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.     

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.     

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.     

MicroRNA‐455 regulates brown adipogenesis via a novel HIF1an‐AMPK‐PGC1α signaling network

Brown adipose tissue (BAT) dissipates chemical energy as heat and can counteract obesity. MicroRNAs are emerging as key regulators in development and disease. Combining microRNA and mRNA microarray profiling followed by bioinformatic analyses, we identified miR‐455 as a new regulator of brown adipogenesis. miR‐455 exhibits a BAT‐specific expression pattern and is induced by cold and the browning inducer BMP7. In vitro gain‐ and loss‐of‐function studies show that miR‐455 regulates brown adipocyte differentiation and thermogenesis. Adipose‐specific miR‐455 transgenic mice display marked browning of subcutaneous white fat upon cold exposure. miR‐455 activates AMPKα1 by targeting HIF1an, and AMPK promotes the brown adipogenic program and mitochondrial biogenesis. Concomitantly, miR‐455 also targets the adipogenic suppressors Runx1t1 and Necdin, initiating adipogenic differentiation. Taken together, the data reveal a novel microRNA‐regulated signaling network that controls brown adipogenesis and may be a potential therapeutic target for human metabolic disorders.     

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.     

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.     

Gillespie eco‐evolutionary models (GEMs) reveal the role of heritable trait variation in eco‐evolutionary dynamics

Heritable trait variation is a central and necessary ingredient of evolution. Trait variation also directly affects ecological processes, generating a clear link between evolutionary and ecological dynamics. Despite the changes in variation that occur through selection, drift, mutation, and recombination, current eco‐evolutionary models usually fail to track how variation changes through time. Moreover, eco‐evolutionary models assume fitness functions for each trait and each ecological context, which often do not have empirical validation. We introduce a new type of model, Gillespie eco‐evolutionary models (GEMs), that resolves these concerns by tracking distributions of traits through time as eco‐evolutionary dynamics progress. This is done by allowing change to be driven by the direct fitness consequences of model parameters within the context of the underlying ecological model, without having to assume a particular fitness function. GEMs work by adding a trait distribution component to the standard Gillespie algorithm – an approach that models stochastic systems in nature that are typically approximated through ordinary differential equations. We illustrate GEMs with the Rosenzweig–MacArthur consumer–resource model. We show not only how heritable trait variation fuels trait evolution and influences eco‐evolutionary dynamics, but also how the erosion of variation through time may hinder eco‐evolutionary dynamics in the long run. GEMs can be developed for any parameter in any ordinary differential equation model and, furthermore, can enable modeling of multiple interacting traits at the same time. We expect GEMs will open the door to a new direction in eco‐evolutionary and evolutionary modeling by removing long‐standing modeling barriers, simplifying the link between traits, fitness, and dynamics, and expanding eco‐evolutionary treatment of a greater diversity of ecological interactions. These factors make GEMs much more than a modeling advance, but an important conceptual advance that bridges ecology and evolution through the central concept of heritable trait variation.     

Population and genetic outcomes 20 years after reintroducing bobcats (Lynx rufus) to Cumberland Island,Georgia USA

In 1988–1989, 32 bobcats Lynx rufus were reintroduced to Cumberland Island (CUIS), Georgia, USA, from which they had previously been extirpated. They were monitored intensively for 3 years immediately post‐reintroduction, but no estimation of the size or genetic diversity of the population had been conducted in over 20 years since reintroduction. We returned to CUIS in 2012 to estimate abundance and effective population size of the present‐day population, as well as to quantify genetic diversity and inbreeding. We amplified 12 nuclear microsatellite loci from DNA isolated from scats to establish genetic profiles to identify individuals. We used spatially explicit capture–recapture population estimation to estimate abundance. From nine unique genetic profiles, we estimate a population size of 14.4 (SE = 3.052) bobcats, with an effective population size (N _e) of 5–8 breeding individuals. This is consistent with predictions of a population viability analysis conducted at the time of reintroduction, which estimated the population would average 12–13 bobcats after 10 years. We identified several pairs of related bobcats (parent‐offspring and full siblings), but ~75% of the pairwise comparisons were typical of unrelated individuals, and only one individual appeared inbred. Despite the small population size and other indications that it has likely experienced a genetic bottleneck, levels of genetic diversity in the CUIS bobcat population remain high compared to other mammalian carnivores. The reintroduction of bobcats to CUIS provides an opportunity to study changes in genetic diversity in an insular population without risk to this common species. Opportunities for natural immigration to the island are limited; therefore, continued monitoring and supplemental bobcat reintroductions could be used to evaluate the effect of different management strategies to maintain genetic diversity and population viability. The successful reintroduction and maintenance of a bobcat population on CUIS illustrates the suitability of translocation as a management tool for re‐establishing felid populations.     

