Functional Characterization of Core Components of the Bacillus subtilis Cyclic-Di-GMP Signaling Pathway

Bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) is an intracellular second messenger that regulates adaptation processes, including biofilm formation, motility, and virulence in Gram-negative bacteria. In this study, we have characterized the core components of a c-di-GMP signaling pathway in the model Gram-positive bacterium Bacillus subtilis. Specifically, we have directly identified and characterized three active diguanylate cyclases, DgcP, DgcK, and DgcW (formerly YtrP, YhcK, and YkoW, respectively), one active c-di-GMP phosphodiesterase, PdeH (formerly YuxH), and a cyclic-diguanylate (c-di-GMP) receptor, DgrA (formerly YpfA). Furthermore, elevation of c-di-GMP levels in B. subtilis led to inhibition of swarming motility, whereas biofilm formation was unaffected. Our work establishes paradigms for Gram-positive c-di-GMP signaling, and we have shown that the concise signaling system identified in B. subtilis serves as a powerful heterologous host for the study of c-di-GMP enzymes from bacteria predicted to possess larger, more-complex signaling systems.     

CRIS—A Novel cAMP-Binding Protein Controlling Spermiogenesis and the Development of Flagellar Bending

The second messengers cAMP and cGMP activate their target proteins by binding to a conserved cyclic nucleotide-binding domain (CNBD). Here, we identify and characterize an entirely novel CNBD-containing protein called CRIS (cyclic nucleotide receptor involved in sperm function) that is unrelated to any of the other members of this protein family. CRIS is exclusively expressed in sperm precursor cells. Cris-deficient male mice are either infertile due to a lack of sperm resulting from spermatogenic arrest, or subfertile due to impaired sperm motility. The motility defect is caused by altered Ca²⁺ regulation of flagellar beat asymmetry, leading to a beating pattern that is reminiscent of sperm hyperactivation. Our results suggest that CRIS interacts during spermiogenesis with Ca²⁺-regulated proteins that—in mature sperm—are involved in flagellar bending.     

Coordination of Division and Development Influences Complex Multicellular Behavior in Agrobacterium tumefaciens

The α-Proteobacterium Agrobacterium tumefaciens has proteins homologous to known regulators that govern cell division and development in Caulobacter crescentus, many of which are also conserved among diverse α-Proteobacteria. In light of recent work demonstrating similarity between the division cycle of C. crescentus and that of A. tumefaciens, the functional conservation for this presumptive control pathway was examined. In C. crescentus the CtrA response regulator serves as the master regulator of cell cycle progression and cell division. CtrA activity is controlled by an integrated pair of multi-component phosphorelays: PleC/DivJ-DivK and CckA-ChpT-CtrA. Although several of the conserved orthologues appear to be essential in A. tumefaciens, deletions in pleC or divK were isolated and resulted in cell division defects, diminished swimming motility, and a decrease in biofilm formation. A. tumefaciens also has two additional pleC/divJ homologue sensor kinases called pdhS1 and pdhS2, absent in C. crescentus. Deletion of pdhS1 phenocopied the ΔpleC and ΔdivK mutants. Cells lacking pdhS2 morphologically resembled wild-type bacteria, but were decreased in swimming motility and elevated for biofilm formation, suggesting that pdhS2 may serve to regulate the motile to non-motile switch in A. tumefaciens. Genetic analysis suggests that the PleC/DivJ-DivK and CckA-ChpT-CtrA phosphorelays in A. tumefaciens are vertically-integrated, as in C. crescentus. A gain-of-function mutation in CckA (Y674D) was identified as a spontaneous suppressor of the ΔpleC motility phenotype. Thus, although the core architecture of the A. tumefaciens pathway resembles that of C. crescentus there are specific differences including additional regulators, divergent pathway architecture, and distinct target functions.     

