Biology subjects

Hansen, K.

Publications and source records attributed to Hansen, K..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

PP1 PNUTS binds the restrictor and dephosphorylates RNA pol II CTD Ser5 to stimulate transcription termination

The restrictor, ZC3H4/WDR82, is the major termination factor for antisense transcription from bidirectional promoters, but its mechanism is poorly understood. We report that ZC3H4/WDR82 co-purifies with PP1 phosphatase and PP1 phosphatase nuclear targeting subunit, PNUTS, which binds directly to the WDR82 subunit of restrictor. AlphaFold predicts a quaternary complex, PPWZ, in which PP1-associated PNUTS and ZC3H4 both contact WDR82. To investigate the role of protein dephosphorylation in PPWZ activity, we expressed a substrate trap comprising inactive PP1H66K linked to the PNUTS C-terminus. PP1H66K-PNUTS binds pol II large subunit and nuclear exosome components. PP1H66K-PNUTS, but not PP1WT-PNUTS, functions as a dominant-negative inhibitor of antisense termination and CTD Ser5 dephosphorylation. Both these activities require the PNUTS WDR82 binding domain that interacts with restrictor. We show that CTD Ser5 hyperphosphorylation is associated with higher processivity and reduced pausing that would counteract termination, and propose that Ser5 dephosphorylation by PPWZ is coupled to termination. In summary, we identify the PP1 phosphatase activity of the PPWZ complex as essential for terminator function and propose that this heterotetramer is the physiologically relevant form of restrictor.

molecular biology↗

bioRxiv2024

Ferroptosis regulates hemolysis in stored murine and human red blood cells

Red blood cell (RBC) metabolism regulates hemolysis during aging in vivo and in the blood bank. Here, we leveraged a diversity outbred mouse population to map the genetic drivers of fresh/stored RBC metabolism and extravascular hemolysis upon storage and transfusion in 350 mice. We identify the ferrireductase Steap3 as a critical regulator of a ferroptosis-like process of lipid peroxidation. Steap3 polymorphisms were associated with RBC iron content, in vitro hemolysis, and in vivo extravascular hemolysis both in mice and 13,091 blood donors from the Recipient Epidemiology and Donor evaluation Study. Using metabolite Quantitative Trait Loci analyses, we identified a network of gene products (FADS1/2, EPHX2 and LPCAT3) - enriched in donors of African descent - associated with oxylipin metabolism in stored human RBCs and related to Steap3 or its transcriptional regulator, the tumor protein TP53. Genetic variants were associated with lower in vivo hemolysis in thousands of single-unit transfusion recipients. HighlightsO_LISteap3 regulates lipid peroxidation and extravascular hemolysis in 350 diversity outbred mice C_LIO_LISteap3 SNPs are linked to RBC iron, hemolysis, vesiculation in 13,091 blood donors C_LIO_LImQTL analyses of oxylipins identified ferroptosis-related gene products FADS1/2, EPHX2, LPCAT3 C_LIO_LIFerroptosis markers are linked to hemoglobin increments in transfusion recipients C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/598512v1_ufig1.gif" ALT="Figure 1"> View larger version (117K): org.highwire.dtl.DTLVardef@150fec2org.highwire.dtl.DTLVardef@859c43org.highwire.dtl.DTLVardef@1d60156org.highwire.dtl.DTLVardef@f1b91e_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Ligand-induced conformational changes in the β1-Adrenergic Receptor Revealed by Hydrogen-Deuterium Exchange Mass Spectrometry

