Regulation of protein function and degradation by heme, heme responsive motifs, and CO.

Heme is an essential biomolecule and cofactor involved in a myriad of biological processes. In this review, we focus on how heme binding to heme regulatory motifs (HRMs), catalytic sites, and gas signaling molecules as well as how changes in the heme redox state regulate protein structure, function, and degradation. We also relate these heme-dependent changes to the affected metabolic processes. We center our discussion on two HRM-containing proteins: human heme oxygenase-2, a protein that binds and degrades heme (releasing Fe2+ and CO) in its catalytic core and binds Fe3+-heme at HRMs located within an unstructured region of the enzyme, and the transcriptional regulator Rev-erbß, a protein that binds Fe3+-heme at an HRM and is involved in CO sensing. We will discuss these and other proteins as they relate to cellular heme composition, homeostasis, and trafficking. In addition, we will discuss the HRM-containing family of proteins and how the stability and activity of these proteins are regulated in a dependent manner through the HRMs. Then, after reviewing CO-mediated protein regulation of heme proteins, we turn our attention to the involvement of heme, HRMs, and CO in circadian rhythms. In sum, we stress the importance of understanding the various roles of heme and the distribution of the different heme pools as they relate to the heme redox state, CO, and heme binding affinities.

Cascade [4 + 1] Annulation through Activation of the C(sp2)-H Bond Enabling Benzothiadiazinoisoindolcarboxylate, Benzothiadiazinoisoindole, and Benzothiadiazinoisoindolepyrrolidinedione as Hybrid Spiro-Heterocyclic Frameworks†.

Two structurally distinct and biologically privileged succinimide and isoindole heteroarenes bearing benzothiadiazinedioxide motif-centered hybrid conjugates are proficiently achieved through Rh(III)-catalyzed sequential C(sp2)-H bond activation, ortho-alkenylation and finally cascade intramolecular cyclization. The significant feature of this developed protocol is that the resulting diversely decorated heterocycles contain a quaternary carbon center and this has been coursed through atypical [4 + 1] annulation ignoring the prevalent [4 + 2]-cyclization pathway and interestingly the applied coupling partners (e.g., maleimide, maleate, and styrene) to materialize the protocol functioned only as C1 synthon. Furthermore, the selective reduction strategy enables to modify the hybrid conjugate of succinimide and benzothiazine dioxide to benzothiazine dioxide-based spirocyclic isoindolopyrrolidinedione skeleton following preferential reduction of one carbonyl group of imide functionality. Overall this methodology emerges to be easily handled, versatile, time-efficient, and manifests relatively unfamiliar spiro-cyclization and good functional group tolerance so easy to grab a library of the entirely new variant of decorated hybrid spiro-heterocyclic scaffolds.

Ferric heme as a CO/NO sensor in the nuclear receptor Rev-Erbß by coupling gas binding to electron transfer.

Rev-Erbß is a nuclear receptor that couples circadian rhythm, metabolism, and inflammation. Heme binding to the protein modulates its function as a repressor, its stability, its ability to bind other proteins, and its activity in gas sensing. Rev-Erbß binds Fe3+-heme more tightly than Fe2+-heme, suggesting its activities may be regulated by the heme redox state. Yet, this critical role of heme redox chemistry in defining the protein's resting state and function is unknown. We demonstrate by electrochemical and whole-cell electron paramagnetic resonance experiments that Rev-Erbß exists in the Fe3+ form within the cell allowing the protein to be heme replete even at low concentrations of labile heme in the nucleus. However, being in the Fe3+ redox state contradicts Rev-Erb's known function as a gas sensor, which dogma asserts must be Fe2+ This paper explains why the resting Fe3+ state is congruent both with heme binding and cellular gas sensing. We show that the binding of CO/NO elicits a striking increase in the redox potential of the Fe3+/Fe2+ couple, characteristic of an EC mechanism in which the unfavorable Electrochemical reduction of heme is coupled to the highly favorable Chemical reaction of gas binding, making the reduction spontaneous. Thus, Fe3+-Rev-Erbß remains heme-loaded, crucial for its repressor activity, and undergoes reduction when diatomic gases are present. This work has broad implications for proteins in which ligand-triggered redox changes cause conformational changes influencing its function or interprotein interactions (e.g., between NCoR1 and Rev-Erbß). This study opens up the possibility of CO/NO-mediated regulation of the circadian rhythm through redox changes in Rev-Erbß.

Ru(II) catalyzed chelation assisted C(sp2)-H bond functionalization along with concomitant (4 + 2) annulation.

