two Staphylococcus epidermidis RNase J paralogs, RNase J1 and RNase J2. RNase J1 is a homodimer with exonuclease activity aided by two metal cofactors at the active site. RNase J2, on the other hand, has endonuclease activity and one metal ion at the active site and is predominantly a monomer. We note that the expression levels of these enzymes vary across Staphylococcal strains. Together, these observations suggest that multiple interacting RNase J paralogs could provide a strategy for functional improvisation utilizing differences in intracellular concentration, quaternary structure, and distinct active site architecture despite overall structural similarity.

α1-antitrypsin (AAT) regulates the activity of multiple proteases in the lungs and liver. A mutant of AAT (E342K) called ATZ forms polymers that are present at only low levels in the serum and induce intracellular protein inclusions, causing lung emphysema and liver cirrhosis. An understanding of factors that can reduce the intracellular accumulation of ATZ is of great interest. We now show that calreticulin (CRT), an endoplasmic reticulum (ER) glycoprotein chaperone, promotes the secretory trafficking of ATZ, enhancing the media:cell ratio. This effect is more pronounced for ATZ than with AAT and is only partially dependent on the glycan-binding site of CRT, which is generally relevant to substrate recruitment and folding by CRT. The CRT-related chaperone calnexin does not enhance ATZ secretory trafficking, despite the higher cellular abundance of calnexin-ATZ complexes. CRT deficiency alters the distributions of ATZ-ER chaperone complexes, increasing ATZ-BiP binding and inclusion body formation and reducing ATZ interactions with components required for ER-Golgi trafficking, coincident with reduced levels of the protein transport protein Sec31A in CRT-deficient cells. These findings indicate a novel role for CRT in promoting the secretory trafficking of a protein that forms polymers and large intracellular inclusions. Inefficient secretory trafficking of ATZ in the absence of CRT is coincident with enhanced accumulation of ER-derived ATZ inclusion bodies. Further understanding of the factors that control the secretory trafficking of ATZ and their regulation by CRT could lead to new therapies for lung and liver diseases linked to AAT deficiency.

Proteins in the α-macroglobulin (αM) superfamily use thiol esters to form covalent conjugation products upon their proteolytic activation. αM protease inhibitors use theirs to conjugate proteases and preferentially react with primary amines (e.g. on lysine side chains), whereas those of αM complement components C3 and C4B have an increased hydroxyl reactivity that is conveyed by a conserved histidine residue and allows conjugation to cell surface glycans. Human α2-macroglobulin–like protein 1 (A2ML1) is a monomeric protease inhibitor but has the hydroxyl reactivity–conveying histidine residue. Here, we have investigated the role of hydroxyl reactivity in a protease inhibitor by comparing recombinant WT A2ML1 and the A2ML1 H1084N mutant in which this histidine is removed. Both of A2ML1s' thiol esters were reactive toward the amine substrate glycine, but only WT A2ML1 reacted with the hydroxyl substrate glycerol, demonstrating that His-1084 increases the hydroxyl reactivity of A2ML1's thiol ester. Although both A2ML1s conjugated and inhibited thermolysin, His-1084 was required for the conjugation and inhibition of acetylated thermolysin, which lacks primary amines. Using MS, we identified an ester bond formed between a thermolysin serine residue and the A2ML1 thiol ester. These results demonstrate that a histidine-enhanced hydroxyl reactivity can contribute to protease inhibition by an αM protein. His-1084 did not improve A2ML1's protease inhibition at pH 5, indicating that A2ML1's hydroxyl reactivity is not an adaption to its acidic epidermal environment.

The actin cytoskeleton is of profound importance to cell shape, division, and intracellular force generation. Profilins bind to globular (G-)actin and regulate actin filament formation. Although profilins are well-established actin regulators, the distinct roles of the dominant profilin, profilin 1 (PFN1), versus the less abundant profilin 2 (PFN2) remain enigmatic. In this study, we use interaction proteomics to discover that PFN2 is an interaction partner of the actin N-terminal acetyltransferase NAA80, and further confirm this by analytical ultracentrifugation. Enzyme assays with NAA80 and different profilins demonstrate that PFN2 binding specifically increases the intrinsic catalytic activity of NAA80. NAA80 binds PFN2 through a proline-rich loop, deletion of which abrogates PFN2 binding. Small-angle X-ray scattering shows that NAA80, actin, and PFN2 form a ternary complex and that NAA80 has partly disordered regions in the N-terminus and the proline-rich loop, the latter of which is partly ordered upon PFN2 binding. Furthermore, binding of PFN2 to NAA80 via the proline-rich loop promotes binding between the globular domains of actin and NAA80, and thus acetylation of actin. However, the majority of cellular NAA80 is stably bound to PFN2 and not to actin, and we propose that this complex acetylates G-actin before it is incorporated into filaments. In conclusion, we reveal a functionally specific role of PFN2 as a stable interactor and regulator of the actin N-terminal acetyltransferase NAA80, and establish the modus operandi for NAA80-mediated actin N-terminal acetylation, a modification with a major impact on cytoskeletal dynamics.