G-Protein Coupled Receptors (GPCRs) constitute the largest family of signalling proteins responsible for translating extracellular stimuli into intracellular functions. When dysregulated, GPCRs drive numerous diseases and are the most targeted proteins in drug discovery. GPCR structural dynamics and activity can be modulated by a wide range of drugs, including full/partial agonists and antagonists. While crucial for developing novel therapeutics targeting GPCRs, the structural dynamics of the receptors associated with their activity upon drug interactions are not yet fully understood. Here, we employ Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS), to characterise the structural dynamics of turkey {beta}1-adrenergic receptor (t{beta}1AR) in complex with nine ligands, including agonists, partial agonists and antagonists. We show that dynamic signatures across the GPCR structure can be grouped by compound modality. Surprisingly, we discovered repeated destabilisation of the intracellular loop 1 (ICL1) upon full agonist binding and stabilisation upon antagonist binding, suggesting that increased dynamics in this region are an essential component for G-protein recruitment. Multiple sequence alignments and molecular dynamics simulations indicate that L72 in ICL1 plays important structural role. Differential HDX-MS experiment of t{beta}1AR and t{beta}1AR L72A construct in complex with miniGs, in response to various ligands, suggests involvement of ICL1 in stabilising the GDP bound state by influencing the stability of HG helix of miniGs. Overall, our results provide a platform for determining drug modality and highlight how HDX-MS can be used to dissect receptor ligand interaction properties and GPCR mechanism. Significance statementRecent advances in hydrogen-deuterium exchange mass spectrometry have allowed probing conformational signatures of challenging membrane protein assemblies. We studied the structural dynamics of a class A GPCR, namely t{beta}1AR, in response to diverse ligands including agonists, antagonists and partial agonists. We demonstrate that the functional effect of compounds can be discerned by simply profiling the dynamics induced across the receptor, without the need for downstream interaction partners. We showed that ICL1 undergoes a significant change in dynamics between activated and inhibited states consistent with a role in downstream signaling pathways in class A GPCRs.

biophysics↗

bioRxiv2024

Aggregation of recount3 RNA-seq data improves the inference of consensus and tissue-specific gene co-expression networks.

BackgroundGene co-expression networks (GCNs) describe relationships among expressed genes key to maintaining cellular identity and homeostasis. However, the small sample size of typical RNA-seq experiments which is several orders of magnitude fewer than the number of genes is too low to infer GCNs reliably. recount3, a publicly available dataset comprised of 316,443 uniformly processed human RNA-seq samples, provides an opportunity to improve power for accurate network reconstruction and obtain biological insight from the resulting networks. ResultsWe compared alternate aggregation strategies to identify an optimal workflow for GCN inference by data aggregation and inferred three consensus networks: a universal network, a non-cancer network, and a cancer network in addition to 27 tissue context-specific networks. Central network genes from our consensus networks were enriched for evolutionarily constrained genes and ubiquitous biological pathways, whereas central context-specific network genes included tissue-specific transcription factors and factorization based on the hubs led to clustering of related tissue contexts. We discovered that annotations corresponding to context-specific networks inferred from aggregated data were enriched for trait heritability beyond known functional genomic annotations and were significantly more enriched when we aggregated over a larger number of samples. ConclusionThis study outlines best practices for network GCN inference and evaluation by data aggregation. We recommend estimating and regressing confounders in each data set before aggregation and prioritizing large sample size studies for GCN reconstruction. Increased statistical power in inferring context-specific networks enabled the derivation of variant annotations that were enriched for concordant trait heritability independent of functional genomic annotations that are context-agnostic. While we observed strictly increasing held-out log-likelihood with data aggregation, we noted diminishing marginal improvements. Future directions aimed at alternate methods for estimating confounders and integrating orthogonal information from modalities such as Hi-C and ChIP-seq can further improve GCN inference.

genomics↗

bioRxiv2023

Selected humanization of yeast U1 snRNP leads to global suppression of pre-mRNA splicing and mitochondrial dysfunction in the budding yeast

