A new class of repression modules is critical for heme regulation of the yeast transcriptional activator Hap1.

Heme plays key regulatory roles in numerous molecular and cellular processes for systems that sense or use oxygen. In the yeast Saccharomyces cerevisiae, oxygen sensing and heme signaling are mediated by heme activator protein 1 (Hap1). Hap1 contains seven heme-responsive motifs (HRMs): six are clustered in the heme domain, and a seventh is near the activation domain. To determine the functional role of HRMs and to define which parts of Hap1 mediate heme regulation, we carried out a systematic analysis of Hap1 mutants with various regions deleted or mutated. Strikingly, the data show that HRM1 to -6, located in the previously designated Hap1 heme domain, have little impact on heme regulation. All seven HRMs are dispensable for Hap1 repression in the absence of heme, but HRM7 is required for Hap1 activation by heme. More importantly, we show that a novel class of repression modules-RPM1, encompassing residues 245 to 278; RPM2, encompassing residues 1061 to 1185; and RPM3, encompassing residues 203 to 244-is critical for Hap1 repression in the absence of heme. Biochemical analysis indicates that RPMs mediate Hap1 repression, at least partly, by the formation of a previously identified higher-order complex termed the high-molecular-weight complex (HMC), while HRMs mediate heme activation by permitting heme binding and the disassembly of the HMC. These findings provide significant new insights into the molecular interactions critical for Hap1 repression in the absence of heme and Hap1 activation by heme.     

Spectroscopic insights into axial ligation and active-site H-bonding in substrate-bound human heme oxygenase-2

Heme oxygenases (HOs) are monooxygenases that catalyze the first step in heme degradation, converting heme to biliverdin with concomitant release of Fe(II) and CO from the porphyrin macrocycle. Two heme oxygenase isoforms, HO-1 and HO-2, exist that differ in several ways, including a complete lack of Cys residues in HO-1 and the presence of three Cys residues as part of heme-regulatory motifs (HRMs) in HO-2. HRMs in other heme proteins are thought to directly bind heme, or to otherwise regulate protein stability or activity; however, it is not currently known how the HRMs exert these effects on HO-2 function. To better understand the properties of this vital enzyme and to elucidate possible roles of its HRMs, various forms of HO-2 possessing distinct alterations to the HRMs were prepared. In this study, variants with Cys265 in a thiol form are compared with those with this residue in an oxidized (part of a disulfide bond or existing as a sulfenate moiety) form. Absorption and magnetic circular dichroism spectroscopic data of these HO-2 variants clearly demonstrate that a new low-spin Fe(III) heme species characteristic of thiolate ligation is formed when Cys265 is reduced. Additionally, absorption, magnetic circular dichroism, and resonance Raman data collected at different temperatures reveal an intriguing temperature dependence of the iron spin state in the heme–HO-2 complex. These findings are consistent with the presence of a hydrogen-bonding network at the heme’s distal side within the active site of HO-2 with potentially significant differences from that observed in HO-1.     

Structural environment dictates the biological significance of heme-responsive motifs and the role of Hsp90 in the activation of the heme activator protein Hap1

Heme-responsive motifs (HRMs) mediate heme regulation of diverse regulatory proteins. The heme activator protein Hap1 contains seven HRMs, but only one of them, HRM7, is essential for heme activation of Hap1. To better understand the molecular basis underlying the biological significance of HRMs, we examined the effects of various mutations of HRM7 on Hap1. We found that diverse mutations of HRM7 significantly diminished the extent of Hap1 activation by heme and moderately enhanced the interaction of Hap1 with Hsp90. Furthermore, deletions of nonregulatory sequences completely abolished heme activation of Hap1 and greatly enhanced the interaction of Hap1 with Hsp90. These results show that the biological functions of HRMs and Hsp90 are highly sensitive to structural changes. The unique role of HRM7 in heme activation stems from its specific structural environment, not its mere presence. Likewise, the role of Hsp90 in Hap1 activation is dictated by the conformational or structural state of Hap1, not by the mere strength of Hap1-Hsp90 interaction. It appears likely that HRM7 and Hsp90 act together to promote the Hap1 conformational changes that are necessary for Hap1 activation. Such fundamental mechanisms of HRM-Hsp90 cooperation may operate in diverse regulatory systems to mediate signal transduction.     