Cation diffusion facilitator (CDF) proteins are a conserved family of divalent transition metal cation transporters. CDF proteins are usually composed of two domains: the transmembrane domain, in which the metal cations are transported through, and a regulatory cytoplasmic C-terminal domain (CTD). Each CDF protein transports either one specific metal or multiple metals from the cytoplasm, and it is not known whether the CTD takes an active regulatory role in metal recognition and discrimination during cation transport. Here, the model CDF protein MamM, an iron transporter from magnetotactic bacteria, was used to probe the role of the CTD in metal recognition and selectivity. Using a combination of biophysical and structural approaches, the binding of different metals to MamM CTD was characterized. Results reveal that different metals bind distinctively to MamM CTD in terms of their binding sites, thermodynamics, and binding-dependent conformations, both in crystal form and in solution, which suggests a varying level of functional discrimination between CDF domains. Furthermore, these results provide the first direct evidence that CDF CTDs play a role in metal selectivity. We demonstrate that MamM's CTD can discriminate against Mn2+, supporting its postulated role in preventing magnetite formation poisoning in magnetotactic bacteria via Mn2+ incorporation.

Connexin (Cx) protein forms hemichannels and gap junctional channels, which play diverse and profound roles in human physiology and diseases. Gap junctions are arrays of intercellular channels formed by the docking of two hemichannels from adjacent cells. Each hexameric hemichannel contains the same or different Cx isoform. Although homomeric Cxs forms have been largely described functionally and structurally, the stoichiometry and arrangement of heteromeric Cx channels remain unknown. The latter, however, are widely expressed in human tissues and variation might have important implications on channel function. Investigating properties of heteromeric Cx channels is challenging considering the high number of potential subunit arrangements and stoichiometries, even when only combining two Cx isoforms. To tackle this problem, we engineered an HA tag onto Cx26 or Cx30 subunits and imaged hemichannels that were liganded by Fab-epitope antibody fragments via atomic force microscopy. For Cx26-HA/Cx30 or Cx30-HA/Cx26 heteromeric channels, the Fab-HA binding distribution was binomial with a maximum of three Fab-HA bound. Furthermore, imaged Cx26/Cx30-HA triple liganded by Fab-HA showed multiple arrangements that can be derived from the law of total probabilities. Atomic force microscopy imaging of ringlike structures of Cx26/Cx30-HA hemichannels confirmed these findings and also detected a polydisperse distribution of stoichiometries. Our results indicate a dominant subunit stoichiometry of 3Cx26:3Cx30 with the most abundant subunit arrangement of Cx26-Cx26-Cx30-Cx26-Cx30-Cx30. To our knowledge, this is the first time that the molecular architecture of heteromeric Cx channels has been revealed, thus providing the basis to explore the functional effect of these channels in biology.

Heme oxygenase-2 (HO2) and -1 (HO1) catalyze heme degradation to biliverdin, CO, and iron, forming an essential link in the heme metabolism network. Tight regulation of the cellular levels and catalytic activities of HO1 and HO2 is important for maintaining heme homeostasis. HO1 expression is transcriptionally regulated; however, HO2 expression is constitutive. How the cellular levels and activity of HO2 are regulated remains unclear. Here, we elucidate the mechanism of post-translational regulation of cellular HO2 levels by heme. We find that, under heme-deficient conditions, HO2 is destabilized and targeted for degradation, suggesting that heme plays a direct role in HO2 regulation. HO2 has three heme binding sites: one at its catalytic site and the others at its two heme regulatory motifs (HRMs). We report that, in contrast to other HRM-containing proteins, the cellular protein level and degradation rate of HO2 are independent of heme binding to the HRMs. Rather, under heme deficiency, loss of heme binding to the catalytic site destabilizes HO2. Consistently, an HO2 catalytic site variant that is unable to bind heme exhibits a constant low protein level and an enhanced protein degradation rate compared with the WT HO2. Finally, HO2 is degraded by the lysosome through chaperone-mediated autophagy, distinct from other HRM-containing proteins and HO1, which are degraded by the proteasome. These results reveal a novel aspect of HO2 regulation and deepen our understanding of HO2's role in maintaining heme homeostasis, paving the way for future investigation into HO2's pathophysiological role in heme deficiency response.

High-throughput sequencing of hematologic malignancies and other cancers has revealed recurrent mis-sense mutations of genes encoding pre-mRNA splicing factors. The essential splicing factor U2AF2 recognizes a polypyrimidine-tract splice-site signal and initiates spliceosome assembly. Here, we investigate representative, acquired U2AF2 mutations, namely N196K or G301D amino acid substitutions associated with leukemia or solid tumors, respectively. We determined crystal structures of the wild-type (WT) compared with N196K- or G301D-substituted U2AF2 proteins, each bound to a prototypical AdML polypyrimidine tract, at 1.5, 1.4, or 1.7 Å resolutions. The N196K residue appears to stabilize the open conformation of U2AF2 with an inter-RNA recognition motif hydrogen bond, in agreement with an increased apparent RNA-binding affinity of the N196K-substituted protein. The G301D residue remains in a similar position as the WT residue, where unfavorable proximity to the RNA phosphodiester could explain the decreased RNA-binding affinity of the G301D-substituted protein. We found that expression of the G301D-substituted U2AF2 protein reduces splicing of a minigene transcript carrying prototypical splice sites. We further show that expression of either N196K- or G301D-substituted U2AF2 can subtly alter splicing of representative endogenous transcripts, despite the presence of endogenous, WT U2AF2 such as would be present in cancer cells. Altogether, our results demonstrate that acquired U2AF2 mutations such as N196K and G301D are capable of dysregulating gene expression for neoplastic transformation.