The Linker for Activation of B Cells (LAB)/Non-T Cell Activation Linker (NTAL) Regulates Triggering Receptor Expressed on Myeloid Cells (TREM)-2 Signaling and Macrophage Inflammatory Responses Independently of the Linker for Activation of T Cells

Triggering receptor expressed on myeloid cells-2 (TREM-2) is rapidly emerging as a key regulator of the innate immune response via its regulation of macrophage inflammatory responses. Here we demonstrate that proximal TREM-2 signaling parallels other DAP12-based receptor systems in its use of Syk and Src-family kinases. However, we find that the linker for activation of T cells (LAT) is severely reduced as monocytes differentiate into macrophages and that TREM-2 exclusively uses the linker for activation of B cells (LAB encoded by the gene Lat2^−/−) to mediate downstream signaling. LAB is required for TREM-2-mediated activation of Erk1/2 and dampens proximal TREM-2 signals through a novel LAT-independent mechanism resulting in macrophages with proinflammatory properties. Thus, Lat2^−/− macrophages have increased TREM-2-induced proximal phosphorylation, and lipopolysaccharide stimulation of these cells leads to increased interleukin-10 (IL-10) and decreased IL-12p40 production relative to wild type cells. Together these data identify LAB as a critical, LAT-independent regulator of TREM-2 signaling and macrophage development capable of controlling subsequent inflammatory responses.     

RNA Binding and Core Complexes Constitute the U-Insertion/Deletion Editosome

Enzymes embedded into the RNA editing core complex (RECC) catalyze the U-insertion/deletion editing cascade to generate open reading frames in trypanosomal mitochondrial mRNAs. The sequential reactions of mRNA cleavage, U-addition or removal, and ligation are directed by guide RNAs (gRNAs). We combined proteomic, genetic, and functional studies with sequencing of total and complex-bound RNAs to define a protein particle responsible for the recognition of gRNAs and pre-mRNA substrates, editing intermediates, and products. This approximately 23-polypeptide tripartite assembly, termed the RNA editing substrate binding complex (RESC), also functions as the interface between mRNA editing, polyadenylation, and translation. Furthermore, we found that gRNAs represent only a subset of small mitochondrial RNAs, and yet an inexplicably high fraction of them possess 3′ U-tails, which correlates with gRNA''s enrichment in the RESC. Although both gRNAs and mRNAs are associated with the RESC, their metabolic fates are distinct: gRNAs are degraded in an editing-dependent process, whereas edited mRNAs undergo 3′ adenylation/uridylation prior to translation. Our results demonstrate that the well-characterized editing core complex (RECC) and the RNA binding particle defined in this study (RESC) typify enzymatic and substrate binding macromolecular constituents, respectively, of the ∼40S RNA editing holoenzyme, the editosome.     

In Vivo Protein Interactions and Complex Formation in the Pectobacterium atrosepticum Subtype I-F CRISPR/Cas System

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated proteins (Cas; CRISPR associated) are a bacterial defense mechanism against extra-chromosomal elements. CRISPR/Cas systems are distinct from other known defense mechanisms insofar as they provide acquired and heritable immunity. Resistance is accomplished in multiple stages in which the Cas proteins provide the enzymatic machinery. Importantly, subtype-specific proteins have been shown to form complexes in combination with small RNAs, which enable sequence-specific targeting of foreign nucleic acids. We used Pectobacterium atrosepticum, a plant pathogen that causes soft-rot and blackleg disease in potato, to investigate protein-protein interactions and complex formation in the subtype I-F CRISPR/Cas system. The P. atrosepticum CRISPR/Cas system encodes six proteins: Cas1, Cas3, and the four subtype specific proteins Csy1, Csy2, Csy3 and Cas6f (Csy4). Using co-purification followed by mass spectrometry as well as directed co-immunoprecipitation we have demonstrated complex formation by the Csy1-3 and Cas6f proteins, and determined details about the architecture of that complex. Cas3 was also shown to co-purify all four subtype-specific proteins, consistent with its role in targeting. Furthermore, our results show that the subtype I-F Cas1 and Cas3 (a Cas2-Cas3 hybrid) proteins interact, suggesting a protein complex for adaptation and a role for subtype I-F Cas3 proteins in both the adaptation and interference steps of the CRISPR/Cas mechanism.     