The recognition of 5 splice site (5 ss) is one of the earliest steps of pre-mRNA splicing. To better understand the mechanism and regulation of 5 ss recognition, we selectively humanized components of the yeast U1 snRNP to reveal the function of these components in 5 ss recognition and splicing. We targeted U1C and Luc7, two proteins that interact with and stabilize the yeast U1 (yU1) snRNA and the 5 ss RNA duplex. We replaced the Zinc-Finger (ZnF) domain of yU1C with its human counterpart, which resulted in cold-sensitive growth phenotype and moderate splicing defects. Next, we added an auxin-inducible degron to yLuc7 protein and found that Luc7-depleted yU1 snRNP resulted in the concomitant loss of PRP40 and Snu71 (two other essential yeast U1 snRNP proteins), and further biochemical analyses suggest a model of how these three proteins interact with each other in the U1 snRNP. The loss of these proteins resulted in a significant growth retardation accompanied by a global suppression of pre-mRNA splicing. The splicing suppression led to mitochondrial dysfunction as revealed by a release of Fe2+ into the growth medium and an induction of mitochondrial reactive oxygen species. Together, these observations indicate that the human U1C ZnF can substitute that of yeast, Luc7 is essential for the incorporation of the Luc7-Prp40-Snu71 trimer into yeast U1 snRNP, and splicing plays a major role in the regulation of mitochondria function in yeast.

biochemistry↗

bioRxiv2023

Palatal segment contributions to midfacial growth in three inbred mouse strains

Anterior-posterior (A-P) elongation of the palate is a critical aspect of integrated midfacial morphogenesis. Reciprocal epithelial-mesenchymal interactions drive secondary palate elongation that is coupled to the periodic formation of signaling centers within the rugae growth zone (RGZ). However, the relationship between RGZ-driven morphogenetic processes, the differentiative dynamics of underlying palatal bone mesenchymal precursors, and the segmental organization of the upper jaw has remained enigmatic. A detailed ontogenetic study of these relationships is important because palatal segment growth is a critical aspect of normal midfacial growth, can produce dysmorphology when altered, and is a likely basis for evolutionary differences in upper jaw morphology. We completed a combined whole mount gene expression and morphometric analysis of normal murine palatal segment growth dynamics and resulting upper jaw morphology. Our results demonstrated that the first formed palatal ruga (ruga 1), found just posterior to the RGZ, maintained an association with important nasal, neurovascular and palatal structures throughout early midfacial development. This suggested that these features are positioned at a proximal source of embryonic midfacial directional growth. Our detailed characterization of midfacial morphogenesis revealed a one-to-one relationship between palatal segments and upper jaw bones during the earliest stages of palatal elongation. Growth of the maxillary anlage within the anterior secondary palate is uniquely coupled to RGZ-driven morphogenesis. This may help drive the unequaled proportional elongation of the anterior secondary palate segment prior to palatal shelf fusion. Our results also demonstrated that the future maxillary-palatine suture, approximated by the position of ruga 1 and consistently associated with the palatine anlage, formed predominantly via the posterior differentiation of the maxilla within the expanding anterior secondary palate. Our ontogenetic analysis provides a novel and detailed picture of the earliest spatiotemporal dynamics of intramembranous midfacial skeletal specification and differentiation within the context of the surrounding palatal segment A-P elongation and associated rugae formation.

developmental biology↗

bioRxiv2023

Biological and Genetic Determinants of Red Blood Cell Glycolysis

Mature red blood cells (RBCs) lack mitochondria, and thus exclusively rely on glycolysis to generate adenosine triphosphate (ATP) during aging in vivo or storage in the blood bank. Here we leveraged 13,029 volunteers from the Recipient Epidemiology and Donor Evaluation Study to identify an association between end-of-storage levels of glycolytic metabolites and donor age, sex, and ancestry-specific genetic polymorphisms in regions encoding phosphofructokinase 1, platelet (detected in mature RBCs), hexokinase 1, ADP-ribosyl cyclase 1 and 2 (CD38/BST1). Gene-metabolite associations were validated in fresh and stored RBCs from 525 Diversity Outbred mice, and via multi-omics characterization of 1,929 samples from 643 human RBC units during storage. ATP and hypoxanthine levels - and the genetic traits linked to them - were associated with hemolysis in vitro and in vivo, both in healthy autologous transfusion recipients and in 5,816 critically ill patients receiving heterologous transfusions, suggesting their potential as markers to improve transfusion outcomes. eTOC and Highlights O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/557250v4_ufig1.gif" ALT="Figure 1"> View larger version (87K): org.highwire.dtl.DTLVardef@1a556b7org.highwire.dtl.DTLVardef@1e02b13org.highwire.dtl.DTLVardef@2bfab1org.highwire.dtl.DTLVardef@15787e3_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIBlood donor age and sex affect glycolysis in stored RBCs from 13,029 volunteers; C_LIO_LIAncestry, genetic polymorphisms in PFKP, HK1, CD38/BST1 influence RBC glycolysis; C_LIO_LIModeled PFKP effects relate to preventing loss of the total AXP pool in stored RBCs; C_LIO_LIATP and hypoxanthine are biomarkers of hemolysis in vitro and in vivo. C_LI