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.     

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.     

Using Africa's protected area network to estimate the global population of a threatened and declining species: a case study of the Critically Endangered White‐headed Vulture Trigonoceps occipitalis

The White‐headed Vulture Trigonoceps occipitalis (WhV) is uncommon and largely restricted to protected areas across its range in sub‐Saharan Africa. We used the World Database on Protected Areas to identify protected areas (PAs) likely to contain White‐headed Vultures. Vulture occurrence on road transects in Southern, East, and West Africa was adjusted to nests per km² using data from areas with known numbers of nests and corresponding road transect data. Nest density was used to calculate the number of WhV nests within identified PAs and from there extrapolated to estimate the global population. Across a fragmented range, 400 PAs are estimated to contain 1893 WhV nests. Eastern Africa is estimated to contain 721 nests, Central Africa 548 nests, Southern Africa 468 nests, and West Africa 156 nests. Including immature and nonbreeding birds, and accounting for data deficient PAs, the estimated global population is 5475 ‐ 5493 birds. The identified distribution highlights are alarming: over 78% (n = 313) of identified PAs contain fewer than five nests. A further 17% (n = 68) of PAs contain 5 ‐ 20 nests and 4% (n = 14) of identified PAs are estimated to contain >20 nests. Just 1% (n = 5) of PAs are estimated to contain >40 nests; none is located in West Africa. Whilst ranging behavior of WhVs is currently unknown, 35% of PAs large enough to hold >20 nests are isolated by more than 100 km from other PAs. Spatially discrete and unpredictable mortality events such as poisoning pose major threats to small localized vulture populations and will accelerate ongoing local extinctions. Apart from reducing the threat of poisoning events, conservation actions promoting linkages between protected areas should be pursued. Identifying potential areas for assisted re‐establishment via translocation offers the potential to expand the range of this species and alleviate risk.     

ApoE4 upregulates the activity of mitochondria‐associated ER membranes

In addition to the appearance of senile plaques and neurofibrillary tangles, Alzheimer''s disease (AD) is characterized by aberrant lipid metabolism and early mitochondrial dysfunction. We recently showed that there was increased functionality of mitochondria‐associated endoplasmic reticulum (ER) membranes (MAM), a subdomain of the ER involved in lipid and cholesterol homeostasis, in presenilin‐deficient cells and in fibroblasts from familial and sporadic AD patients. Individuals carrying the ε4 allele of apolipoprotein E (ApoE4) are at increased risk for developing AD compared to those carrying ApoE3. While the reason for this increased risk is unknown, we hypothesized that it might be associated with elevated MAM function. Using an astrocyte‐conditioned media (ACM) model, we now show that ER–mitochondrial communication and MAM function—as measured by the synthesis of phospholipids and of cholesteryl esters, respectively—are increased significantly in cells treated with ApoE4‐containing ACM as compared to those treated with ApoE3‐containing ACM. Notably, this effect was seen with lipoprotein‐enriched preparations, but not with lipid‐free ApoE protein. These data are consistent with a role of upregulated MAM function in the pathogenesis of AD and may help explain, in part, the contribution of ApoE4 as a risk factor in the disease.