Oleoyl Coenzyme A Regulates Interaction of Transcriptional Regulator RaaS (Rv1219c) with DNA in Mycobacteria

We have recently shown that RaaS (regulator of antimicrobial-assisted survival), encoded by Rv1219c in Mycobacterium tuberculosis and by bcg_1279c in Mycobacterium bovis bacillus Calmette-Guérin, plays an important role in mycobacterial survival in prolonged stationary phase and during murine infection. Here, we demonstrate that long chain acyl-CoA derivatives (oleoyl-CoA and, to lesser extent, palmitoyl-CoA) modulate RaaS binding to DNA and expression of the downstream genes that encode ATP-dependent efflux pumps. Moreover, exogenously added oleic acid influences RaaS-mediated mycobacterial improvement of survival and expression of the RaaS regulon. Our data suggest that long chain acyl-CoA derivatives serve as biological indicators of the bacterial metabolic state. Dysregulation of efflux pumps can be used to eliminate non-growing mycobacteria.     

An Active Immune Defense with a Minimal CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) RNA and without the Cas6 Protein

The prokaryotic immune system CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) is a defense system that protects prokaryotes against foreign DNA. The short CRISPR RNAs (crRNAs) are central components of this immune system. In CRISPR-Cas systems type I and III, crRNAs are generated by the endonuclease Cas6. We developed a Cas6b-independent crRNA maturation pathway for the Haloferax type I-B system in vivo that expresses a functional crRNA, which we termed independently generated crRNA (icrRNA). The icrRNA is effective in triggering degradation of an invader plasmid carrying the matching protospacer sequence. The Cas6b-independent maturation of the icrRNA allowed mutation of the repeat sequence without interfering with signals important for Cas6b processing. We generated 23 variants of the icrRNA and analyzed them for activity in the interference reaction. icrRNAs with deletions or mutations of the 3′ handle are still active in triggering an interference reaction. The complete 3′ handle could be removed without loss of activity. However, manipulations of the 5′ handle mostly led to loss of interference activity. Furthermore, we could show that in the presence of an icrRNA a strain without Cas6b (Δcas6b) is still active in interference.     

A Ruler Protein in a Complex for Antiviral Defense Determines the Length of Small Interfering CRISPR RNAs

Small RNAs undergo maturation events that precisely determine the length and structure required for their function. CRISPRs (clustered regularly interspaced short palindromic repeats) encode small RNAs (crRNAs) that together with CRISPR-associated (cas) genes constitute a sequence-specific prokaryotic immune system for anti-viral and anti-plasmid defense. crRNAs are subject to multiple processing events during their biogenesis, and little is known about the mechanism of the final maturation step. We show that in the Staphylococcus epidermidis type III CRISPR-Cas system, mature crRNAs are measured in a Cas10·Csm ribonucleoprotein complex to yield discrete lengths that differ by 6-nucleotide increments. We looked for mutants that impact this crRNA size pattern and found that an alanine substitution of a conserved aspartate residue of Csm3 eliminates the 6-nucleotide increments in the length of crRNAs. In vitro, recombinant Csm3 binds RNA molecules at multiple sites, producing gel-shift patterns that suggest that each protein binds 6 nucleotides of substrate. In vivo, changes in the levels of Csm3 modulate the crRNA size distribution without disrupting the 6-nucleotide periodicity. Our data support a model in which multiple Csm3 molecules within the Cas10·Csm complex bind the crRNA with a 6-nucleotide periodicity to function as a ruler that measures the extent of crRNA maturation.     

Human ISCA1 Interacts with IOP1/NARFL and Functions in Both Cytosolic and Mitochondrial Iron-Sulfur Protein Biogenesis

Iron-sulfur proteins play an essential role in many biologic processes. Hence, understanding their assembly is an important goal. In Escherichia coli, the protein IscA is a product of the isc (iron-sulfur cluster) operon and functions in the iron-sulfur cluster assembly pathway in this organism. IscA is conserved in evolution, but its function in mammalian cells is not known. Here, we provide evidence for a role for a human homologue of IscA, named IscA1, in iron-sulfur protein biogenesis. We observe that small interfering RNA knockdown of IscA1 in HeLa cells leads to decreased activity of two mitochondrial iron-sulfur enzymes, succinate dehydrogenase and mitochondrial aconitase, as well as a cytosolic iron-sulfur enzyme, cytosolic aconitase. IscA1 is observed both in cytosolic and mitochondrial fractions. We find that IscA1 interacts with IOP1 (iron-only hydrogenase-like protein 1)/NARFL (nuclear prelamin A recognition factor-like), a cytosolic protein that plays a role in the cytosolic iron-sulfur protein assembly pathway. We therefore propose that human IscA1 plays an important role in both mitochondrial and cytosolic iron-sulfur cluster biogenesis, and a notable component of the latter is the interaction between IscA1 and IOP1.     

