Islet transplantation is an emerging therapy for type 1 diabetes (T1D) and hypoglycaemic unawareness. However, a key challenge for islet transplantation is cellular rejection and the requirement for long-term immunosuppression. In this study we established a diabetic-humanized NOD-scidIL2R^null(NSG) mouse model of T cell mediated human islet-allograft rejection and developed a therapeutic regimen of low-dose recombinant human interleukin2(IL-2) combined with low-dose rapamycin to prolong graft survival. NSG-mice that had received renal-subcapsular human islet-allografts and were transfused with 1x10⁷ of human-spleen-mononuclear-cells (hSPMCs), reconstituted human CD45⁺ cells that were predominantly CD3⁺ T cells and rejected their grafts with a median survival time of 27 days. IL-2 alone (0.3x10⁶ IU/m² or 1x10⁶ IU/m²), or rapamycin alone (0.5-1mg/kg) for 3 weeks did not prolong survival. However, the combination of rapamycin with IL-2 for 3 weeks significantly prolonged human islet-allograft survival. Graft survival was associated with expansion of CD4⁺CD25⁺FOXP3⁺ Tregs and enhanced TGF-β production by CD4⁺ T cells. CD8⁺ T cells showed reduced IFN- production and reduced expression of perforin-1. The combination of IL-2 and rapamycin has the potential to inhibit human islet-allograft rejection by expanding CD4⁺FOXP3⁺ Tregs in vivo and supressing effector cell function, and could be the basis of effective tolerance-based regimens.

Milk production may involve a transient development of insulin resistance in non-mammary tissues to support redistribution of maternal macronutrients to match the requirements of the lactating mammary gland. In the present study, adipose and liver metabolic responses were measured in the fasting state and during a 2-step (10 and 20 mU/m²/min) hyperinsulinemic-euglycemic clamp with stable isotopes, in 6-week postpartum women who were lactating (n=12) or formula-feeding (n=6) their infants and who were closely matched for baseline characteristics (e.g., parity, body composition, intrahepatic lipid). When controlling for the low insulin concentrations of both groups, the lactating women exhibited a fasting rate of endogenous glucose production (EGP) that was 2.6-fold greater, and a lipolysis rate that was 2.3-fold greater than the formula-feeding group. During the clamp, the groups exhibited similar suppression rates of EGP and lipolysis. In the lactating women only, higher prolactin concentrations were associated with greater suppression rates of lipolysis, lower intrahepatic lipid and plasma triacylglycerol concentrations. These data suggest that whole-body alterations in glucose transport may be organ specific and facilitate nutrient partitioning during lactation. Recapitulating a shift toward noninsulin-mediated glucose uptake could be an early postpartum strategy to enhance lactation success in women at risk for delayed onset of milk production.

Hepatic steatosis, the excess storage of intrahepatic lipids, is a rampant clinical problem associated with the obesity epidemic. Hepatic steatosis is linked to increased risk for insulin resistance, type 2 diabetes, and cardiovascular and advanced liver disease. Accumulating evidence shows that physical activity, exercise, and aerobic capacity have profound effects on regulating intrahepatic lipids and mediating susceptibility for hepatic steatosis. Moreover, exercise can effectively reduce hepatic steatosis independent of changes in body mass. In this perspective, we highlight 1) the relationship between obesity and metabolic pathways putatively driving hepatic steatosis compared with changes induced by exercise; 2) the impact of physical activity, exercise, and aerobic capacity compared with caloric restriction on regulating intrahepatic lipids and steatosis risk; 3) the effects of exercise training (modalities, volume, intensity) for treatment of hepatic steatosis, and 4) evidence for a sustained protection against steatosis induced by exercise. Overall, evidence clearly indicates that exercise powerfully regulates intrahepatic storage of fat and risk for steatosis.

Diabetes is now a pandemic disease. Moreover, a large number of people with prediabetes are at risk for developing frank diabetes worldwide. Both type 1 and type 2 diabetes increase the risk of atherosclerotic cardiovascular disease (CVD). Even with statin treatment to lower LDL cholesterol, patients with diabetes have a high residual CVD risk. Factors mediating the residual risk are incompletely characterized. An attractive hypothesis is that remnant lipoprotein particles (RLPs), derived by lipolysis from VLDL and chylomicrons, contribute to this residual risk. RLPs constitute a heterogeneous population of lipoprotein particles, varying markedly in size and composition. Although a universally accepted definition is lacking, for the purpose of this review we define RLPs as postlipolytic partially triglyceride-depleted particles derived from chylomicrons and VLDL that are relatively enriched in cholesteryl esters and apolipoprotein (apo)E. RLPs derived from chylomicrons contain apoB48, while those derived from VLDL contain apoB100. Clarity as to the role of RLPs in CVD risk is hampered by lack of a widely accepted definition and a paucity of adequate methods for their accurate and precise quantification. New specific methods for RLP quantification would greatly improve our understanding of their biology and role in promoting atherosclerosis in diabetes and other disorders.

