miR‐9‐5p suppresses pro‐fibrogenic transformation of fibroblasts and prevents organ fibrosis by targeting NOX4 and TGFBR2

Uncontrolled extracellular matrix (ECM) production by fibroblasts in response to injury contributes to fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). Reactive oxygen species (ROS) generation is involved in the pathogenesis of IPF. Transforming growth factor‐β1 (TGF‐β1) stimulates the production of NADPH oxidase 4 (NOX4)‐dependent ROS, promoting lung fibrosis (LF). Dysregulation of microRNAs (miRNAs) has been shown to contribute to LF. To identify miRNAs involved in redox regulation relevant for IPF, we performed arrays in human lung fibroblasts exposed to ROS. miR‐9‐5p was selected as the best candidate and we demonstrate its inhibitory effect on TGF‐β receptor type II (TGFBR2) and NOX4 expression. Increased expression of miR‐9‐5p abrogates TGF‐β1‐dependent myofibroblast phenotypic transformation. In the mouse model of bleomycin‐induced LF, miR‐9‐5p dramatically reduces fibrogenesis and inhibition of miR‐9‐5p and prevents its anti‐fibrotic effect both in vitro and in vivo. In lung specimens from patients with IPF, high levels of miR‐9‐5p are found. In omentum‐derived mesothelial cells (MCs) from patients subjected to peritoneal dialysis (PD), miR‐9‐5p also inhibits mesothelial to myofibroblast transformation. We propose that TGF‐β1 induces miR‐9‐5p expression as a self‐limiting homeostatic response.     

Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1

Mutations in the PTEN‐induced kinase 1 (PINK1) are causative of autosomal recessive Parkinson''s disease (PD). We have previously reported that PINK1 is activated by mitochondrial depolarisation and phosphorylates serine 65 (Ser⁶⁵) of the ubiquitin ligase Parkin and ubiquitin to stimulate Parkin E3 ligase activity. Here, we have employed quantitative phosphoproteomics to search for novel PINK1‐dependent phosphorylation targets in HEK (human embryonic kidney) 293 cells stimulated by mitochondrial depolarisation. This led to the identification of 14,213 phosphosites from 4,499 gene products. Whilst most phosphosites were unaffected, we strikingly observed three members of a sub‐family of Rab GTPases namely Rab8A, 8B and 13 that are all phosphorylated at the highly conserved residue of serine 111 (Ser¹¹¹) in response to PINK1 activation. Using phospho‐specific antibodies raised against Ser¹¹¹ of each of the Rabs, we demonstrate that Rab Ser¹¹¹ phosphorylation occurs specifically in response to PINK1 activation and is abolished in HeLa PINK1 knockout cells and mutant PINK1 PD patient‐derived fibroblasts stimulated by mitochondrial depolarisation. We provide evidence that Rab8A GTPase Ser¹¹¹ phosphorylation is not directly regulated by PINK1 in vitro and demonstrate in cells the time course of Ser¹¹¹ phosphorylation of Rab8A, 8B and 13 is markedly delayed compared to phosphorylation of Parkin at Ser⁶⁵. We further show mechanistically that phosphorylation at Ser¹¹¹ significantly impairs Rab8A activation by its cognate guanine nucleotide exchange factor (GEF), Rabin8 (by using the Ser111Glu phosphorylation mimic). These findings provide the first evidence that PINK1 is able to regulate the phosphorylation of Rab GTPases and indicate that monitoring phosphorylation of Rab8A/8B/13 at Ser¹¹¹ may represent novel biomarkers of PINK1 activity in vivo. Our findings also suggest that disruption of Rab GTPase‐mediated signalling may represent a major mechanism in the neurodegenerative cascade of Parkinson''s disease.     

Glucagon signalling in the dorsal vagal complex is sufficient and necessary for high‐protein feeding to regulate glucose homeostasis in vivo

High‐protein feeding acutely lowers postprandial glucose concentration compared to low‐protein feeding, despite a dichotomous rise of circulating glucagon levels. The physiological role of this glucagon rise has been largely overlooked. We here first report that glucagon signalling in the dorsal vagal complex (DVC) of the brain is sufficient to lower glucose production by activating a Gcgr–PKA–ERK–K_ATP channel signalling cascade in the DVC of rats in vivo. We further demonstrate that direct blockade of DVC Gcgr signalling negates the acute ability of high‐ vs. low‐protein feeding to reduce plasma glucose concentration, indicating that the elevated circulating glucagon during high‐protein feeding acts in the brain to lower plasma glucose levels. These data revise the physiological role of glucagon and argue that brain glucagon signalling contributes to glucose homeostasis during dietary protein intake.     

