Biogenesis

Macrophage interactions with other cells, either locally or at distances, are imperative in both normal and pathological conditions. While soluble means of communication can transmit signals between different cells, it does not account for all long distance macrophage interactions. Recently described tunneling nanotubes (TNTs) are membranous channels that connect cells together and allow for transfer of signals, vesicles, and organelles. However, very little is known about the mechanism by which these structures are formed. Here we investigated the signaling pathways involved in TNT formation by macrophages using multiple imaging techniques including super-resolution microscopy (3D-SIM) and live-cell imaging including the use of FRET-based Rho GTPase biosensors. We found that formation of TNTs required the activity and differential localization of Cdc42 and Rac1. The downstream Rho GTPase effectors mediating actin polymerization through Arp2/3 nucleation, Wiskott-Aldrich syndrome pr...

Discovered over a half-century ago, lipoteichoic acid (LTA) is an abundant polymer found on the surface of Gram-positive bacteria. Although LTA is essential for the survival of many Gram-positive species, knowledge of how LTA contributes to bacterial physiology has remained elusive. Recently, LTA-deficient strains have been generated in some Gram-positive species, including the human oral commensal Streptococcus gordonii . The significance of our research is that we utilized an LTA-deficient strain of S. gordonii to address why LTA is physiologically important to Gram-positive bacteria. We demonstrate that in S. gordonii , LTA plays an important role in the presentation of many cell surface-associated proteins, contributing to cell envelope homeostasis, cell-to-cell interactions in biofilms, and adhesion to eukaryotic cells. These data may broadly reflect a physiological role of LTA in Gram-positive bacteria.

The bacterial pathogen Helicobacter pylori is capable of colonizing the gastric mucosa of the human stomach using a variety of factors associated with or secreted from its outer membrane (OM). Lipopolysaccharide (LPS) and numerous OM proteins have been shown to be involved in adhesion and immune stimulation/evasion. Many of these factors are essential for colonization and/or pathogenesis in a variety of animal models. Despite this wide array of potential targets present on the bacterial surface, the ability of H. pylori to vary its OM profile limits the effectiveness of vaccines or therapeutics that target any single one of these components. However, it has become evident that the proteins comprising the complexes that transport the majority of these molecules to the OM are highly conserved and often essential. The field of membrane biogenesis has progressed remarkably in the last few years, and the possibility now exists for targeting the mechanisms by which β-barrel proteins, lipo...

ABSTRACTThe function of the essential inner membrane protein (IMP) YidC inEscherichia colihas been studied for a limited number of model IMPs and primarily using targeted approaches. These studies suggested that YidC acts at the level of insertion, folding, and quality control of IMPs, both in the context of the Sec translocon and as a separate entity. To further our understanding of YidC's role in IMP biogenesis, we screened a random overexpression library for factors that rescued the growth of cells upon YidC depletion. We found that the overexpression of the GadX and GadY regulators of the glutamate-dependent acid resistance system complemented the growth defect of YidC-depleted cells. Evidence is presented that GadXY overexpression counteracts the deleterious effects of YidC depletion on at least two fronts. First, GadXY prepares the cells for the decrease in respiratory capacity upon the depletion of YidC. Most likely, GadXY-regulated processes reduce the drop in proton-mot...

The reversible state of proliferative arrest known as "cellular quiescence" plays an important role in tissue homeostasis and stem cell biology. By analyzing the expression of miRNAs and miRNA-processing factors during quiescence in primary human fibroblasts, we identified a group of miRNAs that are induced during quiescence despite markedly reduced expression of Exportin-5, a protein required for canonical miRNA biogenesis. The biogenesis of these quiescence-induced miRNAs is independent of Exportin-5 and depends instead on Exportin-1. Moreover, these quiescence-induced primary miRNAs (pri-miRNAs) are modified with a 2,2,7-trimethylguanosine (TMG)-cap, which is known to bind Exportin-1, and knockdown of Exportin-1 or trimethylguanosine synthase 1, responsible for (TMG)-capping, inhibits their biogenesis. Surprisingly, in quiescent cells Exportin-1-dependent pri-miR-34a is present in the cytoplasm together with a small isoform of Drosha, implying the existence of a differe...

Plant Argonaute proteins, chiefly AGO1 and 2, restrict viral infections. AGO1/2 also participate in developmental processes and are tightly regulated by microRNAs. Under viral infections, the regulatory loop comprising miR168/AGO1 is well studied, but much less so the miR403/AGO2 system. We studied both regulatory systems in TuMV-infected Arabidopsis plants. TuMV downregulated miRNAs precursor molecules, but mature miRNAs overaccumulated, without evidence of transcriptional alteration. AGO1 protein remained at basal levels whereas AGO2 overaccumulated. These results are in line with previous reports studying abiotic and biotic impact on microRNA biogenesis and AGO-dependent antiviral defense, expanding our knowledge of the miR403/AGO2 regulatory system.

The TIP49a and TIP49b proteins belong to the family of AAA+ ATPases and play essential roles in vital processes such as transcription, DNA repair, snoRNP biogenesis, and chromatin remodeling. We report the crystal structure of a TIP49b hexamer and the comparative analysis of large-scale conformational flexibility of TIP49a, TIP49b, and TIP49a/TIP49b complexes using molecular modeling and molecular dynamics simulations in a water environment. Our results establish key principles of domain mobility that affect protein conformation and biochemical properties, including a mechanistic basis for the downregulation of ATPase activity upon protein hexamerization. These approaches, applied to the lik-TIP49b mutant reported to possess enhanced DNA-independent ATPase activity, help explain how a three-amino acid insertion remotely affects the structure and conformational dynamics of the ATP binding and hydrolysis pocket while uncoupling ATP hydrolysis from DNA binding. This might be similar to the effects of conformations adopted by TIP49 heterohexamers.