biochemistry↗

bioRxiv2023

RxRx3: Phenomics Map of Biology

The combination of modern genetic perturbation techniques with high content screening has enabled genome-scale cell microscopy experiments that can be leveraged to construct maps of biology. These are built by processing microscopy images to produce readouts in unified and relatable representation space to capture known biological relationships and discover new ones. To further enable the scientific community to develop methods and insights from map-scale data, here we release RxRx3, the first ever public high-content screening dataset combining genome-scale CRISPR knockouts with multiple-concentration screening of small molecules (a set of FDA approved and commercially available bioactive compounds). The dataset contains 6-channel fluorescent microscopy images and associated deep learning embeddings from over 2.2 million wells that span 17,063 CRISPR knockouts and 1,674 compounds at 8 doses each. RxRx3 is one of the largest collections of cellular screening data, and as far as we know, the largest generated consistently via a common experimental protocol within a single laboratory. Our goal in releasing RxRx3 is to demonstrate the benefits of generating consistent data, enable the development of the machine learning methods on this scale of data and to foster research, methods development, and collaboration. For more information about RxRx3 please visit RxRx.ai/rxrx3

systems biology↗

bioRxiv2022

Small molecule inhibition of multiple RNA binding proteins underlies Musashi-2 independent phenotypes

RNA binding proteins (RBPs) are key regulators of gene expression. Small molecules targeting these RBP-RNA interactions are a rapidly emerging class of therapeutics for treating a variety of diseases. Ro-08-2750 (Ro) is a small molecule inhibitor identified as a competitive inhibitor of Musashi(MSI)-RNA interactions. Here we show Ro potently inhibits adrenocortical steroidogenesis and viability independent of MSI2 in multiple cell lines. We identified Ro-interacting proteins using an unbiased proteome-wide approach and discovered it is broadly targeting RBPs. To confirm this finding, we leveraged the large-scale ENCODE data and found a subset of RBPs whose depletion phenocopies Ro inhibition. We conclude that Ro is a promiscuous inhibitor of multiple RBPs, many containing RRM1 domains. Moreover, we provide a general framework for validating the specificity and identifying targets of RBP inhibitors in a cellular context.

molecular biology↗

bioRxiv2022

MyD88-TLR4-dependent choroid plexus activation precedes perilesional inflammation and edema in intracerebral hemorrhage

