• A distributed residue network permits conformational binding specificity in a conserved family of actin remodelers

      Hwang, T.; Parker, S.S.; Hill, S.M.; Ilunga, M.W.; Grant, R.A.; Mouneimne, G.; Keating, A.E.; Department of Cellular and Molecular Medicine, University of Arizona Cancer Center, University of Arizona (eLife Sciences Publications Ltd, 2021)
      Metazoan proteomes contain many paralogous proteins that have evolved distinct functions. The Ena/VASP family of actin regulators consists of three members that share an EVH1 interaction domain with a 100% conserved binding site. A proteome-wide screen revealed photoreceptor cilium actin regulator (PCARE) as a high-affinity ligand for ENAH EVH1. Here, we report the surprising observation that PCARE is ~100-fold specific for ENAH over paralogs VASP and EVL and can selectively bind ENAH and inhibit ENAH-dependent adhesion in cells. Specificity arises from a mechanism whereby PCARE stabilizes a conformation of the ENAH EVH1 domain that is inaccessible to family members VASP and EVL. Structure-based modeling  rapidly identified seven residues distributed throughout EVL that are sufficient to differentiate binding by ENAH vs. EVL. By exploiting the ENAH-specific conformation, we rationally designed the tightest and most selective ENAH binder to date. Our work uncovers a conformational mechanism of interaction specificity that distinguishes highly similar paralogs and establishes tools for dissecting specific Ena/VASP functions in processes including cancer cell invasion. © Hwang et al.
    • Antimicrobials from a feline commensal bacterium inhibit skin infection by drug-resistant S. Pseudintermedius

      O’Neill, A.M.; Worthing, K.A.; Kulkarni, N.; Li, F.; Nakatsuji, T.; McGrosso, D.; Mills, R.H.; Kalla, G.; Cheng, J.Y.; Norris, J.M.; et al. (eLife Sciences Publications Ltd, 2021)
      Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is an important emerging zoonotic pathogen that causes severe skin infections. To combat infections from drug- resistant bacteria, the transplantation of commensal antimicrobial bacteria as a therapeutic has shown clinical promise. We screened a collection of diverse staphylococcus species from domestic dogs and cats for antimicrobial activity against MRSP. A unique strain (S. felis C4) was isolated from feline skin that inhibited MRSP and multiple gram-positive pathogens. Whole genome sequencing and mass spectrometry revealed several secreted antimicrobials including a thiopeptide bacteriocin micrococcin P1 and phenol-soluble modulin beta (PSMP) peptides that exhibited antimicrobial and anti-inflammatory activity. Fluorescence and electron microscopy revealed that S. felis antimicrobials inhibited translation and disrupted bacterial but not eukaryotic cell membranes. Competition experiments in mice showed that S. felis significantly reduced MRSP skin colonization and an antimicrobial extract from S. felis significantly reduced necrotic skin injury from MRSP infection. These findings indicate a feline commensal bacterium that could be utilized in bacteriotherapy against difficult-to-treat animal and human skin infections. © 2021, eLife Sciences Publications Ltd. All rights reserved.
    • Brain endothelial cell trpa1 channels initiate neurovascular coupling

      Thakore, P.; Alvarado, M.G.; Ali, S.; Mughal, A.; Pires, P.W.; Yamasaki, E.; Pritchard, H.A.T.; Isakson, B.E.; Tran, C.H.T.; Earley, S.; et al. (eLife Sciences Publications Ltd, 2021)
      Cerebral blood flow is dynamically regulated by neurovascular coupling to meet the dynamic metabolic demands of the brain. We hypothesized that TRPA1 channels in capillary endothelial cells are stimulated by neuronal activity and instigate a propagating retrograde signal that dilates upstream parenchymal arterioles to initiate functional hyperemia. We find that activation of TRPA1 in capillary beds and post-arteriole transitional segments with mural cell coverage initiates retrograde signals that dilate upstream arterioles. These signals exhibit a unique mode of biphasic propagation. Slow, short-range intercellular Ca2+ signals in the capillary network are converted to rapid electrical signals in transitional segments that propagate to and dilate upstream arterioles. We further demonstrate that TRPA1 is necessary for functional hyperemia and neurovascular coupling within the somatosensory cortex of mice in vivo. These data establish endothelial cell TRPA1 channels as neuronal activity sensors that initiate microvascular vasodilatory responses to redirect blood to regions of metabolic demand. © 2021, eLife Sciences Publications Ltd. All rights reserved.
    • Concerted modification of nucleotides at functional centers of the ribosome revealed by single-molecule RNA modification profiling