Evolution of the B3 DNA Binding Superfamily: New Insights into REM Family Gene Diversification

Background

The B3 DNA binding domain includes five families: auxin response factor (ARF), abscisic acid-insensitive3 (ABI3), high level expression of sugar inducible (HSI), related to ABI3/VP1 (RAV) and reproductive meristem (REM). The release of the complete genomes of the angiosperm eudicots Arabidopsis thaliana and Populus trichocarpa, the monocot Orysa sativa, the bryophyte Physcomitrella patens,the green algae Chlamydomonas reinhardtii and Volvox carteri and the red algae Cyanidioschyzon melorae provided an exceptional opportunity to study the evolution of this superfamily.

Methodology

In order to better understand the origin and the diversification of B3 domains in plants, we combined comparative phylogenetic analysis with exon/intron structure and duplication events. In addition, we investigated the conservation and divergence of the B3 domain during the origin and evolution of each family.

Conclusions

Our data indicate that showed that the B3 containing genes have undergone extensive duplication events, and that the REM family B3 domain has a highly diverged DNA binding. Our results also indicate that the founding member of the B3 gene family is likely to be similar to the ABI3/HSI genes found in C. reinhardtii and V. carteri. Among the B3 families, ABI3, HSI, RAV and ARF are most structurally conserved, whereas the REM family has experienced a rapid divergence. These results are discussed in light of their functional and evolutionary roles in plant development.     

A Fungal Sarcolemmal Membrane-Associated Protein (SLMAP) Homolog Plays a Fundamental Role in Development and Localizes to the Nuclear Envelope,Endoplasmic Reticulum,and Mitochondria

Sarcolemmal membrane-associated protein (SLMAP) is a tail-anchored protein involved in fundamental cellular processes, such as myoblast fusion, cell cycle progression, and chromosomal inheritance. Further, SLMAP misexpression is associated with endothelial dysfunctions in diabetes and cancer. SLMAP is part of the conserved striatin-interacting phosphatase and kinase (STRIPAK) complex required for specific signaling pathways in yeasts, filamentous fungi, insects, and mammals. In filamentous fungi, STRIPAK was initially discovered in Sordaria macrospora, a model system for fungal differentiation. Here, we functionally characterize the STRIPAK subunit PRO45, a homolog of human SLMAP. We show that PRO45 is required for sexual propagation and cell-to-cell fusion and that its forkhead-associated (FHA) domain is essential for these processes. Protein-protein interaction studies revealed that PRO45 binds to STRIPAK subunits PRO11 and SmMOB3, which are also required for sexual propagation. Superresolution structured-illumination microscopy (SIM) further established that PRO45 localizes to the nuclear envelope, endoplasmic reticulum, and mitochondria. SIM also showed that localization to the nuclear envelope requires STRIPAK subunits PRO11 and PRO22, whereas for mitochondria it does not. Taken together, our study provides important insights into fundamental roles of the fungal SLMAP homolog PRO45 and suggests STRIPAK-related and STRIPAK-unrelated functions.     

Preferential Utilization of d-Lactate by Shewanella oneidensis

Shewanella oneidensis couples oxidation of lactate to respiration of many substrates. Here we report that llpR (l-lactate-positive regulator, SO_3460) encodes a positive regulator of l-lactate utilization distinct from previously studied regulators. We also demonstrate d-lactate inhibition of l-lactate utilization in S. oneidensis, resulting in preferential utilization of the d isomer.     