The transcription factor forkhead box P3 (FOXP3) is a biomarker for regulatory T cells and can also be expressed in cancer cells, but its function in cancer appears to be divergent. The role of hepatocyte-expressed FOXP3 in hepatocellular carcinoma (HCC) is unknown. Here, we collected tumor samples and clinical information from 115 HCC patients and used five human cancer cell lines. We examined FOXP3 mRNA sequences for mutations, used a luciferase assay to assess promoter activities of FOXP3's target genes, and employed mouse tumor models to confirm in vitro results. We detected mutations in the FKH domain of FOXP3 mRNAs in 33% of the HCC tumor tissues, but in none of the adjacent nontumor tissues. None of the mutations occurred at high frequency, indicating that they occurred randomly. Notably, the mutations were not detected in the corresponding regions of FOXP3 genomic DNA, and many of them resulted in amino acid substitutions in the FKH region, altering FOXP3's subcellular localization. FOXP3 delocalization from the nucleus to the cytoplasm caused loss of transcriptional regulation of its target genes, inactivated its tumor-inhibitory capability, and changed cellular responses to histone deacetylase (HDAC) inhibitors. More complex FKH mutations appeared to be associated with worse prognosis in HCC patients. We conclude that mutations in the FKH domain of FOXP3 mRNA frequently occur in HCC and that these mutations are caused by errors in transcription and are not derived from genomic DNA mutations. Our results suggest that transcriptional mutagenesis of FOXP3 plays a role in HCC.

Prostate cancer (PCa) cells heavily rely on an active androgen receptor (AR) pathway for their survival. Enzalutamide (MDV3100) is a second-generation antiandrogenic drug that was approved by the Food and Drug Administration in 2012 to treat patients with castration-resistant prostate cancer (CRPC). However, emergence of resistance against this drug is inevitable, and it has been a major challenge to develop interventions that help manage enzalutamide-resistant CRPC. Erythropoietin-producing human hepatocellular (Eph) receptors are targeted by ephrin protein ligands and have a broad range of functions. Increasing evidence indicates that this signaling pathway plays an important role in tumorigenesis. Overexpression of EPH receptor B4 (EPHB4) has been observed in multiple types of cancer, being closely associated with proliferation, invasion, and metastasis of tumors. Here, using RNA-Seq analyses of clinical and preclinical samples, along with several biochemical and molecular methods, we report that enzalutamide-resistant PCa requires an active EPHB4 pathway that supports drug resistance of this tumor type. Using a small kinase inhibitor and RNAi-based gene silencing to disrupt EPHB4 activity, we found that these disruptions re-sensitize enzalutamide-resistant PCa to the drug both in vitro and in vivo. Mechanistically, we found that EPHB4 stimulates the AR by inducing proto-oncogene c-Myc (c-Myc) expression. Taken together, these results provide critical insight into the mechanism of enzalutamide resistance in PCa, potentially offering a therapeutic avenue for enhancing the efficacy of enzalutamide to better manage this common malignancy.

The actin cytoskeleton is extremely dynamic and supports diverse cellular functions in many physiological and pathological processes, including tumorigenesis. However, the mechanisms that regulate the actin-related protein 2/3 (ARP2/3) complex and thereby promote actin polymerization and organization in cancer cells are not well-understood. We previously implicated the proline-rich 11 (PRR11) protein in lung cancer development. In this study, using immunofluorescence staining, actin polymerization assays, and siRNA-mediated gene silencing, we uncovered that cytoplasmic PRR11 is involved in F-actin polymerization and organization. We found that dysregulation of PRR11 expression results in F-actin rearrangement and nuclear instability in non-small cell lung cancer cells. Results from molecular mechanistic experiments indicated that PRR11 associates with and recruits the ARP2/3 complex, facilitates F-actin polymerization, and thereby disrupts the F-actin cytoskeleton, leading to abnormal nuclear lamina assembly and chromatin reorganization. Inhibition of the ARP2/3 complex activity abolished irregular F-actin polymerization, lamina assembly, and chromatin reorganization due to PRR11 overexpression. Notably, experiments with truncated PRR11 variants revealed that PRR11 regulates F-actin through different regions. We found that deletion of either the N or C terminus of PRR11 abrogates its effects on F-actin polymerization and nuclear instability and that deletion of amino acid residues 100–184 or 100–200 strongly induces an F-actin structure called the actin comet tail, not observed with WT PRR11. Our findings indicate that cytoplasmic PRR11 plays an essential role in regulating F-actin assembly and nuclear stability by recruiting the ARP2/3 complex in human non-small cell lung carcinoma cells.