Evidence that the heme regulatory motifs in heme oxygenase-2 serve as a thiol/disulfide redox switch regulating heme binding

Heme oxygenase (HO) catalyzes the O(2)- and NADPH-dependent conversion of heme to biliverdin, CO, and iron. The two forms of HO (HO-1 and HO-2) share similar physical properties but are differentially regulated and exhibit dissimilar physiological roles and tissue distributions. Unlike HO-1, HO-2 contains heme regulatory motifs (HRMs) (McCoubrey, W. K., Jr., Huang, T. J., and Maines, M. D. (1997) J. Biol. Chem. 272, 12568-12574). Here we describe UV-visible, EPR, and differential scanning calorimetry experiments on human HO-2 variants containing single, double, and triple mutations in the HRMs. Oxidized HO-2, which contains an intramolecular disulfide bond linking Cys(265) of HRM1 and Cys(282) of HRM2, binds heme tightly. Reduction of the disulfide bond increases the K(d) for ferric heme from 0.03 to 0.3 microm, which is much higher than the concentration of the free heme pool in cells. Although the HRMs markedly affect the K(d) for heme, they do not alter the k(cat) for heme degradation and do not bind additional hemes. Because HO-2 plays a key role in CO generation and heme homeostasis, reduction of the disulfide bond would be expected to increase intracellular free heme and decrease CO concentrations. Thus, we propose that the HRMs in HO-2 constitute a thiol/disulfide redox switch that regulates the myriad physiological functions of HO-2, including its involvement in the hypoxic response in the carotid body, which involves interactions with a Ca(2+)-activated potassium channel.     

Role of the heme regulatory motif in the heme-mediated inhibition of mitochondrial import of 5-aminolevulinate synthase

5-Aminolevulinate synthase (ALAS) is a mitochondrial enzyme that catalyzes the first step of the heme biosynthetic pathway. The mitochondrial import, as well as the synthesis, of the nonspecific isoform of ALAS (ALAS1) is regulated by heme through a feedback mechanism. A short amino acid sequence, the heme regulatory motif (HRM), is known to be involved in the regulatory function of heme. To determine the role of the HRM in the heme-regulated transport of the nonspecific and erythroid forms of ALAS in vivo, we constructed a series of mutants of rat ALAS1, in which the cysteine residues in the three putative HRMs in the N-terminal region of the enzyme were converted to serine ones by site-directed mutagenesis. The wild-type and mutant enzymes were expressed in quail QT6 fibroblasts through transient transfection, and the mitochondrial import of these enzymes was examined in the presence of hemin. Hemin inhibited the mitochondrial import of wild-type ALAS1, but this inhibition was reversed on the mutation of all three HRMs in the enzyme, indicating that the HRMs are essential for the heme-mediated inhibition of ALAS1 transport in the cell. By contrast, exogenous hemin did not affect the mitochondrial import of the erythroid-specific ALAS isoform (ALAS2) under the same experimental conditions. These results may reflect the difference in the physiological functions of the two ALAS isoforms.     

Recombination rates of human microRNA

The fact that microRNAs play a role in almost all biological processes is well established, as is the importance of recombination in generating genome variability. However, the association between microRNAs and recombination remains largely unknown. In order to investigate the recombination patterns of microRNAs, we performed a comprehensive analysis of the recombination rate of human microRNAs. We observed that microRNAs that are expressed in several tissues tend to have lower recombination rates than tissue-specific microRNAs. Additionally, microRNAs that are associated with a number of diseases are also likely to have lower recombination rates. Furthermore, microRNAs with higher expression levels are found to have fewer recombination events. These findings reveal patterns in recombination rates of microRNAs that could help in understanding the function, evolution, and disease-related roles of microRNAs.     