      Bailey, A.D.; Talkish, J.; Ding, H.; Igel, H.; Duran, A.; Mantripragada, S.; Paten, B.; Ares, M.; Department of Pharmacy Practice & Science, College of Pharmacy, University of Arizona (eLife Sciences Publications Ltd, 2022)
      Nucleotides in RNA and DNA are chemically modified by numerous enzymes that alter their function. Eukaryotic ribosomal RNA (rRNA) is modified at more than 100 locations, particularly at highly conserved and functionally important nucleotides. During ribosome biogenesis, modifications are added at various stages of assembly. The existence of differently modified classes of ribosomes in normal cells is unknown because no method exists to simultaneously evaluate the modification status at all sites within a single rRNA molecule. Using a combination of yeast genetics and nanopore direct RNA sequencing, we developed a reliable method to track the modification status of single rRNA molecules at 37 sites in 18 S rRNA and 73 sites in 25 S rRNA. We use our method to characterize patterns of modification heterogeneity and identify concerted modification of nucleotides found near functional centers of the ribosome. Distinct, undermodified subpopulations of rRNAs accumulate upon loss of Dbp3 or Prp43 RNA helicases, suggesting overlapping roles in ribosome biogenesis. Modification profiles are surprisingly resistant to change in response to many genetic and acute environmental conditions that affect translation, ribosome biogenesis, and pre-mRNA splicing. The ability to capture single-molecule RNA modification profiles provides new insights into the roles of nucleotide modifications in RNA function. © 2022 Bailey et al.
    • Decreased recent adaptation at human mendelian disease genes as a possible consequence of interference between advantageous and deleterious variants

      Di, C.; Moreno, J.M.; Salazar-Tortosa, D.F.; Elise Lauterbur, M.; Enard, D.; University of Arizona (eLife Sciences Publications Ltd, 2021)
      Advances in genome sequencing have improved our understanding of the genetic basis of human diseases, and thousands of human genes have been associated with different diseases. Recent genomic adaptation at disease genes has not been well characterized. Here, we compare the rate of strong recent adaptation in the form of selective sweeps between mendelian, non-infectious disease genes and non-disease genes across distinct human populations from the 1000 Genomes Project. We find that mendelian disease genes have experienced far less selective sweeps compared to non-disease genes especially in Africa. Investigating further the possible causes of the sweep deficit at disease genes, we find that this deficit is very strong at disease genes with both low recombination rates and with high numbers of associated disease variants, but is almost non-existent at disease genes with higher recombination rates or lower numbers of associated disease variants. Because segregating recessive deleterious variants have the ability to interfere with adaptive ones, these observations strongly suggest that adaptation has been slowed down by the presence of interfering recessive deleterious variants at disease genes. These results suggest that disease genes suffer from a transient inability to adapt as fast as the rest of the genome. © 2021, eLife Sciences Publications Ltd. All rights reserved.
    • Injection with toxoplasma gondii protein affects neuron health and survival

