Electrospray ionization (ESI) generates intact gas-phase ions from analytes in solution for mass spectrometric investigations. ESI can proceed via different mechanisms. Low molecular weight analytes ...follow the ion evaporation model (IEM), whereas the charged residue model (CRM) applies to large globular species. A chain ejection model (CEM) has been proposed for disordered polymers.
Electrospray ionization (ESI) of native proteins results in a narrow distribution of low protonation states. ESI for these folded species proceeds via the charged residue mechanism. In contrast, ESI ...of unfolded proteins yields a wide distribution of much higher charge states. The current work develops a model that can account for this effect. Recent molecular dynamics simulations revealed that ESI for unfolded polypeptide chains involves protein ejection from nanodroplets, representing a type of ion evaporation mechanism (IEM). We point out the analogies between this IEM, and the dissociation of gaseous protein complexes after collisional activation. The latter process commences with unraveling of a single subunit, in concert with Coulombically driven proton transfer. The subunit then separates from the residual complex as a highly charged ion. We propose that similar charge equilibration events accompany the IEM of unfolded proteins, thereby causing the formation of high ESI charge states. A bead chain model is used for examining how charge is partitioned as protein and droplet separate. It is shown that protein ejection from differently sized ESI droplets generates a range of protonation states. The predicted behavior agrees well with experimental data.
To derive a global perspective on the transcription of microRNAs (miRNAs) in mammals, we annotated the genomic position and context of this class of noncoding RNAs (ncRNAs) in the human and mouse ...genomes. Of the 232 known mammalian miRNAs, we found that 161 overlap with 123 defined transcription units (TUs). We identified miRNAs within introns of 90 protein-coding genes with a broad spectrum of molecular functions, and in both introns and exons of 66 mRNA-like noncoding RNAs (mlncRNAs). In addition, novel families of miRNAs based on host gene identity were identified. The transcription patterns of all miRNA host genes were curated from a variety of sources illustrating spatial, temporal, and physiological regulation of miRNA expression. These findings strongly suggest that miRNAs are transcribed in parallel with their host transcripts, and that the two different transcription classes of miRNAs ('exonic' and 'intronic') identified here may require slightly different mechanisms of biogenesis.
Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) is a widely used technique for probing protein structure and dynamics. Exposure to D2O induces the deuteration of backbone N-H groups via a ...process that involves transient excursions to partially unfolded protein conformers. The resulting mass shifts can be probed by MS, usually in combination with proteolytic digestion and/or electron-based fragmentation. Studies on protein-ligand complexes represent a particularly important HDX/MS application. The prevailing view is that ligand binding should reduce deuteration rates, and it is often expected that this reduction will be most pronounced in the vicinity of the interaction site. Many protein-ligand systems do indeed behave in a fashion that is consistent with this paradigm. In this review we point out that the opposite effect may be encountered as well. Also, mixed scenarios are possible where ligand binding induces elevated HDX rates in some protein regions, whereas rates in other segments are reduced. We present a framework that links ligand-induced changes in HDX kinetics to alterations in the occupancy of excited protein conformers. Spontaneous ligand binding will always lower the free energy of the ground state. In contrast, the corresponding free energy shifts of excited states are largely unpredictable, giving rise to a range of possible HDX responses. "Type 1" scenarios, characterized by a reduction of HDX rates are just as feasible as "Type 2" behavior where deuteration is accelerated. Even "Type 0" phenomena may be encountered, where HDX rates are unaffected by the presence of ligand. Type 0/1/2 scenarios can coexist in the same protein (these terms are not to be confused with the EX1/EX2 expressions which refer to a different aspect of protein HDX). Allosteric effects and ligand-induced protein-protein contacts can affect the outcome of protein-ligand binding studies as well. In summary, comparative HDX measurements conducted in the presence and in the absence ligand provide a detailed fingerprint of biomolecular interactions. However, protein-ligand interactions can elicit a wide range of responses, and the interpretation of binding site mapping experiments may not always be straightforward.