Nuclear and mitochondrial genome mutations lead to various mitochondrial diseases, many of which affect the mitochondrial respiratory chain. The proteome of the intermembrane space (IMS) of mitochondria consists of several important assembly factors that participate in the biogenesis of mitochondrial respiratory chain complexes. The present study comprehensively analyzed a recently identified IMS protein, RESpiratory chain Assembly 1 (RESA1) factor, or cytochrome c oxidase assembly factor 7 (COA7) that is associated with a rare form of mitochondrial leukoencephalopathy and complex IV deficiency. We found that RESA1 requires the mitochondrial IMS import and assembly (MIA) pathway for efficient accumulation in the IMS. We also found that pathogenic mutant versions of RESA1 are imported slower than the wild type protein, and mislocalized mutant proteins are degraded in the cytosol by proteasome machinery. Interestingly, proteasome inhibition rescued both the mitochondrial localization ...

Prematurity complicates 12% of births, and young adults with a history of prematurity are at risk to develop right ventricular hypertrophy and impairment. The long-term risk for pulmonary vascular disease, as well as mechanisms of right ventricular dysfunction and ventricular-vascular uncoupling following prematurity remain poorly defined. Using an established model of prematurity related lung disease, pups from timed-pregnant Sprague Dawley rats were randomized to normoxia or hyperoxia (FIO2 0.85) exposure for the first 14 days of life. After aging to one year in standard conditions, rats underwent hemodynamic assessment followed by tissue harvest for biochemical and histological evaluation. Aged hyperoxia-exposed rats developed significantly greater right ventricular hypertrophy, associated with a 40% increase in right ventricular systolic pressures. Although cardiac index was similar, hyperoxia-exposed rats demonstrated a reduced right ventricular ejection fraction and significan...

Small RNAs are important regulators of gene expression. In maize, adaxial/abaxial (dorsoventral) leaf polarity is established by an abaxial gradient of microRNA166 (miR166), which spatially restricts the expression domain of class III homeodomain leucine zipper (HD-ZIPIII) transcription factors that specify adaxial/upper fate. Here, we show that leafbladeless1 encodes a key component in the trans-acting small interfering RNA (ta-siRNA) biogenesis pathway that acts on the adaxial side of developing leaves and demarcates the domains of hd-zipIII and miR166 accumulation. Our findings indicate that tasiR-ARF, a ta-siRNA, and miR166 establish opposing domains along the adaxial–abaxial axis, thus revealing a novel mechanism of pattern formation.

MicroRNAs and trans-acting siRNAs (ta-siRNAs) have important regulatory roles in development. Unlike other developmentally important regulatory molecules, small RNAs are not known to act as mobile signals during development. Here, we show that low-abundant, conserved ta-siRNAs, termed tasiR-ARFs, move intercellularly from their defined source of biogenesis on the upper (adaxial) side of leaves to the lower (abaxial) side to create a gradient of small RNAs that patterns the abaxial determinant AUXIN RESPONSE FACTOR3. Our observations have important ramifications for the function of small RNAs and suggest they can serve as mobile, instructive signals during development.

Telomerase is a specialized reverse transcriptase that uses an intrinsic RNA subunit as the template for telomeric DNA synthesis. Biogenesis of human telomerase requires its RNA subunit (hTR) to fold into a multi-domain architecture that includes the template-containing pseudoknot (t/PK) and the three-way junction (CR4/5). These two hTR domains bind the telomerase reverse transcriptase (hTERT) protein and are thus essential for telomerase catalytic activity. Here, we probe the structure of hTR in living cells using dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) and ensemble deconvolution analysis. Unexpectedly, approximately 15% of the steady state population of hTR has a CR4/5 conformation lacking features required for hTERT binding. Mutagenesis demonstrates that stabilization of the alternative CR4/5 conformation is detrimental to telomerase assembly and activity. We propose that this misfolded portion of the cellular hTR pool is either slowly refolded or degra...

The nucleolus is a subnuclear compartment critical in ribosome biogenesis and cellular stress responses. These mechanisms are governed by a complex interplay of proteins, including NOC1, a member of the NOC family of nucleolar proteins responsible for controlling rRNA processing and ribosomal maturation. This study reveals a novel relationship between NOC1 and MYC transcription factor, known for its crucial role in controlling ribosomal biogenesis, cell growth, and proliferation. Here, we demonstrate that NOC1 functions as a direct target of MYC, as it is transcriptionally induced through a functional MYC-binding E-box sequence in the NOC1 promoter region. Furthermore, protein interactome analysis reveals that NOC1-complex includes the nucleolar proteins NOC2 and NOC3 and other nucleolar components such as Nucleostemin1 Ns1 transporters of ribosomal subunits and components involved in rRNA processing and maturation. In response to MYC, NOC1 expression and localization within the nuc...

Peptide/protein hormones could be stored as non-toxic amyloid-like structures in pituitary secretory granules. ACTH and β-endorphin are two of the important peptide hormones that get co-stored in the pituitary secretory granules. Here, we study molecular interactions between ACTH and β-endorphin and their colocalization in the form of amyloid aggregates. Although ACTH is known to be a part of ACTH-β-endorphin aggregate, ACTH alone cannot aggregate into amyloid under various plausible conditions. Using all atom molecular dynamics simulation we investigate the early molecular interaction events in the ACTH-β-endorphin system, β-endorphin-only system and ACTH-only system. We find that β-endorphin and ACTH formed an interacting unit, whereas negligible interactions were observed between ACTH molecules in ACTH-only system. Our data suggest that ACTH is not only involved in interaction with β-endorphin but also enhances the stability of mixed oligomers of the entire system.