Integrative genomics positions MKRN1 as a novel ribonucleoprotein within the embryonic stem cell gene regulatory network

In embryonic stem cells (ESCs), gene regulatory networks (GRNs) coordinate gene expression to maintain ESC identity; however, the complete repertoire of factors regulating the ESC state is not fully understood. Our previous temporal microarray analysis of ESC commitment identified the E3 ubiquitin ligase protein Makorin‐1 (MKRN1) as a potential novel component of the ESC GRN. Here, using multilayered systems‐level analyses, we compiled a MKRN1‐centered interactome in undifferentiated ESCs at the proteomic and ribonomic level. Proteomic analyses in undifferentiated ESCs revealed that MKRN1 associates with RNA‐binding proteins, and ensuing RIP‐chip analysis determined that MKRN1 associates with mRNAs encoding functionally related proteins including proteins that function during cellular stress. Subsequent biological validation identified MKRN1 as a novel stress granule‐resident protein, although MKRN1 is not required for stress granule formation, or survival of unstressed ESCs. Thus, our unbiased systems‐level analyses support a role for the E3 ligase MKRN1 as a ribonucleoprotein within the ESC GRN.     

Systematic discovery of linear binding motifs targeting an ancient protein interaction surface on MAP kinases

Mitogen‐activated protein kinases (MAPK) are broadly used regulators of cellular signaling. However, how these enzymes can be involved in such a broad spectrum of physiological functions is not understood. Systematic discovery of MAPK networks both experimentally and in silico has been hindered because MAPKs bind to other proteins with low affinity and mostly in less‐characterized disordered regions. We used a structurally consistent model on kinase‐docking motif interactions to facilitate the discovery of short functional sites in the structurally flexible and functionally under‐explored part of the human proteome and applied experimental tools specifically tailored to detect low‐affinity protein–protein interactions for their validation in vitro and in cell‐based assays. The combined computational and experimental approach enabled the identification of many novel MAPK‐docking motifs that were elusive for other large‐scale protein–protein interaction screens. The analysis produced an extensive list of independently evolved linear binding motifs from a functionally diverse set of proteins. These all target, with characteristic binding specificity, an ancient protein interaction surface on evolutionarily related but physiologically clearly distinct three MAPKs (JNK, ERK, and p38). This inventory of human protein kinase binding sites was compared with that of other organisms to examine how kinase‐mediated partnerships evolved over time. The analysis suggests that most human MAPK‐binding motifs are surprisingly new evolutionarily inventions and newly found links highlight (previously hidden) roles of MAPKs. We propose that short MAPK‐binding stretches are created in disordered protein segments through a variety of ways and they represent a major resource for ancient signaling enzymes to acquire new regulatory roles.     

Africanization of a feral honey bee (Apis mellifera) population in South Texas: does a decade make a difference?

The arrival to the United States of the Africanized honey bee, a hybrid between European subspecies and the African subspecies Apis mellifera scutellata, is a remarkable model for the study of biological invasions. This immigration has created an opportunity to study the dynamics of secondary contact of honey bee subspecies from African and European lineages in a feral population in South Texas. An 11‐year survey of this population (1991–2001) showed that mitochondrial haplotype frequencies changed drastically over time from a resident population of eastern and western European maternal ancestry, to a population dominated by the African haplotype. A subsequent study of the nuclear genome showed that the Africanization process included bidirectional gene flow between European and Africanized honey bees, giving rise to a new panmictic mixture of A. m. scutellata‐ and European‐derived genes. In this study, we examined gene flow patterns in the same population 23 years after the first hybridization event occurred. We found 28 active colonies inhabiting 92 tree cavities surveyed in a 5.14 km² area, resulting in a colony density of 5.4 colonies/km². Of these 28 colonies, 25 were of A. m. scutellata maternal ancestry, and three were of western European maternal ancestry. No colonies of eastern European maternal ancestry were detected, although they were present in the earlier samples. Nuclear DNA revealed little change in the introgression of A. m. scutellata‐derived genes into the population compared to previous surveys. Our results suggest this feral population remains an admixed swarm with continued low levels of European ancestry and a greater presence of African‐derived mitochondrial genetic composition.     

