BNP facilitates NMB-encoded histaminergic itch via NPRC-NMBR crosstalk

Histamine-dependent and -independent itch is conveyed by parallel peripheral neural pathways that express gastrin-releasing peptide (GRP) and neuromedin B (NMB), respectively, to the spinal cord of mice. B-type natriuretic peptide (BNP) has been proposed to transmit both types of itch via its receptor NPRA encoded by Npr1. However, BNP also binds to its cognate receptor, NPRC encoded by Npr3 with equal potency. Moreover, natriuretic peptides (NP) signal through the Gi-couped inhibitory cGMP pathway that is supposed to inhibit neuronal activity, raising the question of how BNP may transmit itch information. Here we report that Npr3 expression in laminae I-II of the dorsal horn partially overlaps with NMB receptor (NMBR) that transmits histaminergic itch via Gq-couped PLCb-Ca2+ signaling pathway. Functional studies indicate that NPRC is required for itch evoked by histamine but not chloroquine (CQ), a nonhistaminergic pruritogen. Importantly, BNP significantly facilitates scratching behaviors mediated by NMB, but not GRP. Consistently, BNP evoked Ca2+ responses in NMBR/NPRC HEK 293 cells and NMBR/NPRC dorsal horn neurons. These results reveal a previously unknown mechanism by which BNP facilitates NMB-encoded itch through a novel NPRC-NMBR cross-signaling in mice. Our studies uncover distinct modes of action for neuropeptides in transmission and modulation of itch in mice.

Multiple phage resistance systems inhibit infection via SIR2-dependent NAD+ depletion

Defense-associated sirtuins (DSR) comprise a family of proteins that defend bacteria from phage infection via an unknown mechanism. These proteins are common in bacteria and harbor an N-terminal sirtuin (SIR2) domain. In this study we report that DSR proteins degrade nicotinamide adenine dinucleotide (NAD+) during infection, depleting the cell of this essential molecule and aborting phage propagation. Our data show that one of these proteins, DSR2, directly identifies phage tail tube proteins and then becomes an active NADase in Bacillus subtilis. Using a phage mating methodology that promotes genetic exchange between pairs of DSR2-sensitive and DSR2-resistant phages, we further show that some phages express anti-DSR2 proteins that bind and repress DSR2. Finally, we demonstrate that the SIR2 domain serves as an effector NADase in a diverse set of phage defense systems outside the DSR family. Our results establish the general role of SIR2 domains in bacterial immunity against phages.

Reversible bacteriophage resistance by shedding the bacterial cell wall

Phages are highly abundant in the environment and a major threat for bacteria. Therefore, bacteria have evolved sophisticated defense systems to withstand phage attacks. Here, we describe a previously unknown mechanism by which mono- and diderm bacteria survive infection with diverse lytic phages. Phage exposure leads to a rapid and near complete conversion of walled cells to a cell wall-deficient state, which remain viable in osmoprotective conditions and can revert to the walled state. While shedding the cell wall dramatically reduces the number of progeny phages produced by the host, it does not always preclude phage infection. Altogether, these results show that the formation of cell wall-deficient cells prevents complete eradication of the bacterial population and suggest that cell wall-deficiency may limit the efficacy of phage therapy, especially in highly osmotic environments or when used together with antibiotics that target the cell wall.

Coordinated conformational changes in the V1 complex during V-ATPase reversible dissociation

Vacuolar-type ATPases (V-ATPases) are rotary enzymes that acidify intracellular compartments in eukaryotic cells. These multi-subunit complexes consist of a cytoplasmic V1 region that hydrolyzes ATP and a membrane-embedded VO region that transports protons. V-ATPase activity is regulated by reversible dissociation of the two regions, with the isolated V1 and VO complexes becoming autoinhibited upon disassembly and subunit C subsequently detaching from V1. In yeast, assembly of the V1 and VO regions is mediated by the RAVE complex through an unknown mechanism. We used cryoEM of yeast V-ATPase to determine structures of the intact enzyme, the dissociated but complete V1 complex, and the V1 complex lacking subunit C. Upon separation, V1 undergoes a dramatic conformational rearrangement, with its rotational state becoming incompatible for reassembly with VO. Loss of subunit C allows V1 to match the rotational state of VO, suggesting how RAVE could reassemble V1 and VO by recruiting subunit C.