High-affinity binding and catalytic activity of His/Tyr-based sequences: Extending heme-regulatory motifs beyond CP

Background & motivationPeptides and proteins can interact with heme through His, Tyr, or Cys in heme-regulatory motifs (HRMs). The Cys-Pro dipeptide is a well investigated HRM, but for His and Tyr such a distinct motif is currently unknown. In addition, many heme-peptide complexes, such as heme-amyloid β, can display a peroxidase-like activity, albeit there is little understanding of how the local primary and secondary coordination environment influences catalytic activity. We thus systematically evaluated a series of His- and Tyr-based peptides to identify sequence features for high-affinity heme binding and their impact on the catalytic activity of heme.MethodsWe employed solid-phase peptide synthesis to produce 58 nonapeptides, which were investigated by UV/vis, resonance Raman, and 2D NMR spectroscopy. A chromogenic assay was used to determine the catalytic activity of the heme-peptide complexes.ResultsHeme-binding affinity and binding mode were found to be dependent on the coordinating amino acid and spacer length between multiple potential coordination sites in a motif. In particular, HXH and HXXXH motifs showed strong heme binding. Analysis of the peroxidase-like activity revealed that some of these peptides and also HXXXY motifs enhance the catalytic activity of heme significantly.ConclusionsWe identify HXH, HXXXH, and HXXXY as potential new HRMs with functional properties. Several peptides displayed a strikingly high peroxidase-like activity.General significanceThe identification of HRMs allows to discover yet unknown heme-regulated proteins, and consequently, enhances our current understanding of pathologies involving labile heme.     

Emerging Role of microRNAs in Dementia

MicroRNAs are small non-coding RNAs regulating mRNA translation. They play a crucial role in regulating homeostasis in neurons, especially in regulating local and stimulation dependent protein synthesis. Since activity-mediated protein synthesis in neurons is critical for memory and cognition, microRNAs have become key players in modulating these processes. Dementia is a broad term used for symptoms involving decline of memory and cognition. Several studies have implicated the dysregulation of microRNAs in many brain diseases like neurodegenerative diseases, neurodevelopmental disorders, brain injuries and dementia. In this review, we give an overview of microRNA-mediated regulation of proteins and cellular processes affected in dementia pathology, hence illustrating the importance of microRNAs in normal functioning. We also focus on a relatively less explored area in dementia pathology—the importance of activity-mediated protein synthesis at the synapse and the role of microRNAs in modulating this. Overall, this review will be helpful in looking at the significance of microRNAs in dementia from the perspective of defective regulation of protein synthesis and synaptic dysfunction.     

The solution structure and heme binding of the presequence of murine 5-aminolevulinate synthase

The mitochondrial import of 5-aminolevulinate synthase (ALAS), the first enzyme of the mammalian heme biosynthetic pathway, requires the N-terminal presequence. The 49 amino acid presequence transit peptide (psALAS) for murine erythroid ALAS was chemically synthesized, and circular dichroism and (1)H nuclear magnetic resonance (NMR) spectroscopies used to determine structural elements in trifluoroethanol/H(2)O solutions and micellar environments. A well defined amphipathic alpha-helix, spanning L22 to F33, was present in psALAS in 50% trifluoroethanol. Further, a short alpha-helix, defined by A5-L8, was also apparent in the 26 amino acid N-terminus peptide, when its structure was determined in sodium dodecyl sulfate. Heme inhibition of ALAS mitochondrial import has been reported to be mediated through cysteine residues in presequence heme regulatory motifs (HRMs). A UV/visible and (1)H NMR study of hemin and psALAS indicated that a heme-peptide interaction occurs and demonstrates, for the first time, that heme interacts with the HRMs of psALAS.     

Dysregulation of cellular microRNAs by human oncogenic viruses – Implications for tumorigenesis

Infection with certain animal and human viruses, often referred to as tumor viruses, induces oncogenic processes in their host. These viruses can induce tumorigenesis through direct and/or indirect mechanisms, and the regulation of microRNAs expression has been shown to play a key role in this process. Some human oncogenic viruses can express their own microRNAs; however, they all can dysregulate the expression of cellular microRNAs, facilitating their respective life cycles. The modulation of cellular microRNAs expression brings consequences to the host cells that may lead to malignant transformation, since microRNAs regulate the expression of genes involved in oncogenic pathways. This review focus on the mechanisms used by each human oncogenic virus to dysregulate the expression of cellular microRNAs, and their impact on tumorigenesis.     