Over the last several years it has become clear that higher order assemblies on membranes, exemplified by signalosomes, are a paradigm for the regulation of many membrane signaling processes. We have recently combined two-color direct stochastic optical reconstruction microscopy (dSTORM) with the (Clus-DoC) algorithm that combines cluster detection and colocalization analysis to observe the organization of 5-lipoxygenase (5-LO) and 5-lipoxygenase–activating protein (FLAP) into higher order assemblies on the nuclear envelope of mast cells; these assemblies were linked to leukotriene (LT) C4 production. In this study we investigated whether higher order assemblies of 5-LO and FLAP included cytosolic phospholipase A2 (cPLA2) and were linked to LTB4 production in murine neutrophils. Using two- and three-color dSTORM supported by fluorescence lifetime imaging microscopy we identified higher order assemblies containing 40 molecules (median) (IQR: 23, 87) of 5-LO, and 53 molecules (62, 156) of FLAP monomer. 98 (18, 154) molecules of cPLA2 were clustered with 5-LO, and 77 (33, 114) molecules of cPLA2 were associated with FLAP. These assemblies were tightly linked to LTB4 formation. The activation-dependent close associations of cPLA2, FLAP, and 5-LO in higher order assemblies on the nuclear envelope support a model in which arachidonic acid is generated by cPLA2 in apposition to FLAP, facilitating its transfer to 5-LO to initiate LT synthesis.

Accumulating evidence suggests that brown adipose tissue (BAT) is a potential therapeutic target for managing obesity and related diseases. PGAM family member 5, mitochondrial serine/threonine protein phosphatase (PGAM5), is a protein phosphatase that resides in the mitochondria and regulates many biological processes, including cell death, mitophagy, and immune responses. Because BAT is a mitochondria-rich tissue, we have hypothesized that PGAM5 has a physiological function in BAT. We previously reported that PGAM5-knockout (KO) mice are resistant to severe metabolic stress. Importantly, lipid accumulation is suppressed in PGAM5-KO BAT, even under unstressed conditions, raising the possibility that PGAM5 deficiency stimulates lipid consumption. However, the mechanism underlying this observation is undetermined. Here, using an array of biochemical approaches, including quantitative RT-PCR, immunoblotting, and oxygen consumption assays, we show that PGAM5 negatively regulates energy expenditure in brown adipocytes. We found that PGAM5-KO brown adipocytes have an enhanced oxygen consumption rate and increased expression of uncoupling protein 1 (UCP1), a protein that increases energy consumption in the mitochondria. Mechanistically, we found that PGAM5 phosphatase activity and intramembrane cleavage are required for suppression of UCP1 activity. Furthermore, utilizing a genome-wide siRNA screen in HeLa cells to search for regulators of PGAM5 cleavage, we identified a set of candidate genes, including phosphatidylserine decarboxylase (PISD), which catalyzes the formation of phosphatidylethanolamine at the mitochondrial membrane. Taken together, these results indicate that PGAM5 suppresses mitochondrial energy expenditure by down-regulating UCP1 expression in brown adipocytes and that its phosphatase activity and intramembrane cleavage are required for UCP1 suppression.

The developing nervous system is remarkably sensitive to environmental signals, including disruptive toxins, such as polybrominated diphenyl ethers (PBDEs). PBDEs are an environmentally pervasive class of brominated flame retardants whose neurodevelopmental toxicity mechanisms remain largely unclear. Using dissociated cortical neurons from embryonic Rattus norvegicus, we found here that chronic exposure to 6-OH–BDE-47, one of the most prevalent hydroxylated PBDE metabolites, suppresses both spontaneous and evoked neuronal electrical activity. On the basis of our previous work on mitogen-activated protein kinase (MAPK)/extracellular signal-related kinase (ERK) (MEK) biology and our observation that 6-OH–BDE-47 is structurally similar to kinase inhibitors, we hypothesized that certain hydroxylated PBDEs mediate neurotoxicity, at least in part, by impairing the MEK–ERK axis of MAPK signal transduction. We tested this hypothesis on three experimental platforms: 1) in silico, where modeling ligand–protein docking suggested that 6-OH–BDE-47 is a promiscuous ATP-competitive kinase inhibitor; 2) in vitro in dissociated neurons, where 6-OH–BDE-47 and another specific hydroxylated BDE metabolite similarly impaired phosphorylation of MEK/ERK1/2 and activity-induced transcription of a neuronal immediate early gene; and 3) in vivo in Drosophila melanogaster, where developmental exposures to 6-OH–BDE-47 and a MAPK inhibitor resulted in offspring displaying similarly increased frequency of mushroom-body β–lobe midline crossing, a metric of axonal guidance. Taken together, our results support that certain ortho-hydroxylated PBDE metabolites are promiscuous kinase inhibitors and can cause disruptions of critical neurodevelopmental processes, including neuronal electrical activity, pre-synaptic functions, MEK–ERK signaling, and axonal guidance.