Mammalian Asn-linked glycans are extensively processed as they transit the secretory pathway to generate diverse glycans on cell surface and secreted glycoproteins. Additional modification of the glycan core by α-1,6-fucose addition to the innermost GlcNAc residue (core fucosylation) is catalyzed by an α-1,6-fucosyltransferase (FUT8). The importance of core fucosylation can be seen in the complex pathological phenotypes of FUT8 null mice, which display defects in cellular signaling, development, and subsequent neonatal lethality. Elevated core fucosylation has also been identified in several human cancers. However, the structural basis for FUT8 substrate specificity remains unknown.Here, using various crystal structures of FUT8 in complex with a donor substrate analog, and with four distinct glycan acceptors, we identify the molecular basis for FUT8 specificity and activity. The ordering of three active site loops corresponds to an increased occupancy for bound GDP, suggesting an induced-fit folding of the donor-binding subsite. Structures of the various acceptor complexes were compared with kinetic data on FUT8 active site mutants and with specificity data from a library of glycan acceptors to reveal how binding site complementarity and steric hindrance can tune substrate affinity. The FUT8 structure was also compared with other known fucosyltransferases to identify conserved and divergent structural features for donor and acceptor recognition and catalysis. These data provide insights into the evolution of modular templates for donor and acceptor recognition among GT-B fold glycosyltransferases in the synthesis of diverse glycan structures in biological systems.

The bacterial enhancer-binding protein (bEBP) FlrC, controls motility and colonization of Vibrio cholerae by regulating the transcription of class-III flagellar genes in σ54-dependent manner. However, the mechanism by which FlrC regulates transcription is not fully elucidated. Although, most bEBPs require nucleotides to stimulate the oligomerization necessary for function, our previous study showed that the central domain of FlrC (FlrCC) forms heptamer in a nucleotide-independent manner. Furthermore, heptameric FlrCC binds ATP in “cis-mediated” style without any contribution from sensor I motif 285REDXXYR291 of the trans protomer. This atypical ATP binding raises the question of whether heptamerization of FlrC is solely required for transcription regulation, or if it is also critical for ATPase activity. ATPase assays and size exclusion chromatography of the trans-variants FlrCC-Y290A and FlrCC-R291A showed destabilization of heptameric assembly with concomitant abrogation of ATPase activity. Crystal structures showed that in the cis-variant FlrCC-R349A drastic shift of Walker A encroached ATP-binding site, whereas the site remained occupied by ADP in FlrCC-Y290A. We postulated that FlrCC heptamerizes through concentration-dependent cooperativity for maximal ATPase activity and upon heptamerization, packing of trans-acting Tyr290 against cis-acting Arg349 compels Arg349 to maintain proper conformation of Walker A. Finally, a Trp quenching study revealed binding of cyclic-di-GMP with FlrCC. Excess cyclic-di-GMP repressed ATPase activity of FlrCC through destabilization of heptameric assembly, especially at low concentration of protein. Systematic phylogenetic analysis allowed us to propose similar regulatory mechanisms for FlrCs of several Vibrio species and a set of monotrichous Gram-negative bacteria.

CD81 plays a central role in a variety of physiological and pathological processes. Recent structural analysis of CD81 indicates that it contains an intramembrane cholesterol-binding pocket and that interaction with cholesterol may regulate a conformational switch in the large extracellular domain of CD81. Therefore, CD81 possesses a potential cholesterol-sensing mechanism; however, its relevance for protein function is thus far unknown. In this study we investigate CD81 cholesterol sensing in the context of its activity as a receptor for hepatitis C virus (HCV). Structure-led mutagenesis of the cholesterol-binding pocket reduced CD81–cholesterol association but had disparate effects on HCV entry, both reducing and enhancing CD81 receptor activity. We reasoned that this could be explained by alterations in the consequences of cholesterol binding. To investigate this further we performed molecular dynamic simulations of CD81 with and without cholesterol; this identified a potential allosteric mechanism by which cholesterol binding regulates the conformation of CD81. To test this, we designed further mutations to force CD81 into either the open (cholesterol-unbound) or closed (cholesterol-bound) conformation. The open mutant of CD81 exhibited reduced HCV receptor activity, whereas the closed mutant enhanced activity. These data are consistent with cholesterol sensing switching CD81 between a receptor active and inactive state. CD81 interactome analysis also suggests that conformational switching may modulate the assembly of CD81–partner protein networks. This work furthers our understanding of the molecular mechanism of CD81 cholesterol sensing, how this relates to HCV entry, and CD81's function as a molecular scaffold; these insights are relevant to CD81's varied roles in both health and disease.