The functional neurological outcome of patients with intracerebral hemorrhage (ICH) strongly relates to the degree of secondary brain injury (ICH-SBI) evolving within days after the initial bleeding. Different mechanisms including the incitement of inflammatory pathways, dysfunction of the blood-brain barrier (BBB), activation of resident microglia, and an influx of blood-borne immune cells, have been hypothesized to contribute to ICH-SBI. Yet, the spatiotemporal interplay of specific inflammatory processes within different brain compartments has not been sufficiently characterized, limiting potential therapeutic interventions to prevent and treat ICH-SBI. Using a whole-blood injection model in mice, we systematically characterized the spatial and temporal dynamics of inflammatory processes after ICH using 7-Tesla magnetic resonance imaging (MRI), spatial RNA sequencing (spRNAseq), functional BBB assessment, and immunofluorescence average-intensity-mapping. We identified a pronounced early response of the choroid plexus (CP) peaking at 12 to 24h, that was characterized by inflammatory cytokine expression, epithelial and endothelial expression of leukocyte adhesion molecules, and the accumulation of leukocytes. In contrast, we observed a delayed secondary reaction pattern at the injection site (striatum) peaking at 96h, defined by gene expression corresponding to perilesional leukocyte infiltration and correlating to the delayed signal alteration seen on MRI. Pathway analysis revealed a dependence of the early inflammatory reaction in the CP on toll-like receptor 4 (TLR4) signaling via myeloid differentiation factor 88 (MyD88). TLR4 and MyD88 knockout mice corroborated this observation, lacking the early upregulation of adhesion molecules and leukocyte infiltration within the CP 24h after whole-blood injection. In conclusion, we report a biphasic brain reaction pattern after ICH with a MyD88-TLR4-dependent early inflammatory response of the CP, preceding inflammation, edema and leukocyte infiltration at the lesion site. Pharmacological targeting of the early CP-activation might harbor the potential to modulate the development of ICH-SBI. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/506660v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@195554corg.highwire.dtl.DTLVardef@1130d16org.highwire.dtl.DTLVardef@1c03678org.highwire.dtl.DTLVardef@381ff4_HPS_FORMAT_FIGEXP M_FIG C_FIG Akeret, Buzzi et al. characterized the spatiotemporal dynamics after striatal whole blood injection in mice using magnetic resonance imaging (MRI), spatial RNA sequencing (spRNAseq), functional blood-brain barrier (BBB) assessment, and immunofluorescence average intensity mapping (IF). They report a biphasic brain reaction pattern with an early MyD88-TLR4-dependent inflammatory response of the CP, which preceded secondary inflammation and leukocyte infiltration at the perilesional site.

neuroscience↗

bioRxiv2022

A common cis-regulatory variant impacts normal-range and disease-associated human facial shape through regulation of PKDCC during chondrogenesis

Genome-wide association studies (GWAS) identified thousands of genetic variants linked to phenotypic traits and disease risk. However, mechanistic understanding of how GWAS variants influence complex morphological traits and can, in certain cases, simultaneously confer normal-range phenotypic variation and disease predisposition, is still largely lacking. Here, we focus on rs6740960, a single nucleotide polymorphism (SNP) at the 2p21 locus, which in GWAS studies has been associated both with normal-range variation in jaw shape and with an increased risk of non-syndromic orofacial clefting. Using in vitro derived embryonic cell types relevant for human facial morphogenesis, we show that this SNP resides in an enhancer that regulates chondrocytic expression of PKDCC - a gene encoding a tyrosine kinase involved in chondrogenesis and skeletal development. In agreement, rs6740960 SNP is sufficient to confer a large difference in acetylation of its cognate enhancer preferentially in chondrocytes. By deploying dense landmark morphometric analysis of skull elements in mice, we show that changes in Pkdcc dosage are associated with quantitative changes in maxilla, mandible, and palatine bone shape that are concordant with the facial phenotypes and disease predisposition seen in humans. We further demonstrate that the frequency of the rs6740960 variant strongly deviated among different human populations, and that the activity of its cognate enhancer diverged in hominids. Our study provides a mechanistic explanation of how a common SNP can mediate normal-range and disease-associated morphological variation, with implications for the evolution of human facial features.

developmental biology↗

bioRxiv2022

Cryo-EM structure and functional landscape of an RNA polymerase ribozyme

The emergence of an RNA replicase capable of self-replication is considered an important stage in the origin of life. RNA polymerase ribozymes (PR) including a variant that uses trinucleotide triphosphates (triplets) as substrates have been created by in vitro evolution and are the closest functional analogues of the replicase but the structural basis for their function is poorly understood. Here, we leverage single-particle cryo-EM and high-throughput mutation analysis to obtain the structure of a triplet polymerase ribozyme (TPR) apoenzyme and map its functional landscape. The TPR cryo-EM structure at 5-[A] resolution reveals an RNA heterodimer comprising a catalytic and an inactive accessory subunit, where the complex resembles a left hand with thumb and fingers at a 70{degrees} angle. The two subunits are connected by two distinct kissing-loop (KL) interactions that are essential for polymerase function. Our combined structural and functional data suggest a model for templated RNA synthesis by the TPR holoenzyme whereby heterodimer formation and KL interactions preorganize the TPR for optimal template binding and templated RNA synthesis activity. These results provide a foundation for a better understanding RNAs potential for self-replication.