Kindlins are focal adhesion proteins that regulate integrin activation and outside-in signaling. The kindlin family consists of three members, kindlin-1, -2, and -3. Kindlin-2 is widely expressed in multiple cell types, except those from the hematopoietic lineage. A previous study has reported that the Drosophila Fit1 protein (an ortholog of kindlin-2) prevents abnormal spindle assembly; however, the mechanism remains unknown. Here, we show that kindlin-2 maintains spindle integrity in mitotic human cells. The human neuroblastoma SH-SY5Y cell line expresses only kindlin-2, and we found that when SH-SY5Y cells are depleted of kindlin-2, they exhibit pronounced spindle abnormalities and delayed mitosis. Of note, acetylation of α-tubulin, which maintains microtubule flexibility and stability, was diminished in the kindlin-2–depleted cells. Mechanistically, we found that kindlin-2 maintains α-tubulin acetylation by inhibiting the microtubule-associated deacetylase histone deacetylase 6 (HDAC6) via a signaling pathway involving AKT Ser/Thr kinase (AKT)/glycogen synthase kinase 3β (GSK3β) or paxillin. We also provide evidence that prolonged hypoxia down-regulates kindlin-2 expression, leading to spindle abnormalities not only in the SH-SY5Y cell line, but also cell lines derived from colon and breast tissues. The findings of our study highlight that kindlin-2 regulates mitotic spindle assembly and that this process is perturbed in cancer cells in a hypoxic environment.

We previously reported that overexpression of cytochrome P450 family 24 subfamily A member 1 (CYP24A1) increases lung cancer cell proliferation by activating RAS signaling and that CYP24A1 knockdown inhibits tumor growth. However, the mechanism of CYP24A1-mediated cancer cell proliferation remains unclear. Here, we conducted cell synchronization and biochemical experiments in lung adenocarcinoma cells, revealing a link between CYP24A1 and anaphase-promoting complex (APC), a key cell cycle regulator. We demonstrate that CYP24A1 expression is cell cycle–dependent; it was higher in the G2-M phase and diminished upon G1 entry. CYP24A1 has a functional destruction box (D-box) motif that allows binding with two APC adaptors, CDC20-homologue 1 (CDH1) and cell division cycle 20 (CDC20). Unlike other APC substrates, however, CYP24A1 acted as a pseudo-substrate, inhibiting CDH1 activity and promoting mitotic progression. Conversely, overexpression of a CYP24A1 D-box mutant compromised CDH1 binding, allowing CDH1 hyperactivation, thereby hastening degradation of its substrates cyclin B1 and CDC20, and accumulation of the CDC20 substrate p21, prolonging mitotic exit. These activities also occurred with a CYP24A1 isoform 2 lacking the catalytic cysteine (Cys-462), suggesting that CYP24A1's oncogenic potential is independent of its catalytic activity. CYP24A1 degradation reduced clonogenic survival of mutant KRAS-driven lung cancer cells, and calcitriol treatment increased CYP24A1 levels and tumor burden in Lsl-KRASG12D mice. These results disclose a catalytic activity-independent growth-promoting role of CYP24A1 in mutant KRAS-driven lung cancer. This suggests that CYP24A1 could be therapeutically targeted in lung cancers in which its expression is high.

Arrestin-1 is the arrestin family member responsible for inactivation of the G protein–coupled receptor rhodopsin in photoreceptors. Arrestin-1 is also well-known to interact with additional protein partners and to affect other signaling cascades beyond phototransduction. In this study, we investigated one of these alternative arrestin-1 binding partners, the glycolysis enzyme enolase-1, to map the molecular contact sites between these two proteins and investigate how the binding of arrestin-1 affects the catalytic activity of enolase-1. Using fluorescence quench protection of strategically placed fluorophores on the arrestin-1 surface, we observed that arrestin-1 primarily engages enolase-1 along a surface that is opposite of the side of arrestin-1 that binds photoactivated rhodopsin. Using this information, we developed a molecular model of the arrestin-1–enolase-1 complex, which was validated by targeted substitutions of charge-pair interactions. Finally, we identified the likely source of arrestin's modulation of enolase-1 catalysis, showing that selective substitution of two amino acids in arrestin-1 can completely remove its effect on enolase-1 activity while still remaining bound to enolase-1. These findings open up opportunities for examining the functional effects of arrestin-1 on enolase-1 activity in photoreceptors and their surrounding cells.