      Mendez, O.A.; Machado, E.F.; Lu, J.; Koshy, A.A.; Graduate Interdisciplinary Program in Neuroscience, University of Arizona; BIO5 Institute, University of Arizona; College of Nursing, University of Arizona; Department of Immunobiology, University of Arizona; Department of Neurology, University of Arizona (eLife Sciences Publications Ltd, 2021)
      Toxoplasma gondii is an intracellular parasite that causes a long-term latent infection of neurons. Using a custom MATLAB-based mapping program in combination with a mouse model that allows us to permanently mark neurons injected with parasite proteins, we found that Toxoplasma-injected neurons (TINs) are heterogeneously distributed in the brain, primarily localizing to the cortex followed by the striatum. In addition, we determined that cortical TINs are commonly (>50%) excitatory neurons (FoxP2+) and that striatal TINs are often (>65%) medium spiny neurons (MSNs) (FoxP2+). By performing single neuron patch-clamping on striatal TINs and neighboring uninfected MSNs, we discovered that TINs have highly aberrant electrophysiology. As approximately 90% of TINs will die by 8 weeks post-infection, this abnormal physiology suggests that injection with Toxoplasma protein— either directly or indirectly— affects neuronal health and survival. Collectively, these data offer the first insights into which neurons interact with Toxoplasma and how these interactions alter neuron physiology in vivo. © 2021, eLife Sciences Publications Ltd. All rights reserved.
    • Molecular basis for functional connectivity between the voltage sensor and the selectivity filter gate in shaker k+ channels

      Bassetto, C.A.Z., Jr; Carvalho-De-souza, J.L.; Bezanilla, F.; Department of Anesthesiology, University of Arizona (eLife Sciences Publications Ltd, 2021)
      In Shaker K+ channels, the S4-S5 linker couples the voltage sensor (VSD) and pore domain (PD). Another coupling mechanism is revealed using two W434F-containing channels: L361R:W434F and L366H:W434F. In L361R:W434F, W434F affects the L361R VSD seen as a shallower Q-V curve that crosses the G-V. In L366H:W434F, L366H relieves the W434F effect converting a non-conductive channel in a conductive one. We report a chain of residues connecting the VSD (S4) to the selectivity filter (SF) in the PD of an adjacent subunit as the molecular basis for voltage-sensor selectivity filter gate (VS-SF) coupling. Single alanine substitutions in this region (L409A, S411A, S412A or F433A) are enough to disrupt the VS-SF coupling, shown by the absence of Q-V and G-V crossing in L361R:W434F mutant and by the lack of ionic conduction in the L366H:W434F mutant. This residue chain defines a new coupling between the VSD and the PD in voltage-gated channels. © 2021, eLife Sciences Publications Ltd. All rights reserved.
    • Native proline-rich motifs exploit sequence context to target actin-remodeling Ena/VASP protein ENAH

      Hwang, T.; Parker, S.S.; Hill, S.M.; Grant, R.A.; Ilunga, M.W.; Sivaraman, V.; Mouneimne, G.; Keating, A.E.; Department of Cellular & Molecular Medicine, University of Arizona (eLife Sciences Publications Ltd, 2022)
      The human proteome is replete with short linear motifs (SLiMs) of four to six residues that are critical for protein-protein interactions, yet the importance of the sequence surrounding such motifs is underexplored. We devised a proteomic screen to examine the influence of SLiM sequence context on protein-protein interactions. Focusing on the EVH1 domain of human ENAH, an actin regulator that is highly expressed in invasive cancers, we screened 36-residue proteome-derived peptides and discovered new interaction partners of ENAH and diverse mechanisms by which context influences binding. A pocket on the ENAH EVH1 domain that has diverged from other Ena/VASP paralogs recognizes extended SLiMs and favors motif-flanking proline residues. Many high-affinity ENAH binders that contain two proline-rich SLiMs use a noncanonical site on the EVH1 domain for binding and display a thermodynamic signature consistent with the two-motif chain engaging a single domain. We also found that photoreceptor cilium actin regulator (PCARE) uses an extended 23-residue region to obtain a higher affinity than any known ENAH EVH1-binding motif. Our screen provides a way to uncover the effects of proteomic context on motif-mediated binding, revealing diverse mechanisms of control over EVH1 interactions and establishing that SLiMs can’t be fully understood outside of their native context. © Hwang et al.
    • Pp2a/b55a substrate recruitment as defined by the retinoblastoma-related protein p107