biochemistry↗

bioRxiv2022

Multi-omics analysis of sarcospan overexpression in mdx skeletal muscle reveals compensatory remodeling of cytoskeleton-matrix interactions that promote mechanotransduction pathways

BackgroundThe dystrophin-glycoprotein complex (DGC) is a critical adhesion complex of the muscle cell membrane, providing a mechanical link between the extracellular matrix (ECM) and the cortical cytoskeleton that stabilizes the sarcolemma during repeated muscle contractions. One integral component of the DGC is the transmembrane protein, sarcospan (SSPN). Overexpression of SSPN in the skeletal muscle of mdx mice (murine model of DMD) restores muscle fiber attachment to the ECM in part through an associated increase in utrophin and integrin adhesion complexes at the cell membrane, protecting the muscle from contraction-induced injury. In this study, we utilized transcriptomic and ECM protein-optimized proteomics data sets from wild-type, mdx, and mdx transgenic (mdx TG) skeletal muscle tissues to identify pathways and proteins driving the compensatory action of SSPN overexpression. MethodsThe tibialis anterior and quadriceps muscles were isolated from wild-type, mdx, and mdx TG mice and subjected to bulk RNA-Seq and global proteomics analysis using methods to enhance capture of ECM proteins. Data sets were further analyzed through the Ingenuity Pathway Analysis (QIAGEN) and integrative gene set enrichment to identify candidate networks, signaling pathways, and upstream regulators. ResultsThrough our multi-omics approach, we identified 3 classes of differentially expressed genes and proteins in mdx TG muscle, included those that were: 1) unrestored (significantly different from wild-type, but not from mdx), 2) restored (significantly different from mdx, but not from wild-type), and 3) compensatory (significantly different from both wild-type and mdx). We identified signaling pathways that may contribute to the rescue phenotype, most notably cytoskeleton and ECM organization pathways. ECM optimized-proteomics revealed an increased abundance of collagens II, V, and XI, along with {beta}-spectrin in mdx TG samples. Using Ingenuity Pathway Analysis, we identified upstream regulators that are computationally predicted to drive compensatory changes, revealing a possible mechanism of SSPN rescue through a rewiring of cell-ECM bidirectional communication. We found that SSPN overexpression results in upregulation of key signaling molecules associated with regulation of cytoskeleton organization and mechanotransduction, including Rho, RAC, and Wnt. ConclusionsOur findings indicate that SSPN overexpression rescues dystrophin deficiency partially through mechanotransduction signaling cascades mediated through components of the ECM and the cortical cytoskeleton.

molecular biology↗

bioRxiv2021

Heme-stress activated NRF2 signaling skews fate trajectories of bone marrow cells from dendritic cells towards red pulp-like macrophages