Site-specific recombinases, such as Cre, are a widely used tool for genetic lineage tracing in the fields of developmental biology, neural science, stem cell biology, and regenerative medicine. However, nonspecific cell labeling by some genetic Cre tools remains a technical limitation of this recombination system, which has resulted in data misinterpretation and led to many controversies in the scientific community. In the past decade, to enhance the specificity and precision of genetic targeting, researchers have used two or more orthogonal recombinases simultaneously for labeling cell lineages. Here, we review the history of cell-tracing strategies and then elaborate on the working principle and application of a recently developed dual genetic lineage-tracing approach for cell fate studies. We place an emphasis on discussing the technical strengths and caveats of different methods, with the goal to develop more specific and efficient tracing technologies for cell fate mapping. Our review also provides several examples for how to use different types of DNA recombinase–mediated lineage-tracing strategies to improve the resolution of the cell fate mapping in order to probe and explore cell fate–related biological phenomena in the life sciences.

Sperm head shaping is a key event in spermiogenesis and is tightly controlled via the acrosome–manchette network. Linker of nucleoskeleton and cytoskeleton (LINC) complexes consist of Sad1 and UNC84 domain–containing (SUN) and Klarsicht/ANC-1/Syne-1 homology (KASH) domain proteins and form conserved nuclear envelope bridges implicated in transducing mechanical forces from the manchette to sculpt sperm nuclei into a hook-like shape. However, the role of LINC complexes in sperm head shaping is still poorly understood. Here we assessed the role of SUN3, a testis-specific LINC component harboring a conserved SUN domain, in spermiogenesis. We show that CRISPR/Cas9-generated Sun3 knockout male mice are infertile, displaying drastically reduced sperm counts and a globozoospermia-like phenotype, including a missing, mislocalized, or fragmented acrosome, as well as multiple defects in sperm flagella. Further examination revealed that the sperm head abnormalities are apparent at step 9 and that the sperm nuclei fail to elongate because of the absence of manchette microtubules and perinuclear rings. These observations indicate that Sun3 deletion likely impairs the ability of the LINC complex to transduce the cytoskeletal force to the nuclear envelope, required for sperm head elongation. We also found that SUN3 interacts with SUN4 in mouse testes and that the level of SUN4 proteins is drastically reduced in Sun3-null mice. Altogether, our results indicate that SUN3 is essential for sperm head shaping and male fertility, providing molecular clues regarding the underlying pathology of the globozoospermia-like phenotype.

Lethal infections by strains of the highly-pathogenic avian influenza virus (HPAIV) H5N1 pose serious threats to both the poultry industry and public health worldwide. A lack of confirmed HPAIV epitopes recognized by cytotoxic T lymphocytes (CTLs) has hindered the utilization of CD8+ T-cell–mediated immunity and has precluded the development of effectively diversified epitope-based vaccination approaches. In particular, an HPAIV H5N1 CTL-recognized epitope based on the peptide MHC-I–β2m (pMHC-I) complex has not yet been designed. Here, screening a collection of selected peptides of several HPAIV strains against a specific pathogen-free pMHC-I (pBF2*1501), we identified a highly-conserved HPAIV H5N1 CTL epitope, named HPAIV–PA123–130. We determined the structure of the BF2*1501–PA123–130 complex at 2.1 Å resolution to elucidate the molecular mechanisms of a preferential presentation of the highly-conserved PA123–130 epitope in the chicken B15 lineage. Conformational characteristics of the PA123–130 epitope with a protruding Tyr-7 residue indicated that this epitope has great potential to be recognized by specific TCRs. Moreover, significantly increased numbers of CD8+ T cells specific for the HPAIV–PA123–130 epitope in peptide-immunized chickens indicated that a repertoire of CD8+ T cells can specifically respond to this epitope. We anticipate that the identification and structural characterization of the PA123–130 epitope reported here could enable further studies of CTL immunity against HPAIV H5N1. Such studies may aid in the development of vaccine development strategies using well-conserved internal viral antigens in chickens.

Heme-regulatory motifs (HRMs) are present in many proteins that are involved in diverse biological functions. The C-terminal tail region of human heme oxygenase-2 (HO2) contains two HRMs whose cysteine residues form a disulfide bond; when reduced, these cysteines are available to bind Fe3+-heme. Heme binding to the HRMs occurs independently of the HO2 catalytic active site in the core of the protein, where heme binds with high affinity and is degraded to biliverdin. Here, we describe the reversible, protein-mediated transfer of heme between the HRMs and the HO2 core. Using hydrogen-deuterium exchange (HDX)-MS to monitor the dynamics of HO2 with and without Fe3+-heme bound to the HRMs and to the core, we detected conformational changes in the catalytic core only in one state of the catalytic cycle—when Fe3+-heme is bound to the HRMs and the core is in the apo state. These conformational changes were consistent with transfer of heme between binding sites. Indeed, we observed that HRM-bound Fe3+-heme is transferred to the apo-core either upon independent expression of the core and of a construct spanning the HRM-containing tail or after a single turnover of heme at the core. Moreover, we observed transfer of heme from the core to the HRMs and equilibration of heme between the core and HRMs. We therefore propose an Fe3+-heme transfer model in which HRM-bound heme is readily transferred to the catalytic site for degradation to facilitate turnover but can also equilibrate between the sites to maintain heme homeostasis.