For appropriate development, tissue and organ system morphogenesis and maturation must occur in synchrony with the overall developmental requirements of the host. Mistiming of such developmental ...events often results in disease. The hematopoietic system matures from the fetal state, characterized by robust erythrocytic output that supports prenatal growth in the hypoxic intrauterine environment, to the postnatal state wherein granulocytes predominate to provide innate immunity. Regulation of the developmental timing of these myeloerythroid states is not well understood. In this study, we find that expression of the heterochronic factor Lin28b decreases in common myeloid progenitors during hematopoietic maturation to adulthood in mice. This decrease in Lin28b coincides with accumulation of mature let-7 microRNAs, whose biogenesis is regulated by Lin28 proteins. We find that inhibition of let-7 in the adult hematopoietic system recapitulates fetal erythroid-dominant hematopoiesis. Conversely, deletion of Lin28b or ectopic activation of let-7 microRNAs in the fetal state induces a shift toward adult-like myeloid-dominant output. Furthermore, we identify Hmga2 as an effector of this genetic switch. These studies provide the first detailed analysis of the roles of endogenous Lin28b and let-7 in the timing of hematopoietic states during development.
microRNA-155 (miR-155) is expressed by cells of the immune system after activation and has been shown to be required for antibody production after vaccination with attenuated
Salmonella. Here we show ...the intrinsic requirement for miR-155 in B cell responses to thymus-dependent and -independent antigens. B cells lacking miR-155 generated reduced extrafollicular and germinal center responses and failed to produce high-affinity IgG1 antibodies. Gene-expression profiling of activated B cells indicated that miR-155 regulates an array of genes with diverse function, many of which are predicted targets of miR-155. The transcription factor Pu.1 is validated as a direct target of miR155-mediated inhibition. When Pu.1 is overexpressed in wild-type B cells, fewer IgG1 cells are produced, indicating that loss of Pu.1 regulation is a contributing factor to the miR-155-deficient phenotype. Our results implicate post-transcriptional regulation of gene expression for establishing the terminal differentiation program of B cells.
Delineating the role of microRNAs (miRNAs) in the posttranscriptional gene regulation offers new insights into how the heart adapts to pathological stress. We developed a knockout of miR-22 in mice ...and investigated its function in the heart.
Here, we show that miR-22-deficient mice are impaired in inotropic and lusitropic response to acute stress by dobutamine. Furthermore, the absence of miR-22 sensitized mice to cardiac decompensation and left ventricular dilation after long-term stimulation by pressure overload. Calcium transient analysis revealed reduced sarcoplasmic reticulum Ca(2+) load in association with repressed sarcoplasmic reticulum Ca(2+) ATPase activity in mutant myocytes. Genetic ablation of miR-22 also led to a decrease in cardiac expression levels for Serca2a and muscle-restricted genes encoding proteins in the vicinity of the cardiac Z disk/titin cytoskeleton. These phenotypes were attributed in part to inappropriate repression of serum response factor activity in stressed hearts. Global analysis revealed increased expression of the transcriptional/translational repressor purine-rich element binding protein B, a highly conserved miR-22 target implicated in the negative control of muscle expression.
These data indicate that miR-22 functions as an integrator of Ca(2+) homeostasis and myofibrillar protein content during stress in the heart and shed light on the mechanisms that enhance propensity toward heart failure.