Efficacious protocols have been established to synthesize a structurally privileged Π-extended coumarin-fused pyridone nucleus by activating the vinylic C(sp2)-H bond of coumarin-3-carboxamide under the influence of inexpensive Ru(II)-metal. Here an N-methoxy carboxamide entity has been exploited as the chelating fragment to manifest C(sp2)-H bond functionalization with a concomitant (4 + 2) annulation reaction, resulting in heterocyclic ring-forming protocols along with sulfoxonium ylide and iodonium ylide as representative bench-stable carbene surrogates. This diverse heterocycle formation via carbene insertion strategies, is further expanded to activate the ortho-C(sp2)-H bonds of different heterocycles by employing the sp2-N moiety as the directing group to develop acyl-alkylated/alkenylated quinazolines, isoxazoles and highly fluorescent pyridone-N-oxides. Intriguingly, during an evaluation of the versatility of the current protocols, a one-pot double C-H activation has been rationalized in the presence of iodonium ylide, which results in biologically potent benzimidazole-fused coumarin-centered bridge-headed polycyclic heteroarenes. Furthermore, a chemo-selective late-stage synthetic transformation is being designed to develop differently substituted pyridone analogues by switching the nature of the reducing agent. In addition, a photophysical experiment was done on one pyridine-N-oxide compound (7e) and delightfully it exhibited fluorescence quenching activity selectively in the presence of Al3+ ions, which appears to be a unique feature of our methodology. Finally, upon correlation of the merit of the developed pathways, the iodonium ylide mediated strategy appears to be superior.

Manganese is a Deinococcus radiodurans growth limiting factor in rich culture medium.

To understand the effects triggered by Mn2+ on Deinococcus radiodurans, the proteome patterns associated with different growth phases were investigated. In particular, under physiological conditions we tested the growth rate and the biomass yield of D. radiodurans cultured in rich medium supplemented or not with MnCl2. The addition of 2.5-5.0 µM MnCl2 to the medium neither altered the growth rate nor the lag phase, but significantly increased the biomass yield. When higher MnCl2 concentrations were used (10-250 µM), biomass was again found to be positively affected, although we did observe a concentration-dependent lag phase increase. The in vivo concentration of Mn2+ was determined in cells grown in rich medium supplemented or not with 5 µM MnCl2. By atomic absorption spectroscopy, we estimated 0.2 and 0.75 mM Mn2+ concentrations in cells grown in control and enriched medium, respectively. We qualitatively confirmed this observation using a fluorescent turn-on sensor designed to selectively detect Mn2+in vivo. Finally, we investigated the proteome composition of cells grown for 15 or 19 h in medium to which 5 µM MnCl2 was added, and we compared these proteomes with those of cells grown in the control medium. The presence of 5 µM MnCl2 in the culture medium was found to alter the pI of some proteins, suggesting that manganese affects post-translational modifications. Further, we observed that Mn2+ represses enzymes linked to nucleotide recycling, and triggers overexpression of proteases and enzymes linked to the metabolism of amino acids.

A Sensitive Water-Soluble Reversible Optical Probe for Hg2+ Detection.

We report the serendipitous discovery of an optical mercury sensor while trying to develop a water-soluble manganese probe. The sensor is based on a pentaaza macrocycle conjugated to a hemicyanine dye. The pentaaza macrocycle earlier designed in our group was used to develop photoinduced electron transfer (PET)-based "turn-on" fluorescent sensors for manganese. (1) In an attempt to increase the water-solubility of the manganese sensors we changed the dye from BODIPY to hemicyanine. The resultant molecule qHCM afforded a distinct reversible change in the absorption features and a concomitant visible color change upon binding to Hg2+ ions, leading to a highly water-soluble mercury sensor with a 10 ppb detection limit. The molecule acts as a reversible "ON-OFF" fluorescent sensor for Hg2+ with a 35 times decrease in the emission intensity in the presence of 1 equiv of Hg2+ ions. We have demonstrated the applicability of the probe for detecting Hg2+ ions in living cells and in live zebrafish larvae using confocal fluorescence microscopy with visible excitation. High selectivity and sensitivity toward Hg2+ detection make qHCM an attractive probe for detecting Hg2+ in contaminated water sources, which is a major environmental toxicity concern. We have scrutinized the altered metal-ion selectivity of the probe using density functional theory (DFT) and time-dependent DFT calculations, which show that a PET-based metal-sensing scheme is not operational in qHCM. 1H NMR studies and DFT calculations indicate that Hg2+ ions coordinate to oxygen-donor atoms from both the chromophore and macrocycle, leading to sensitive mercury detection.