Heme is an erythrocyte-derived toxin that drives disease progression in hemolytic anemias, such as sickle cell disease. During hemolysis, specialized bone marrow-derived macrophages with a high heme-metabolism capacity orchestrate disease adaptation by removing damaged erythrocytes and heme-protein complexes from the blood and supporting iron recycling for erythropoiesis. Since chronic heme-stress is noxious for macrophages, erythrophagocytes in the spleen are continuously replenished from bone marrow-derived progenitors. Here, we hypothesized that adaptation to heme stress progressively shifts differentiation trajectories of BM progenitors to expand the capacity of heme-handling monocyte-derived macrophages at the expense of the homeostatic generation of dendritic cells, which emerge from shared myeloid precursors. This heme-induced redirection of differentiation trajectories may contribute to hemolysis-induced secondary immunodeficiency. We performed single-cell RNA sequencing with directional RNA velocity analysis of GM-CSF-supplemented mouse bone marrow cultures to assess myeloid differentiation under heme stress. We found that heme-activated NRF2 signaling shifted the differentiation of bone marrow cells towards antioxidant, iron-recycling macrophages, suppressing the generation of dendritic cells in heme-exposed bone marrow cultures. Heme eliminated the capacity of GM-CSF-supplemented bone marrow cultures to activate antigen-specific CD4 T cells. The generation of functionally competent dendritic cells was restored by NRF2 loss. The heme-induced phenotype of macrophage expansion with concurrent dendritic cell depletion was reproduced in hemolytic mice with sickle cell disease and spherocytosis and associated with reduced dendritic cell functions in the spleen. Our data provide a novel mechanistic underpinning of hemolytic stress as a driver of hyposplenism-related secondary immunodeficiency. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/454342v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1995a26org.highwire.dtl.DTLVardef@eda1b8org.highwire.dtl.DTLVardef@11441c3org.highwire.dtl.DTLVardef@e9a95c_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2021

Proteolytic Activation of Human-specific Olduvai Domains by the Furin Protease

Olduvai protein domains (formerly DUF1220) show the greatest human-specific increase in copy number of any coding region in the genome and are highly correlated with human brain evolution and cognitive disease. The majority of human copies are found within four NBPF genes organized in a variable number of a tandemly arranged three-domain blocks called Olduvai triplets. Here we show that these human-specific Olduvai domains are posttranslationally processed by the furin protease, with a cleavage site occurring once at each triplet. These findings suggest that all expanded human-specific NBPF genes encode proproteins consisting of many independent Olduvai triplet proteins which are activated by furin processing. The exceptional correlation of Olduvai copy number and brain size taken together with our new furin data, indicates the ultimate target of selection was a rapid increase in dosage of autonomously functioning Olduvai triplet proteins, and that these proteins are the primary active agent underlying Olduvais role in human brain expansion.

evolutionary biology↗

bioRxiv2021

Pro-regenerative Extracellular Matrix Hydrogel Prevents and Mitigates Pathological Alterations of Pelvic Muscles Following Birth Injury

Pelvic floor disorders, which include pelvic organ prolapse, and urinary and fecal incontinence, affect millions of women globally and represent a major public health concern. Pelvic floor muscle (PFM) dysfunction has been identified as one of the leading risk factors for the development of these morbid conditions. Even though childbirth, specifically vaginal delivery, has been long recognized as the most important potentially modifiable risk factor for PFM injury, the precise mechanisms of PFM dysfunction following childbirth remain elusive. In this study we demonstrate that PFMs undergo atrophy and severe fibrosis in parous women with symptomatic pelvic organ prolapse compared to age-matched nulliparous cadaveric donors without history of pelvic floor disorders. These pathological alterations are recapitulated in the pre-clinical rat model of simulated birth injury. The transcriptional signature of PFMs post-injury demonstrates a sustained inflammatory response, impairment in muscle anabolism, and persistent expression of extracellular matrix (ECM) remodeling genes. Next, we evaluated the administration of acellular injectable skeletal muscle extracellular matrix hydrogel for the prevention and mitigation of these pathological alterations. Treatment of PFMs with the biomaterial either at the time of birth injury or 4 weeks post-injury reduced muscle atrophy and mitigated fibrotic degeneration. By evaluating gene expression, we demonstrate that these changes are mainly driven by the hydrogel-induced modulation of the immune response and intramuscular fibrosis, as well as enhancement of the endogenous myogenesis. This work furthers our understanding of PFM birth injury and demonstrates proof-of-concept for a new pragmatic pro-regenerative biomaterial approach for treating injured PFMs.

bioengineering↗

bioRxiv2021

Quantification of biologically and chemically bound phosphorus in activated sludge from full-scale plants with biological P-removal