Environmental air irritants including nanosized carbon black (nCB) can drive systemic inflammation, promoting chronic obstructive pulmonary disease (COPD) and emphysema development. The
microRNA (
...miRNA) family is associated with IL-17-driven T cell inflammation, a canonical signature of lung inflammation. Recent evidence suggests the
family is downregulated in patients with COPD, however, whether this repression conveys a functional consequence on emphysema pathology has not been elucidated. Here, we show that overall expression of the
clusters,
and
, are reduced in the lungs and T cells of smokers with emphysema as well as in mice with cigarette smoke (CS)- or nCB-elicited emphysema. We demonstrate that loss of the
cluster in T cells predisposed mice to exaggerated CS- or nCB-elicited emphysema. Furthermore, ablation of the
cluster enhanced CD8
IL17a
T cells (Tc17) formation in emphysema development in mice. Additionally, transgenic mice overexpressing
in T cells are resistant to Tc17 and CD4
IL17a
T cells (Th17) development when exposed to nCB. Mechanistically, our findings reveal the master regulator of Tc17/Th17 differentiation, RAR-related orphan receptor gamma t (RORγt), as a direct target of
in T cells. Overall, our findings shed light on the
RORγt axis with
acting as a molecular brake in the generation of Tc17 cells and suggest a novel therapeutic approach for tempering the augmented IL-17-mediated response in emphysema.
Increasing evidence suggests that microRNAs are intimately involved in the pathophysiology of heart failure. MicroRNA-22 (miR-22) is a muscle-enriched miRNA required for optimum cardiac gene ...transcription and adaptation to hemodynamic stress by pressure overload in mice. Recent evidence also suggests that miR-22 induces hypertrophic growth and it is oftentimes upregulated in end stage heart failure. However the scope of mRNA targets and networks of miR-22 in the heart failure remained unclear. We analyzed transgenic mice with enhanced levels of miR-22 expression in adult cardiomyocytes to identify important pathophysiologic targets of miR-22. Our data shows that forced expression of miR-22 induces a pro-hypertrophic gene expression program, and it elicits contractile dysfunction leading to cardiac dilation and heart failure. Increased expression of miR-22 impairs the Ca(2+) transient, Ca(2+) loading into the sarcoplasmic reticulum plus it interferes with transcription of estrogen related receptor (ERR) and PPAR downstream genes. Mechanistically, miR-22 postranscriptionally inhibits peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α), PPARα and sirtuin 1 (SIRT1) expression via a synergistic circuit, which may account for deleterious actions of unchecked miR-22 expression on the heart.
Accumulating evidence suggests that microRNAs (miRNAs) contribute to a myriad of kidney diseases. However, the regulatory role of miRNAs on the key molecules implicated in kidney fibrosis remains ...poorly understood. Bone morphogenetic protein-7 (BMP-7) and its related BMP-6 have recently emerged as key regulators of kidney fibrosis. Using the established unilateral ureteral obstruction (UUO) model of kidney fibrosis as our experimental model, we examined the regulatory role of miRNAs on BMP-7/6 signaling. By analyzing the potential miRNAs that target BMP-7/6 in silica, we identified miR-22 as a potent miRNA targeting BMP-7/6. We found that expression levels of BMP-7/6 were significantly elevated in the kidneys of the miR-22 null mouse. Importantly, mice with targeted deletion of miR-22 exhibited attenuated renal fibrosis in the UUO model. Consistent with these in vivo observations, primary renal fibroblast isolated from miR-22-deficient UUO mice demonstrated a significant increase in BMP-7/6 expression and their downstream targets. This phenotype could be rescued when cells were transfected with miR-22 mimics. Interestingly, we found that miR-22 and BMP-7/6 are in a regulatory feedback circuit, whereby not only miR-22 inhibits BMP-7/6, but miR-22 by itself is induced by BMP-7/6. Finally, we identified two BMP-responsive elements in the proximal region of miR-22 promoter. These findings identify miR-22 as a critical miRNA that contributes to renal fibrosis on the basis of its pivotal role on BMP signaling cascade.
Background: BMPs have emerged as key regulators of kidney fibrosis.
Results: Deletion of miR-22 attenuated renal fibrosis via direct targeting of BMP-7/6. BMP-7/6 in turn transcriptionally regulates miR-22 expression.
Conclusion: miR-22 and BMP-7/6 form a negative feedback circuit.
Significance: The present study is the first to report the effect of miR-22 on BMP signaling and kidney fibrosis.
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