Cell-surface signaling (CSS) in Gram-negative bacteria involves highly conserved regulatory pathways that optimize gene expression by transducing extracellular environmental signals to the cytoplasm via inner-membrane sigma regulators. The molecular details of ferric siderophore-mediated activation of the iron import machinery through a sigma regulator are unclear. Here, we present the 1.56 Å resolution structure of the periplasmic complex of the C-terminal CSS domain (CCSSD) of PupR, the sigma regulator in the Pseudomonas capeferrum pseudobactin BN7/8 transport system, and the N-terminal signaling domain (NTSD) of PupB, an outer-membrane TonB-dependent transducer. The structure revealed that the CCSSD consists of two subdomains: a juxta-membrane subdomain, which has a novel all-β-fold, followed by a secretin/TonB, short N-terminal subdomain at the C terminus of the CCSSD, a previously unobserved topological arrangement of this domain. Using affinity pulldown assays, isothermal titration calorimetry, and thermal denaturation CD spectroscopy, we show that both subdomains are required for binding the NTSD with micromolar affinity and that NTSD binding improves CCSSD stability. Our findings prompt us to present a revised model of CSS wherein the CCSSD:NTSD complex forms prior to ferric-siderophore binding. Upon siderophore binding, conformational changes in the CCSSD enable regulated intramembrane proteolysis of the sigma regulator, ultimately resulting in transcriptional regulation.

In cyanobacteria, metabolic pathways that use the nitrogen-rich amino acid arginine play a pivotal role in nitrogen storage and mobilization. The N-terminal domains of two recently identified bacterial enzymes: ArgZ from Synechocystis and AgrE from Anabaena, have been found to contain an arginine dihydrolase. This enzyme provides catabolic activity that converts arginine to ornithine, resulting in concomitant release of CO2 and ammonia. In Synechocystis, the ArgZ-mediated ornithine–ammonia cycle plays a central role in nitrogen storage and remobilization. The C-terminal domain of AgrE contains an ornithine cyclodeaminase responsible for the formation of proline from ornithine and ammonia production, indicating that AgrE is a bifunctional enzyme catalyzing two sequential reactions in arginine catabolism. Here, the crystal structures of AgrE in three different ligation states revealed that it has a tetrameric conformation, possesses a binding site for the arginine dihydrolase substrate l-arginine and product l-ornithine, and contains a binding site for the coenzyme NAD(H) required for ornithine cyclodeaminase activity. Structure–function analyses indicated that the structure and catalytic mechanism of arginine dihydrolase in AgrE are highly homologous with those of a known bacterial arginine hydrolase. We found that in addition to other active-site residues, Asn-71 is essential for AgrE's dihydrolase activity. Further analysis suggested the presence of a passage for substrate channeling between the two distinct AgrE active sites, which are situated ∼45 Å apart. These results provide structural and functional insights into the bifunctional arginine dihydrolase–ornithine cyclodeaminase enzyme AgrE required for arginine catabolism in Anabaena.

Mammalian cytochrome P450 enzymes often metabolize many pharmaceuticals and other xenobiotics, a feature that is valuable in a biotechnology setting. However, extant P450 enzymes are typically relatively unstable, with T50 values of ∼30–40 °C. Reconstructed ancestral cytochrome P450 enzymes tend to have variable substrate selectivity compared with related extant forms, but they also have higher thermostability and therefore may be excellent tools for commercial biosynthesis of important intermediates, final drug molecules, or drug metabolites. The mammalian ancestor of the cytochrome P450 1B subfamily was herein characterized structurally and functionally, revealing differences from the extant human CYP1B1 in ligand binding, metabolism, and potential molecular contributors to its thermostability. Whereas extant human CYP1B1 has one molecule of α-naphthoflavone in a closed active site, we observed that subtle amino acid substitutions outside the active site in the ancestor CYP1B enzyme yielded an open active site with four ligand copies. A structure of the ancestor with 17β-estradiol revealed only one molecule in the active site, which still had the same open conformation. Detailed comparisons between the extant and ancestor forms revealed increases in electrostatic and aromatic interactions between distinct secondary structure elements in the ancestral forms that may contribute to their thermostability. To the best of our knowledge, this represents the first structural evaluation of a reconstructed ancestral cytochrome P450, revealing key features that appear to contribute to its thermostability.