Molecular transport and packing underlie increasing ribosome productivity in faster growing cells

Faster growing cells must make proteins more quickly. This occurs in part through increasing total ribosome abundance. However, the productivity of individual ribosomes also increases, almost doubling via an unknown mechanism. To investigate, we model both physical transport and chemical reactions among ensembles of individual molecules involved in translation elongation in Escherichia coli. We predict that the Damkohler number, the ratio of transport latency to reaction latency, for translation elongation is ~4; physical transport of individual ternary complexes accounts for ~80% of elongation latency. We also model how molecules pack closer together as growth quickens. Although denser cytoplasm both decreases transport distances and hinders motion, we predict that decreasing distance wins out, offering a simple mechanism for how individual elongating ribosomes become more productive as growth quickens. We also quantify how crowding imposes a physical limit on the performance of self-mixing molecular systems and likely undergirds cellular behavior more broadly.

Staphylococcus aureus Lipase 3 (SAL3) is a surface-associated lipase that hydrolyzes short chain fatty acids

Bacterial lipases play important roles during infection. The Staphylococcus aureus genome contains several genes that encode well-characterized lipases and several genes predicted to encode lipases or esterases for which the function has not yet been established. In this study, we sought to define the function of an uncharacterized S. aureus protein, and we propose the annotation S. aureus lipase 3 (SAL3) (SAUSA300_0641). We confirmed that SAL3 is a lipase and that it is surface associated and secreted through an unknown mechanism. We determined that SAL3 specifically hydrolyzes short chain (4-carbon and fewer) fatty acids and specifically binds negatively charged lipids including phosphatidic acid, phosphatidylinositol phosphate, and phosphatidylglycerol, which is the most abundant lipid in the staphylococcal cell membrane. Mutating the catalytic triad S66-A, D167-A, S168-A, and H301-A in the recombinant protein abolished lipase activity without altering binding to host lipid substrates. Taken together we report the discovery of a novel lipase from S. aureus specific to short chain fatty acids with yet to be determined roles in host pathogen interactions.

A bipartite chromatophore transit peptide and N-terminal processing of protein in the Paulinella chromatophore

The cercozoan amoeba Paulinella chromatophora contains photosynthetic organelles - termed chromatophores - that evolved from a cyanobacterium ~100 million years ago, independently from plastids in plants and algae. Despite its more recent origin, at least one third of the chromatophore proteome consists of nucleus-encoded proteins that are imported by an unknown mechanism across the chromatophore double envelope membranes. Chromatophore-targeted proteins fall into two classes. Proteins exceeding 250 amino acids carry a conserved N-terminal sequence extension, termed the 'chromatophore transit peptide' (crTP), that is presumably involved in guiding these proteins into the chromatophore. Short imported proteins do not carry discernable targeting signals. To explore whether the import of protein is accompanied by their N-terminal processing, here we used a mass spectrometry-based approach to determine protein N-termini in Paulinella chromatophora and identified N-termini of 208 chromatophore-localized proteins. Our study revealed extensive N-terminal modifications by acetylation and proteolytic processing in both, the nucleus and chromatophore-encoded fraction of the chromatophore proteome. Mature N-termini of 37 crTP-carrying proteins were identified, of which 30 were cleaved in a common processing region. Our results imply that the crTP mediates trafficking through the Golgi, is bipartite and surprisingly only the N-terminal third ('part 1') becomes cleaved upon import, whereas the rest ('part 2') remains at the mature proteins. In contrast, short imported proteins remain largely unprocessed. Finally, this work sheds light on N-terminal processing of proteins encoded in an evolutionary-early-stage photosynthetic organelle and suggests host-derived post-translationally acting factors involved in dynamic regulation of the chromatophore-encoded chromatophore proteome.