Pdsg1 and Pdsg2, Novel Proteins Involved in Developmental Genome Remodelling in Paramecium

The epigenetic influence of maternal cells on the development of their progeny has long been studied in various eukaryotes. Multicellular organisms usually provide their zygotes not only with nutrients but also with functional elements required for proper development, such as coding and non-coding RNAs. These maternally deposited RNAs exhibit a variety of functions, from regulating gene expression to assuring genome integrity. In ciliates, such as Paramecium these RNAs participate in the programming of large-scale genome reorganization during development, distinguishing germline-limited DNA, which is excised, from somatic-destined DNA. Only a handful of proteins playing roles in this process have been identified so far, including typical RNAi-derived factors such as Dicer-like and Piwi proteins. Here we report and characterize two novel proteins, Pdsg1 and Pdsg2 (Paramecium protein involved in Development of the Somatic Genome 1 and 2), involved in Paramecium genome reorganization. We show that these proteins are necessary for the excision of germline-limited DNA during development and the survival of sexual progeny. Knockdown of PDSG1 and PDSG2 genes affects the populations of small RNAs known to be involved in the programming of DNA elimination (scanRNAs and iesRNAs) and chromatin modification patterns during development. Our results suggest an association between RNA-mediated trans-generational epigenetic signal and chromatin modifications in the process of Paramecium genome reorganization.     

Ankyrin Repeat Domain Protein 2 and Inhibitor of DNA Binding 3 Cooperatively Inhibit Myoblast Differentiation by Physical Interaction

Ankyrin repeat domain protein 2 (ANKRD2) translocates from the nucleus to the cytoplasm upon myogenic induction. Overexpression of ANKRD2 inhibits C2C12 myoblast differentiation. However, the mechanism by which ANKRD2 inhibits myoblast differentiation is unknown. We demonstrate that the primary myoblasts of mdm (muscular dystrophy with myositis) mice (pMB^mdm) overexpress ANKRD2 and ID3 (inhibitor of DNA binding 3) proteins and are unable to differentiate into myotubes upon myogenic induction. Although suppression of either ANKRD2 or ID3 induces myoblast differentiation in mdm mice, overexpression of ANKRD2 and inhibition of ID3 or vice versa is insufficient to inhibit myoblast differentiation in WT mice. We identified that ANKRD2 and ID3 cooperatively inhibit myoblast differentiation by physical interaction. Interestingly, although MyoD activates the Ankrd2 promoter in the skeletal muscles of wild-type mice, SREBP-1 (sterol regulatory element binding protein-1) activates the same promoter in the skeletal muscles of mdm mice, suggesting the differential regulation of Ankrd2. Overall, we uncovered a novel pathway in which SREBP-1/ANKRD2/ID3 activation inhibits myoblast differentiation, and we propose that this pathway acts as a critical determinant of the skeletal muscle developmental program.     

Therapeutic Benefits of Induced Pluripotent Stem Cells in Monocrotaline-Induced Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is characterized by progressive increases in vascular resistance and the remodeling of pulmonary arteries. The accumulation of inflammatory cells in the lung and elevated levels of inflammatory cytokines in the bloodstream suggest that inflammation may play a role in PAH. In this study, the benefits of induced pluripotent stem cells (iPSCs) and iPSC-conditioned medium (iPSC CM) were explored in monocrotaline (MCT)-induced PAH rats. We demonstrated that both iPSCs and iPSC CM significantly reduced the right ventricular systolic pressure and ameliorated the hypertrophy of the right ventricle in MCT-induced PAH rats in models of both disease prevention and disease reversal. In the prevention of MCT-induced PAH, iPSC-based therapy led to the decreased accumulation of inflammatory cells and down-regulated the expression of the IL-1β, IL-6, IL-12α, IL-12β, IL-23 and IFNγ genes in lung specimens, which implied that iPSC-based therapy may be involved in the regulation of inflammation. NF-κB signaling is essential to the inflammatory cascade, which is activated via the phosphorylation of the NF-κB molecule. Using the chemical inhibitor specifically blocked the phosphorylation of NF-κB, and in vitro assays of cultured human M1 macrophages implied that the anti-inflammation effect of iPSC-based therapy may contribute to the disturbance of NF-κB activation. Here, we showed that iPSC-based therapy could restore the hemodynamic function of right ventricle with benefits for preventing the ongoing inflammation in the lungs of MCT-induced PAH rats by regulating NF-κB phosphorylation.