The transfer of a phosphate from ATP to a protein substrate, a modification known as protein phosphorylation, is catalyzed by protein kinases. Protein kinases play a crucial role in virtually every cellular activity. Recent studies of atypical protein kinases have highlighted the structural similarity of the kinase superfamily despite notable differences in primary amino acid sequence. Here, using a bioinformatics screen, we searched for putative protein kinases in the intracellular bacterial pathogen Legionella pneumophila and identified the type 4 secretion system effector Lpg2603 as a remote member of the protein kinase superfamily. Employing an array of biochemical and structural biology approaches, including in vitro kinase assays and isothermal titration calorimetry, we show that Lpg2603 is an active protein kinase with several atypical structural features. Importantly, we found that the eukaryote-specific host signaling molecule inositol hexakisphosphate (IP6) is required for Lpg2603 kinase activity. Crystal structures of Lpg2603 in the apo-form and when bound to IP6 revealed an active-site rearrangement that allows for ATP binding and catalysis. Our results on the structure and activity of Lpg2603 reveal a unique mode of regulation of a protein kinase, provide the first example of a bacterial kinase that requires IP6 for its activation, and may aid future work on the function of this effector during Legionella pathogenesis.

Tau is a microtubule-associated protein that plays a major role in Alzheimer's disease (AD) and other tauopathies. Recent reports indicate that, in the presence of crowding agents, tau can undergo liquid–liquid phase separation (LLPS), forming highly dynamic liquid droplets. Here, using recombinantly expressed proteins, turbidimetry, fluorescence microscopy imaging, and fluorescence recovery after photobleaching (FRAP) assays, we show that the divalent transition metal zinc strongly promotes this process, shifting the equilibrium phase boundary to lower protein or crowding agent concentrations. We observed no tau LLPS-promoting effect for any other divalent transition metal ions tested, including Mn2+, Fe2+, Co2+, Ni2+, and Cu2+. We also demonstrate that multiple zinc-binding sites on tau are involved in the LLPS-promoting effect and provide insights into the mechanism of this process. Zinc concentration is highly elevated in AD brains, and this metal ion is believed to be an important player in the pathogenesis of this disease. Thus, the present findings bring a new dimension to understanding the relationship between zinc homeostasis and the pathogenic process in AD and related neurodegenerative disorders.

Fucosylation of the innermost GlcNAc of N-glycans by fucosyltransferase 8 (FUT8) is an important step in the maturation of complex and hybrid N-glycans. This simple modification can dramatically affect the activities and half-lives of glycoproteins, effects that are relevant to understanding the invasiveness of some cancers, development of mAb therapeutics, and the etiology of a congenital glycosylation disorder. The acceptor substrate preferences of FUT8 are well-characterized and provide a framework for understanding N-glycan maturation in the Golgi; however, the structural basis of these substrate preferences and the mechanism through which catalysis is achieved remain unknown. Here we describe several structures of mouse and human FUT8 in the apo state and in complex with GDP, a mimic of the donor substrate, and with a glycopeptide acceptor substrate at 1.80–2.50 Å resolution. These structures provide insights into a unique conformational change associated with donor substrate binding, common strategies employed by fucosyltransferases to coordinate GDP, features that define acceptor substrate preferences, and a likely mechanism for enzyme catalysis. Together with molecular dynamics simulations, the structures also revealed how FUT8 dimerization plays an important role in defining the acceptor substrate-binding site. Collectively, this information significantly builds on our understanding of the core fucosylation process.

Type IV filaments (T4F), which are helical assemblies of type IV pilins, constitute a superfamily of filamentous nanomachines virtually ubiquitous in prokaryotes that mediate a wide variety of functions. The competence (Com) pilus is a widespread T4F, mediating DNA uptake (the first step in natural transformation) in bacteria with one membrane (monoderms), an important mechanism of horizontal gene transfer. Here, we report the results of genomic, phylogenetic, and structural analyses of ComGC, the major pilin subunit of Com pili. By performing a global comparative analysis, we show that Com pili genes are virtually ubiquitous in Bacilli, a major monoderm class of Firmicutes. This also revealed that ComGC displays extensive sequence conservation, defining a monophyletic group among type IV pilins. We further report ComGC solution structures from two naturally competent human pathogens, Streptococcus sanguinis (ComGCSS) and Streptococcus pneumoniae (ComGCSP), revealing that this pilin displays extensive structural conservation. Strikingly, ComGCSS and ComGCSP exhibit a novel type IV pilin fold that is purely helical. Results from homology modeling analyses suggest that the unusual structure of ComGC is compatible with helical filament assembly. Because ComGC displays such a widespread distribution, these results have implications for hundreds of monoderm species.