      Fowle, H.; Zhao, Z.; Xu, Q.; Wasserman, J.S.; Wang, X.; Adeyemi, M.; Feiser, F.; Kurimchak, A.; Atar, D.; McEwan, B.C.; et al. (eLife Sciences Publications Ltd, 2021)
      Protein phosphorylation is a reversible post-translation modification essential in cell signaling. This study addresses a long-standing question as to how the most abundant serine/threonine Protein Phosphatase 2 (PP2A) holoenzyme, PP2A/B55a, specifically recognizes substrates and presents them to the enzyme active site. Here, we show how the PP2A regulatory subunit B55a recruits p107, a pRB-related tumor suppressor and B55a substrate. Using molecular and cellular approaches, we identified a conserved region 1 (R1, residues 615-626) encompassing the strongest p107 binding site. This enabled us to identify an “HxRVxxV619-625” short linear motif (SLiM) in p107 as necessary for B55a binding and dephosphorylation of the proximal pSer-615 in vitro and in cells. Numerous B55a/PP2A substrates, including TAU, contain a related SLiM C- terminal from a proximal phosphosite, “p[ST]-P-x(4,10)-[RK]-V-x-x-[VI]-R”. Mutation of conserved SLiM residues in TAU dramatically inhibits dephosphorylation by PP2A/B55a, validating its generality. A data-guided computational model details the interaction of residues from the conserved p107 SLiM, the B55a groove, and phosphosite presentation. Altogether these data provide key insights into PP2A/B55a mechanisms of substrate recruitment and active site engagement, and also facilitate identification and validation of new substrates, a key step towards understanding PP2A/B55a role in multiple cellular processes. © 2021, eLife Sciences Publications Ltd. All rights reserved.
    • Shore crabs reveal novel evolutionary attributes of the mushroom body

      Strausfeld, N.; Sayre, M.E.; Department of Neuroscience, University of Arizona (eLife Sciences Publications Ltd, 2021)
      Neural organization of mushroom bodies is largely consistent across insects, whereas the ancestral ground pattern diverges broadly across crustacean lineages resulting in successive loss of columns and the acquisition of domed centers retaining ancestral Hebbian-like networks and aminergic connections. We demonstrate here a major departure from this evolutionary trend in Brachyura, the most recent malacostracan lineage. In the shore crab Hemigrapsus nudus, instead of occupying the rostral surface of the lateral protocerebrum, mushroom body calyces are buried deep within it with their columns extending outwards to an expansive system of gyri on the brain’s surface. The organization amongst mushroom body neurons reaches extreme elaboration throughout its constituent neuropils. The calyces, columns, and especially the gyri show DC0 immunoreactivity, an indicator of extensive circuits involved in learning and memory. © Strausfeld and Sayre.
    • Systematic analysis of naturally occurring insertions and deletions that alter transcription factor spacing identifies tolerant and sensitive transcription factor pairs

      Shen, Z.; Li, R.Z.; Prohaska, T.A.; Hoeksema, M.A.; Spann, N.J.; Tao, J.; Fonseca, G.J.; Le, T.; Stolze, L.; Sakai, M.; et al. (eLife Sciences Publications Ltd, 2022)
      Regulation of gene expression requires the combinatorial binding of sequence-specific transcription factors (TFs) at promoters and enhancers. Prior studies showed that alterations in the spacing between TF binding sites can influence promoter and enhancer activity. However, the relative importance of TF spacing alterations resulting from naturally occurring insertions and deletions (InDels) has not been systematically analyzed. To address this question, we first characterized the genome-wide spacing relationships of 73 TFs in human K562 cells as determined by ChIP-seq. We found a dominant pattern of a relaxed range of spacing between collaborative factors, including 45 TFs exclusively exhibiting relaxed spacing with their binding partners. Next, we exploited millions of InDels provided by genetically diverse mouse strains and human individuals to investigate the effects of altered spacing on TF binding and local histone acetylation. These analyses suggested that spacing alterations resulting from naturally occurring InDels are generally tolerated in comparison to genetic variants directly affecting TF binding sites. To experimentally validate this prediction, we introduced synthetic spacing alterations between PU.1 and C/EBPβ binding sites at six endogenous genomic loci in a macrophage cell line. Remarkably, collaborative binding of PU.1 and C/EBPβ at these locations tolerated changes in spacing ranging from 5-bp increase to >30-bp decrease. Collectively, these findings have implications for understanding mechanisms underlying enhancer selection and for the interpretation of non-coding genetic variation. © 2022, eLife Sciences Publications Ltd. All rights reserved.
    • Targeting a cell surface vitamin D receptor on tumor-associated macrophages in triple-negative breast cancer