Transcription factor Lhx2 is necessary and sufficient to suppress astrogliogenesis and promote neurogenesis in the developing hippocampus.

The sequential production of neurons and astrocytes from neuroepithelial precursors is a fundamental feature of central nervous system development. We report that LIM-homeodomain (LIM-HD) transcription factor Lhx2 regulates this transition in the developing hippocampus. Disrupting Lhx2 function in the embryonic hippocampus by in utero electroporation and in organotypic slice culture caused the premature production of astrocytes at stages when neurons are normally generated. Lhx2 function is therefore necessary to suppress astrogliogenesis during the neurogenic period. Furthermore, Lhx2 overexpression was sufficient to suppress astrogliogenesis and prolong the neurogenic period. We provide evidence that Lhx2 overexpression can counteract the instructive astrogliogenic effect of Notch activation. Lhx2 overexpression was also able to override and suppress the activation of the GFAP promoter by Nfia, a Notch-regulated transcription factor that is required for gliogenesis. Thus, Lhx2 appears to act as a "brake" on Notch/Nfia-mediated astrogliogenesis. This critical role for Lhx2 is spatially restricted to the hippocampus, because loss of Lhx2 function in the neocortex did not result in premature astrogliogenesis at the expense of neurogenesis. Our results therefore place Lhx2 as a central regulator of the neuron-glia cell fate decision in the hippocampus and reveal a striking regional specificity of this fundamental function within the dorsal telencephalon.

NFIA controls telencephalic progenitor cell differentiation through repression of the Notch effector Hes1.

The balance between self-renewal and differentiation of neural progenitor cells is an absolute requirement for the correct formation of the nervous system. Much is known about both the pathways involved in progenitor cell self-renewal, such as Notch signaling, and the expression of genes that initiate progenitor differentiation. However, whether these fundamental processes are mechanistically linked, and specifically how repression of progenitor self-renewal pathways occurs, is poorly understood. Nuclear factor I A (Nfia), a gene known to regulate spinal cord and neocortical development, has recently been implicated as acting downstream of Notch to initiate the expression of astrocyte-specific genes within the cortex. Here we demonstrate that, in addition to activating the expression of astrocyte-specific genes, Nfia also downregulates the activity of the Notch signaling pathway via repression of the key Notch effector Hes1. These data provide a significant conceptual advance in our understanding of neural progenitor differentiation, revealing that a single transcription factor can control both the activation of differentiation genes and the repression of the self-renewal genes, thereby acting as a pivotal regulator of the balance between progenitor and differentiated cell states.

N,N-Dimethyl-ethane-1,2-diaminium bis-(3-hy-droxy-benzoate).

In the title compound, C(4)H(14)N(2) (2+)·2C(7)H(5)O(3) (-), both the N,N-dimethyl-ethylenediamine N atoms are protonated and two 3-hy-droxy-benzoate anions act as counter-ions. In the crystal, anions and cations are linked by a network of N-H⋯O and O-H⋯O hydrogen bonds.

Tris(dicyclo-hexyl-ammonium) hydrogen [1-hy-droxy-2-(1H-imidazol-1-yl)-1-phospho-natoethane]-phospho-nate ethanol monosolvate mono-hydrate.

In the title compound, 3C(12)H(24)N(+)·C(5)H(7)N(2)O(7)P(2) (3-)·C(2)H(6)O·H(2)O, the zoledronic acid mol-ecule is singly protonated and stabilized by an intra-molecular O-H⋯O inter-action. The three-dimensional crystal structure is stabilized by inter-molecular O-H⋯O, O-H⋯N and N-H⋯O inter-actions. The ethanol solvent mol-ecule is disordered over two positions; the site-occupancy factor of the major component is 0.510 (4).

A Physiological Instability Displayed in Hippocampal Neurons Derived From Lithium-Nonresponsive Bipolar Disorder Patients.

BACKGROUND: We recently reported a hyperexcitability phenotype displayed in dentate gyrus granule neurons derived from patients with bipolar disorder (BD) as well as a hyperexcitability that appeared only in CA3 pyramidal hippocampal neurons that were derived from patients with BD who responded to lithium treatment (lithium responders) and not in CA3 pyramidal hippocampal neurons that were derived from patients with BD who did not respond to lithium (nonresponders). METHODS: Here we used our measurements of currents in neurons derived from 4 control subjects, 3 patients with BD who were lithium responders, and 3 patients with BD who were nonresponders. We changed the conductances of simulated dentate gyrus and CA3 hippocampal neurons according to our measurements to derive a numerical simulation for BD neurons. RESULTS: The computationally simulated BD dentate gyrus neurons had a hyperexcitability phenotype similar to the experimental results. Only the simulated BD CA3 neurons derived from lithium responder patients were hyperexcitable. Interestingly, our computational model captured a physiological instability intrinsic to hippocampal neurons that were derived from nonresponder patients that we also observed when re-examining our experimental results. This instability was caused by a drastic reduction in the sodium current, accompanied by an increase in the amplitude of several potassium currents. These baseline alterations caused nonresponder BD hippocampal neurons to drastically shift their excitability with small changes to their sodium currents, alternating between hyperexcitable and hypoexcitable states. CONCLUSIONS: Our computational model of BD hippocampal neurons that was based on our measurements reproduced the experimental phenotypes of hyperexcitability and physiological instability. We hypothesize that the physiological instability phenotype strongly contributes to affective lability in patients with BD.