Arrestin-1 is the arrestin family member responsible for inactivation of the G protein–coupled receptor rhodopsin in photoreceptors. Arrestin-1 is also well-known to interact with additional protein partners and to affect other signaling cascades beyond phototransduction. In this study, we investigated one of these alternative arrestin-1 binding partners, the glycolysis enzyme enolase-1, to map the molecular contact sites between these two proteins and investigate how the binding of arrestin-1 affects the catalytic activity of enolase-1. Using fluorescence quench protection of strategically placed fluorophores on the arrestin-1 surface, we observed that arrestin-1 primarily engages enolase-1 along a surface that is opposite of the side of arrestin-1 that binds photoactivated rhodopsin. Using this information, we developed a molecular model of the arrestin-1–enolase-1 complex, which was validated by targeted substitutions of charge-pair interactions. Finally, we identified the likely source of arrestin's modulation of enolase-1 catalysis, showing that selective substitution of two amino acids in arrestin-1 can completely remove its effect on enolase-1 activity while still remaining bound to enolase-1. These findings open up opportunities for examining the functional effects of arrestin-1 on enolase-1 activity in photoreceptors and their surrounding cells.

Andrew S. Kimball
Sep 1, 2017; 66:2459-2471
Immunology and Transplantation

David J. Hodson
May 1, 2020; 69:864-866
Commentaries

Maria Sörhede Winzell
Dec 1, 2004; 53:S215-S219
Section V: The Incretin Pathway

Luigi Fontana
Apr 1, 2007; 56:1010-1013
Metabolism

Jens Juul Holst
Dec 1, 2004; 53:S197-S204
Section V: The Incretin Pathway

Mariam Alatrach
Apr 1, 2020; 69:681-688
Pathophysiology

Errol B. Marliss
Feb 1, 2002; 51:S271-S283
Section 6: Pusatile and Phasic Insulin Release in Normal and Diabetic Men

Edwin A.M. Gale
Dec 1, 2002; 51:3353-3361
Perspectives in Diabetes

Mary C. Gannon
Sep 1, 2004; 53:2375-2382
Pathophysiology

Marlou L. Dirks
Oct 1, 2016; 65:2862-2875
Metabolism

Patrick E. MacDonald
Dec 1, 2002; 51:S434-S442
Section 5: Beta-Cell Stimulus-Secretion Coupling: Hormonal and Pharmacological Modulators

Elisabeth F.C. van Rossum
Oct 1, 2002; 51:3128-3134
Genetics

VOLUME 285 (2010) PAGES 13742–13747In Fig. 1E, passage 10, the splicing of a non-adjacent lane from the same immunoblot was not marked. This error has now been corrected and does not affect the results or conclusions of this work.jbc;295/16/5533/F1F1F1Figure 1E.

VOLUME 295 (2020) PAGES 4079–4092There was an error in the abstract. “The pyridinium cation hampers uptake of OPs into the central nervous system (CNS)” should read as “The pyridinium cation hampers uptake into the central nervous system (CNS).”

VOLUME 287 (2012) PAGES 44508–44517In Fig. 1A, the wrong image for the control group was presented. The authors inadvertently cropped the control images in Fig. 1, A and E, from the same raw image. Fig. 1A has now been corrected and does not affect the results or conclusions of the work. The authors sincerely apologize for their mistake during figure preparation and for any inconvenience this may have caused readers.jbc;295/19/6781/F1F1F1Figure 1A.

Buster Posey plans to be on the field when Giants pitchers and catchers begin their first workout at Scottsdale Stadium on Wednesday, and if his rehab from right hip surgery continues to go well, he expects the same for Opening Day.

It's been less than a year since the Giants selected Joey Bart with the second overall pick in the 2018 MLB Draft, but the 22-year-old catcher is already generating plenty of excitement within the organization.

All 30 Major League teams will wear special "MLB 150" patches on their uniforms for the entire 2019 season in honor of the 150th anniversary of the 1869 Cincinnati Red Stockings, the first openly all-salaried professional baseball team.

Less than two hours after meeting Madison Bumgarner, top Giants prospect Joey Bart found himself catching one of Bumgarner's bullpen sessions after the veteran left-hander approached him and said, "Let's go on No. 1."

The Giants have expressed a desire to add multiple veteran outfielders throughout the offseason, and they made their first notable acquisition earlier this week after signing Gerardo Parra to a Minor League contract.

Bruce Bochy isn't sure what his next step will be after he retires from managing the Giants at the end of the season, but it's safe to assume that a trip to Cooperstown is in his near future.

Bruce Bochy, who guided the Giants to three World Series championships in 2010, '12 and '14, announced Monday that he will retire at the end of the 2019 season, capping a celebrated 25-year managerial career in the Majors.

Bruce Bochy announced on Monday that his 25th season as a manager -- his 13th in an absurdly successful run with the Giants -- would be his last. In making the announcement, Bochy is going to give all of us the opportunity to say thanks during a 2019 season that will be something of a victory lap whether he likes it or not.

Buster Posey has known only one manager since making his debut with the Giants a decade ago. While it's hard for him to envision playing for someone other than Bruce Bochy, he wasn't surprised to hear about his longtime manager's plans to retire after the 2019 season.