CPAP promotes timely cilium disassembly to maintain neural progenitor pool

A mutation in the centrosomal‐P4.1‐associated protein (CPAP) causes Seckel syndrome with microcephaly, which is suggested to arise from a decline in neural progenitor cells (NPCs) during development. However, mechanisms of NPCs maintenance remain unclear. Here, we report an unexpected role for the cilium in NPCs maintenance and identify CPAP as a negative regulator of ciliary length independent of its role in centrosome biogenesis. At the onset of cilium disassembly, CPAP provides a scaffold for the cilium disassembly complex (CDC), which includes Nde1, Aurora A, and OFD1, recruited to the ciliary base for timely cilium disassembly. In contrast, mutated CPAP fails to localize at the ciliary base associated with inefficient CDC recruitment, long cilia, retarded cilium disassembly, and delayed cell cycle re‐entry leading to premature differentiation of patient iPS‐derived NPCs. Aberrant CDC function also promotes premature differentiation of NPCs in Seckel iPS‐derived organoids. Thus, our results suggest a role for cilia in microcephaly and its involvement during neurogenesis and brain size control.     

Hypoxia and loss of PHD2 inactivate stromal fibroblasts to decrease tumour stiffness and metastasis

Cancer‐associated fibroblasts (CAFs) interact with tumour cells and promote growth and metastasis. Here, we show that CAF activation is reversible: chronic hypoxia deactivates CAFs, resulting in the loss of contractile force, reduced remodelling of the surrounding extracellular matrix and, ultimately, impaired CAF‐mediated cancer cell invasion. Hypoxia inhibits prolyl hydroxylase domain protein 2 (PHD2), leading to hypoxia‐inducible factor (HIF)‐1α stabilisation, reduced expression of αSMA and periostin, and reduced myosin II activity. Loss of PHD2 in CAFs phenocopies the effects of hypoxia, which can be prevented by simultaneous depletion of HIF‐1α. Treatment with the PHD inhibitor DMOG in an orthotopic breast cancer model significantly decreases spontaneous metastases to the lungs and liver, associated with decreased tumour stiffness and fibroblast activation. PHD2 depletion in CAFs co‐injected with tumour cells similarly prevents CAF‐induced metastasis to lungs and liver. Our data argue that reversion of CAFs towards a less active state is possible and could have important clinical implications.     

Little or no gene flow despite F1 hybrids at two interspecific contact zones

Hybridization can create the selective force that promotes assortative mating but hybridization can also select for increased hybrid fitness. Gene flow resulting from hybridization can increase genetic diversity but also reduce distinctiveness. Thus the formation of hybrids has important implications for long‐term species coexistence. This study compares the interaction between the tree wētā Hemideina thoracica and its two neighboring species; H. crassidens and H. trewicki. We examined the ratio of parent and hybrid forms in natural areas of sympatry. Individuals with intermediate phenotype were confirmed as first generation hybrids using nine independent genetic markers. Evidence of gene flow from successful hybridization was sought from the distribution of morphological and genetic characters. Both species pairs appear to be largely retaining their own identity where they live in sympatry, each with a distinct karyotype. Hemideina thoracica and H. trewicki are probably reproductively isolated, with sterile F₁ hybrids. This species pair shows evidence of niche differences with adult size and timing of maturity differing where Hemideina thoracica is sympatric with H. trewicki. In contrast, evidence of a low level of introgression was detected in phenotypes and genotypes where H. thoracica and H. crassidens are sympatric. We found no evidence of size divergence although color traits in combination with hind tibia spines reliably distinguish the two species. This species pair show a bimodal hybrid zone in the absence of assortative mating and possible sexual exclusion by H. thoracica males in the formation of F₁ hybrids.     