Mechanisms Underlying the Hyperexcitability of CA3 and Dentate Gyrus Hippocampal Neurons Derived From Patients With Bipolar Disorder.

BACKGROUND: Approximately 1 in every 50 to 100 people is affected with bipolar disorder (BD), making this disease a major economic burden. The introduction of induced pluripotent stem cell methodology enabled better modeling of this disorder. METHODS: Having previously studied the phenotype of dentate gyrus granule neurons, we turned our attention to studying the phenotype of CA3 hippocampal pyramidal neurons of 6 patients with BD compared with 4 control individuals. We used patch clamp and quantitative polymerase chain reaction to measure electrophysiological features and RNA expression by specific channel genes. RESULTS: We found that BD CA3 neurons were hyperexcitable only when they were derived from patients who responded to lithium; they featured sustained activity with large current injections and a large, fast after-hyperpolarization, similar to what we previously reported in dentate gyrus neurons. The higher amplitudes and faster kinetics of fast potassium currents correlated with this hyperexcitability. Further supporting the involvement of potassium currents, we observed an overexpression of KCNC1 and KCNC2 in hippocampal neurons derived from lithium responders. Applying specific potassium channel blockers diminished the hyperexcitability. Long-term lithium treatment decreased the hyperexcitability observed in the CA3 neurons derived from lithium responders while increasing sodium currents and reducing fast potassium currents. When differentiating this cohort into spinal motor neurons, we did not observe any changes in the excitability of BD motor neurons compared with control motor neurons. CONCLUSIONS: The hyperexcitability of BD neurons is neuronal type specific with the involvement of altered potassium currents that allow for a sustained, continued firing activity.

Specific glial populations regulate hippocampal morphogenesis.

The hippocampus plays an integral role in spatial navigation, learning and memory, and is a major site for adult neurogenesis. Critical to these functions is the proper organization of the hippocampus during development. Radial glia are known to regulate hippocampal formation, but their precise function in this process is yet to be defined. We find that in Nuclear Factor I b (Nfib)-deficient mice, a subpopulation of glia from the ammonic neuroepithelium of the hippocampus fail to develop. This results in severe morphological defects, including a failure of the hippocampal fissure, and subsequently the dentate gyrus, to form. As in wild-type mice, immature nestin-positive glia, which encompass all types of radial glia, populate the hippocampus in Nfib-deficient mice at embryonic day 15. However, these fail to mature into GLAST- and GFAP-positive glia, and the supragranular glial bundle is absent. In contrast, the fimbrial glial bundle forms, but alone is insufficient for proper hippocampal morphogenesis. Dentate granule neurons are present in the mutant hippocampus but their migration is aberrant, likely resulting from the lack of the complete radial glial scaffold usually provided by both glial bundles. These data demonstrate a role for Nfib in hippocampal fissure and dentate gyrus formation, and that distinct glial bundles are critical for correct hippocampal morphogenesis.

Emerging chemical tools and techniques for tracking biological manganese.

Recent developments in Mn biology have added new physiological and pathophysiological roles of this essential metal ion to the already existing repertoire of indispensable biological roles of Mn ions. Notably, the discovery of Mn2+ specific transporters, maladies related to mutations in these transporters, and evidence of the role of labile Mn2+ species as anti-oxidants have initiated studies targeted at elucidating Mn ion regulation and pathways implicated in pathological conditions. Closely inter-linked with the quest for understanding metal ion homeostasis are basic questions like "How are metal ions installed in their correct biological addresses where they need to function?" and "Are dynamic changes in metal ion distribution functionally relevant?" These questions become more critical in the context of Mn2+ ions, which have inherently low binding affinities toward most ligands and hence would always face competing metal ions in the biological milieu. In the emerging context of functional roles of the labile Mn2+ ion pool, the development of chemical tools and techniques that can provide information on the location, distribution and dynamic changes in these parameters under physiological and pathophysiological conditions becomes imperative. In this frontier article, we discuss the challenges that had left Mn2+ ions lagging behind in the race for the development of chemical tools and recent approaches that addressed these challenges to develop tools and techniques that can illuminate Mn ions in living systems.