microRNAs与流感病毒感染

MicroRNAs是一类数目庞大,而且可以广泛参与到生命活动各个进程的非编码RNA分子,在病毒感染宿主过程中存在着复杂的microRNAs与病毒的相互作用。流感病毒感染可以引起宿主microRNAs表达谱的明显变化,流感病毒能通过调控某些microRNAs的表达来实现免疫逃逸等增强其感染能力;同时,宿主也可以通过某些microRNAs的变化启动相应的抗流感病毒反应。本文主要针对流感病毒感染过程中宿主-病毒二者在microRNA水平的相互作用进行综述,以期更好的了解流感病毒的致病机制,为抗流感病毒的新药研制提供新的思路。     

MicroRNAs in melanocyte and melanoma biology

The importance of microRNAs as key molecular components of cellular processes is now being recognized. Recent reports have shown that microRNAs regulate processes as diverse as protein expression and nuclear functions inside cells and are able to signal extracellularly, delivered via exosomes, to influence cell fate at a distance. The versatility of microRNAs as molecular tools inspires the design of novel strategies to control gene expression, protein stability, DNA repair and chromatin accessibility that may prove very useful for therapeutic approaches due to the extensive manageability of these small molecules. However, we still lack a comprehensive understanding of the microRNA network and its interactions with the other layers of regulatory elements in cellular and extracellular functions. This knowledge may be necessary before we exploit microRNA versatility in therapeutic settings. To identify rules of interactions between microRNAs and other regulatory systems, we begin by reviewing microRNA activities in a single cell type: the melanocyte, from development to disease.     

MicroRNA regulation of gene expression in plants

It has only been a few years since we began to appreciate that microRNAs provide an unanticipated level of gene regulation in both plants and metazoans. The high level of complementarity between plant microRNAs and their target mRNAs has allowed rapid progress towards the elucidation of their varied biological functions. MicroRNAs have been shown to regulate diverse developmental processes, including organ separation, polarity, and identity, and to modulate their own biogenesis and function. Recently, they have also been implicated in some processes outside of plant development.     

Mechanisms and role of microRNA deregulation in cancer onset and progression

MicroRNAs are key regulators of various fundamental biological processes and, although representing only a small portion of the genome, they regulate a much larger population of target genes. Mature microRNAs (miRNAs) are single-stranded RNA molecules of 20-23 nucleotide (nt) length that control gene expression in many cellular processes. These molecules typically reduce the stability of mRNAs, including those of genes that mediate processes in tumorigenesis, such as inflammation, cell cycle regulation, stress response, differentiation, apoptosis and invasion. MicroRNA targeting is mostly achieved through specific base-pairing interactions between the 5' end ('seed' region) of the miRNA and sites within coding and untranslated regions (UTRs) of mRNAs; target sites in the 3' UTR diminish mRNA stability. Since miRNAs frequently target hundreds of mRNAs, miRNA regulatory pathways are complex. Calin and Croce were the first to demonstrate a connection between microRNAs and increased risk of developing cancer, and meanwhile the role of microRNAs in carcinogenesis has definitively been evidenced. It needs to be considered that the complex mechanism of gene regulation by microRNAs is profoundly influenced by variation in gene sequence (polymorphisms) of the target sites. Thus, individual variability could cause patients to present differential risks regarding several diseases. Aiming to provide a critical overview of miRNA dysregulation in cancer, this article reviews the growing number of studies that have shown the importance of these small molecules and how these microRNAs can affect or be affected by genetic and epigenetic mechanisms.     

Distinctive microRNA signature associated of neoplasms with the Wnt/β-catenin signaling pathway

As the crucial biological regulators, microRNAs that act by suppressing their target genes are involved in a variety of pathophysiological processes. It is generally accepted that microRNAs are often dysregulated in many types of neoplasm and other human diseases. In neoplasm, microRNAs may function as oncogenes or tumor suppressors. As constitutive activation of the Wnt signaling pathway is a common feature of neoplasm and contributes to its development, progression and metastasis in various cancers, numerous studies have revealed that microRNA-mediated gene regulation are interconnected with the Wnt/β-catenin signaling pathway, forming a Wnt/β-catenin–microRNA regulatory network, which is critical to successful targeting of the Wnt/β-catenin pathway for oncotherapy. In this review, we aim to accumulate recent advances on microRNAs that work in tandem with Wnt/β-catenin signaling in tumorigenesis, with particular focus on how microRNAs affect Wnt/β-catenin activity as well as how microRNAs are regulated through the Wnt/β-catenin pathway.