Summer temperature can predict the distribution of wild yeast populations

The wine yeast, Saccharomyces cerevisiae, is the best understood microbial eukaryote at the molecular and cellular level, yet its natural geographic distribution is unknown. Here we report the results of a field survey for S. cerevisiae,S. paradoxus and other budding yeast on oak trees in Europe. We show that yeast species differ in their geographic distributions, and investigated which ecological variables can predict the isolation rate of S. paradoxus, the most abundant species. We find a positive association between trunk girth and S. paradoxus abundance suggesting that older trees harbor more yeast. S. paradoxus isolation frequency is also associated with summer temperature, showing highest isolation rates at intermediate temperatures. Using our statistical model, we estimated a range of summer temperatures at which we expect high S. paradoxus isolation rates, and show that the geographic distribution predicted by this optimum temperature range is consistent with the worldwide distribution of sites where S. paradoxus has been isolated. Using laboratory estimates of optimal growth temperatures for S. cerevisiae relative to S. paradoxus, we also estimated an optimum range of summer temperatures for S. cerevisiae. The geographic distribution of these optimum temperatures is consistent with the locations where wild S. cerevisiae have been reported, and can explain why only human‐associated S. cerevisiae strains are isolated at northernmost latitudes. Our results provide a starting point for targeted isolation of S. cerevisiae from natural habitats, which could lead to a better understanding of climate associations and natural history in this important model microbe.     

TssA forms a gp6‐like ring attached to the type VI secretion sheath

The type VI secretion system (T6SS) is a supra‐molecular bacterial complex that resembles phage tails. It is a killing machine which fires toxins into target cells upon contraction of its TssBC sheath. Here, we show that TssA1 is a T6SS component forming dodecameric ring structures whose dimensions match those of the TssBC sheath and which can accommodate the inner Hcp tube. The TssA1 ring complex binds the T6SS sheath and impacts its behaviour in vivo. In the phage, the first disc of the gp18 sheath sits on a baseplate wherein gp6 is a dodecameric ring. We found remarkable sequence and structural similarities between TssA1 and gp6 C‐termini, and propose that TssA1 could be a baseplate component of the T6SS. Furthermore, we identified similarities between TssK1 and gp8, the former interacting with TssA1 while the latter is found in the outer radius of the gp6 ring. These observations, combined with similarities between TssF and gp6N‐terminus or TssG and gp53, lead us to propose a comparative model between the phage baseplate and the T6SS.     

Structure and function of the N‐terminal domain of the human mitochondrial calcium uniporter

The mitochondrial calcium uniporter (MCU) is responsible for mitochondrial calcium uptake and homeostasis. It is also a target for the regulation of cellular anti‐/pro‐apoptosis and necrosis by several oncogenes and tumour suppressors. Herein, we report the crystal structure of the MCU N‐terminal domain (NTD) at a resolution of 1.50 Å in a novel fold and the S92A MCU mutant at 2.75 Å resolution; the residue S92 is a predicted CaMKII phosphorylation site. The assembly of the mitochondrial calcium uniporter complex (uniplex) and the interaction with the MCU regulators such as the mitochondrial calcium uptake‐1 and mitochondrial calcium uptake‐2 proteins (MICU1 and MICU2) are not affected by the deletion of MCU NTD. However, the expression of the S92A mutant or a NTD deletion mutant failed to restore mitochondrial Ca²⁺ uptake in a stable MCU knockdown HeLa cell line and exerted dominant‐negative effects in the wild‐type MCU‐expressing cell line. These results suggest that the NTD of MCU is essential for the modulation of MCU function, although it does not affect the uniplex formation.     

A comparative approach to testing hypotheses for the evolution of sex‐biased dispersal in bean beetles

Understanding the selective forces that shape dispersal strategies is a fundamental goal of evolutionary ecology and is increasingly important in changing, human‐altered environments. Sex‐biased dispersal (SBD) is common in dioecious taxa, and understanding variation in the direction and magnitude of SBD across taxa has been a persistent challenge. We took a comparative, laboratory‐based approach using 16 groups (species or strains) of bean beetles (genera Acanthoscelides, Callosobruchus, and Zabrotes, including 10 strains of one species) to test two predictions that emerge from dominant hypotheses for the evolution of SBD: (1) groups that suffer greater costs of inbreeding should exhibit greater SBD in favor of either sex (inbreeding avoidance hypothesis) and (2) groups with stronger local mate competition should exhibit greater male bias in dispersal (kin competition avoidance hypothesis). We used laboratory experiments to quantify SBD in crawling dispersal, the fitness effects of inbreeding, and the degree of polygyny (number of female mates per male), a proxy for local mate competition. While we found that both polygyny and male‐biased dispersal were common across bean beetle groups, consistent with the kin competition avoidance hypothesis, quantitative relationships between trait values did not support the predictions. Across groups, there was no significant association between SBD and effects of inbreeding nor SBD and degree of polygyny, using either raw values or phylogenetically independent contrasts. We discuss possible limitations of our experimental approach for detecting the predicted relationships, as well as reasons why single‐factor hypotheses may be too simplistic to explain the evolution of SBD.