Author Correction: L1-associated genomic regions are deleted in somatic cells of the healthy human brain.

In the version of this article initially published, NIH grant U01 MH106882 to F.H.G. was missing from the Acknowledgments. The error has been corrected in the HTML and PDF versions of the article.

Efficient Generation of CA3 Neurons from Human Pluripotent Stem Cells Enables Modeling of Hippocampal Connectivity In Vitro.

Despite widespread interest in using human induced pluripotent stem cells (hiPSCs) in neurological disease modeling, a suitable model system to study human neuronal connectivity is lacking. Here, we report a comprehensive and efficient differentiation paradigm for hiPSCs that generate multiple CA3 pyramidal neuron subtypes as detected by single-cell RNA sequencing (RNA-seq). This differentiation paradigm exhibits characteristics of neuronal network maturation, and rabies virus tracing revealed synaptic connections between stem cell-derived dentate gyrus (DG) and CA3 neurons in vitro recapitulating the neuronal connectivity within the hippocampus. Because hippocampal dysfunction has been implicated in schizophrenia, we applied DG and CA3 differentiation paradigms to schizophrenia-patient-derived hiPSCs. We detected reduced activity in DG-CA3 co-culture and deficits in spontaneous and evoked activity in CA3 neurons from schizophrenia-patient-derived hiPSCs. Our approach offers critical insights into the network activity aspects of schizophrenia and may serve as a promising tool for modeling diseases with hippocampal vulnerability. VIDEO ABSTRACT.

Synaptic activity: An emerging player in schizophrenia.

Schizophrenia is a polygenic disorder with a complex etiology. While the genetic and molecular underpinnings of the disease are poorly understood, variations in genes encoding synaptic pathways are consistently implicated. Although its impact is still an open question, a deficit in synaptic activity provides an attractive model to explain the cognitive etiology of schizophrenia. Recent advances in high-throughput imaging and functional studies bring new hope for the application of in vitro disease modeling with patient-derived neurons to empirically ascertain the extent to which these synaptic pathways are involved in the disease. In addition, the emergent avenue of research targeted to probe neuronal connections is revealing critical insight into circuitry and may influence how we think about psychiatric disorders in the near future. This article is part of a Special Issue entitled SI: Exploiting human neurons.

Functional Implications of miR-19 in the Migration of Newborn Neurons in the Adult Brain.

Altered microRNA profiles have been implicated in human brain disorders. However, the functional contribution of individual microRNAs to neuronal development and function is largely unknown. Here, we report biological functions for miR-19 in adult neurogenesis. We determined that miR-19 is enriched in neural progenitor cells (NPCs) and downregulated during neuronal development in the adult hippocampus. By manipulating miR-19 in NPCs for gain- and loss-of-function studies, we discovered that miR-19 regulates cell migration by directly targeting Rapgef2. Concordantly, dysregulation of miR-19 in NPCs alters the positioning of newborn neurons in the adult brain. Furthermore, we found abnormal expression of miR-19 in human NPCs generated from schizophrenic patient-derived induced pluripotent stem cells (iPSCs) that have been described as displaying aberrant migration. Our study demonstrates the significance of posttranscriptional gene regulation by miR-19 in preventing the irregular migration of adult-born neurons that may contribute to the etiology of schizophrenia.

L1-associated genomic regions are deleted in somatic cells of the healthy human brain.

The healthy human brain is a mosaic of varied genomes. Long interspersed element-1 (LINE-1 or L1) retrotransposition is known to create mosaicism by inserting L1 sequences into new locations of somatic cell genomes. Using a machine learning-based, single-cell sequencing approach, we discovered that somatic L1-associated variants (SLAVs) are composed of two classes: L1 retrotransposition insertions and retrotransposition-independent L1-associated variants. We demonstrate that a subset of SLAVs comprises somatic deletions generated by L1 endonuclease cutting activity. Retrotransposition-independent rearrangements in inherited L1s resulted in the deletion of proximal genomic regions. These rearrangements were resolved by microhomology-mediated repair, which suggests that L1-associated genomic regions are hotspots for somatic copy number variants in the brain and therefore a heritable genetic contributor to somatic mosaicism. We demonstrate that SLAVs are present in crucial neural genes, such as DLG2 (also called PSD93), and affect 44-63% of cells of the cells in the healthy brain.