Oestrogen Activates the MAP3K1 Cascade and β-Catenin to Promote Granulosa-Like Cell Fate in a Human Testis-Derived Cell Line

Sex determination triggers the differentiation of the bi-potential gonad into either an ovary or testis. In non-mammalian vertebrates, the presence or absence of oestrogen dictates gonad differentiation, while in mammals, this mechanism has been supplanted by the testis-determining gene SRY. Exogenous oestrogen can override this genetic trigger to shift somatic cell fate in the gonad towards ovarian developmental pathways by limiting the bioavailability of the key testis factor SOX9 within somatic cells. Our previous work has implicated the MAPK pathway in mediating the rapid cellular response to oestrogen. We performed proteomic and phosphoproteomic analyses to investigate the precise mechanism through which oestrogen impacts these pathways to activate β-catenin—a factor essential for ovarian development. We show that oestrogen can activate β-catenin within 30 min, concomitant with the cytoplasmic retention of SOX9. This occurs through changes to the MAP3K1 cascade, suggesting this pathway is a mechanism through which oestrogen influences gonad somatic cell fate. We demonstrate that oestrogen can promote the shift from SOX9 pro-testis activity to β-catenin pro-ovary activity through activation of MAP3K1. Our findings define a previously unknown mechanism through which oestrogen can promote a switch in gonad somatic cell fate and provided novel insights into the impacts of exogenous oestrogen exposure on the testis.

A histone variant condenses flowering plant sperm via chromatin phase separation

Sperm chromatin is typically transformed by protamines into a compact and transcriptionally inactive state. Flowering plant sperm cells lack protamines, yet have small, transcriptionally active nuclei with chromatin condensed by an unknown mechanism. Here we show that a histone variant, H2B.8, mediates sperm chromatin and nuclear condensation in Arabidopsis thaliana. Loss of H2B.8 causes enlarged sperm nuclei with dispersed chromatin, whereas ectopic expression in somatic cells produces smaller nuclei with aggregated chromatin, demonstrating that H2B.8 is sufficient for chromatin condensation. H2B.8 aggregates transcriptionally inactive AT-rich chromatin into phase-separated condensates, thus achieving nuclear compaction without reducing transcription. H2B.8 also intermixes inactive AT-rich chromatin and GC-rich pericentromeric heterochromatin, altering higher-order chromatin architecture. Altogether, our results reveal a novel mechanism of nuclear compaction via global aggregation of unexpressed chromatin. We propose that H2B.8 is a flowering plant evolutionary innovation that achieves nuclear condensation compatible with active transcription.

Intracellular Pathways of Holothuroid Oocyte Maturation Induced by the Thioredoxin Trx-REES

In holothuroids, oocyte maturation is stopped in ovaries at the prophase I stage of meiosis. In natural conditions, the blockage is removed during the spawning by an unknown mechanism. When oocytes are isolated by dissection, the meiotic release can be successfully induced by a natural inducer, the REES (i.e., Rough Extract of Echinoid Spawn) that is used in aquaculture to obtain viable larvae in mass. A thioredoxin has recently been identified in the REES as the molecule responsible for holothuroid oocyte maturation. As a redox-active protein, thioredoxin is thought to reduce target proteins within the oocyte membrane and initiate an intracellular reaction cascade that leads to the unblocking of the oocyte meiosis. Our results allow us to understand additional steps in the intracellular reaction cascade induced by the action of thioredoxin on oocytes. Pharmacological agents known to have activating or inhibiting actions on oocyte maturation have been used (Forskolin, Isobutylmethylxanthine, Hypoxanthine, 6-dimethyaminopurine, Lavendustin, Genistein, Roscovitine, Cycloheximide). The effects of these agents were analysed on oocytes of the holothuroid Holothuria tubulosa incubated with or without REES and were compared to those obtained with another reducing agent, the dithiothreitol. Our results demonstrated that, at the opposite of dithiothreitol-induced oocyte maturation, thioredoxin-induced oocyte maturation is cAMP independent, but dependent of the presence of calcium in the seawater. Both pathways of induction require the activation of protein serine/threonine kinases.