Large amounts of phosphorus (P) are present in activated sludge from municipal wastewater treatment plants, where it exists in the form of metal salt precipitates or biologically bound into the biomass as nucleic acids, cell membrane components, and the extracellular polymeric substances or, in special polyphosphate-accumulating organisms (PAOs), as intracellular polyphosphate. Only recently, methods that reliably allow an absolute quantification of the different P-fractions, such as sequential extraction, Raman microspectroscopy, solid-state 31P magic angle spinning (MAS) NMR, and solution state 31P NMR have been developed. This study combines these techniques to obtain a comprehensive P mass-balance of activated sludge from four wastewater treatment plants with enhanced biological phosphate removal (EBPR). The total content of P and various cations was measured by chemical analysis (ICP-OES), and different P fractions were extracted for chemical characterization. Chemically bound P constituted 38-69% of total P, most likely in the form of Fe, Mg, or Al minerals, while organically bound P constituted 7-9%. By using Raman microspectroscopy and solution state 31P NMR and 31P MAS NMR spectroscopy before and after anaerobic P-release experiments, poly-P was quantified and constituted 22-54% of total P in the activated sludges and was found in approx. 25% of all bacterial cells. Moreover, Raman microspectroscopy in combination with fluorescence in situ hybridization (FISH) was used to quantify the species-specific intracellular poly-P of known PAO genera (Tetrasphaera, Ca. Accumulibacter, Dechloromonas) and other microorganisms known to possess high level of poly-P, such as the filamentous Ca. Microthrix. They were all abundant, as measured by quantitative-FISH and amplicon sequencing, and accumulated large amount of poly-P, depending on their cell-size, contributing substantially to the P-removal. Interestingly, in all four EBPR plants investigated, only 1-13% of total poly-P was stored by unidentified PAO, highlighting that most PAOs in the full-scale EBPR plants investigated are now known. HighlightsO_LIExhaustive P mass-balance of main organic and inorganic P-species in four EBPR plants C_LIO_LIQuantification of poly-P of FISH-defined PAO and other species with high P content C_LIO_LITotal P content was 36-50 mgP/gSS of which 31-62% was in biomass and as poly-P C_LIO_LIA high fraction of all cells (25-30%) contained a high content of poly-P C_LIO_LIKnown PAOs contained almost all poly-P in the EBPR plants investigated C_LI

microbiology↗

bioRxiv2020

The interactome of the N-terminus of band 3 regulates red blood cell metabolism and storage quality

Band 3 (anion exchanger 1 - AE1) is the most abundant membrane protein in red blood cells (RBCs), the most abundant cell in the human body. A compelling model, based on indirect evidence, posits that - at high oxygen saturation - the N-term cytosolic domain of AE1 binds to and inhibits glycolytic enzymes, thus diverting metabolic fluxes to the pentose phosphate pathway to generate reducing equivalents. Dysfunction of this mechanism occurs during RBC aging or storage under blood bank conditions, suggesting a role for AE1 in the regulation of blood storage quality and efficacy of transfusion - a life-saving intervention for millions of recipients worldwide. Here we leverage two murine models carrying genetic ablations of AE1 to provide the first direct mechanistic evidence of its role in metabolic regulation and blood storage quality. Observations in mice phenocopied those in a human subject lacking expression of AE11-11 (band 3 Neapolis), while common polymorphisms in the region coding for AE11-56 increased susceptibility to osmotic hemolysis in healthy blood donors. Through thermal proteome profiling and cross-linking proteomics, we provide the first comprehensive analysis of the RBC interactome, with a focus on AE11-56 and validate recombinant AE1 interactions with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Finally, we show that incubation with a cell-penetrating AE11-56 peptide can rescue the metabolic defect in glutathione recycling and boost post-transfusion recoveries of stored RBCs from healthy human donors and genetically ablated mice, paving the way for the in vivo metabolic manipulation of RBCs facing oxidant stress - a landmark of many diseases. Key pointsO_LIGenetic ablation of N-term of band 3 results in significant metabolic aberrations and poor post-transfusion recoveries in mice and humans; C_LIO_LIStructural studies on the N-term of band 3 reveal a complex interactome with several enzymes, including GAPDH; C_LI

biochemistry↗