      Staquicini, F.I.; Hajitou, A.; Driessen, W.H.; Proneth, B.; Cardó-Vila, M.; Staquicini, D.I.; Markosian, C.; Hoh, M.; Cortez, M.; Hooda-Nehra, A.; et al. (eLife Sciences Publications Ltd, 2021)
      Triple-negative breast cancer (TNBC) is an aggressive tumor with limited treatment options and poor prognosis. We applied the in vivo phage display technology to isolate peptides homing to the immunosuppressive cellular microenvironment of TNBC as a strategy for non-malignant target discovery. We identified a cyclic peptide (CSSTRESAC) that specifically binds to a vitamin D receptor, protein disulfide-isomerase A3 (PDIA3) expressed on the cell surface of tumor-associated macrophages (TAM), and targets breast cancer in syngeneic TNBC, non-TNBC xenograft, and transgenic mouse models. Systemic administration of CSSTRESAC to TNBC-bearing mice shifted the cytokine profile toward an antitumor immune response and delayed tumor growth. Moreover, CSSTRESAC enabled ligand-directed theranostic delivery to tumors and a mathematical model confirmed our experimental findings. Finally, in silico analysis showed PDIA3-expressing TAM in TNBC patients. This work uncovers a functional interplay between a cell surface vitamin D receptor in TAM and antitumor immune response that could be therapeutically exploited. © 2021, Staquicini et al.
    • Traumatic injury compromises nucleocytoplasmic transport and leads to TDP-43 pathology

      Anderson, E.N.; Morera, A.A.; Kour, S.; Cherry, J.D.; Ramesh, N.; Gleixner, A.; Schwartz, J.C.; Ebmeier, C.; Old, W.; Donnelly, C.J.; et al. (eLife Sciences Publications Ltd, 2021)
      Traumatic brain injury (TBI) is a predisposing factor for many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), and chronic traumatic encephalopathy (CTE). Although defects in nucleocytoplasmic transport (NCT) is reported ALS and other neurodegenerative diseases, whether defects in NCT occur in TBI remains unknown. We performed proteomic analysis on Drosophila exposed to repeated TBI and identified resultant alterations in several novel molecular pathways. TBI upregulated nuclear pore complex (NPC) and nucleocytoplasmic transport (NCT) proteins as well as alter nucleoporin stability. Traumatic injury disrupted RanGAP1 and NPC protein distribution in flies and a rat model and led to coaggregation of NPC components and TDP-43. In addition, trauma-mediated NCT defects and lethality are rescued by nuclear export inhibitors. Importantly, genetic upregulation of nucleoporins in vivo and in vitro triggered TDP-43 cytoplasmic mislocalization, aggregation, and altered solubility and reduced motor function and lifespan of animals. We also found NUP62 pathology and elevated NUP62 concentrations in postmortem brain tissues of patients with mild or severe CTE as well as co-localization of NUP62 and TDP-43 in CTE. These findings indicate that TBI leads to NCT defects, which potentially mediate the TDP-43 pathology in CTE. © 2021, Anderson et al.
    • Unifying the known and unknown microbial coding sequence space

      Vanni, C.; Schechter, M.S.; Acinas, S.G.; Barberán, A.; Buttigieg, P.L.; Casamayor, E.O.; Delmont, T.O.; Duarte, C.M.; Eren, A.M.; Finn, R.D.; et al. (eLife Sciences Publications Ltd, 2022)
      Genes of unknown function are among the biggest challenges in molecular biology, especially in microbial systems, where 40%-60% of the predicted genes are unknown. Despite previous attempts, systematic approaches to include the unknown fraction into analytical workflows are still lacking. Here, we present a conceptual framework, its translation into the computational workflow AGNOSTOS and a demonstration on how we can bridge the known-unknown gap in genomes and metagenomes. By analyzing 415,971,742 genes predicted from 1,749 metagenomes and 28,941 bacterial and archaeal genomes, we quantify the extent of the unknown fraction, its diversity, and its relevance across multiple organisms and environments. The unknown sequence space is exceptionally diverse, phylogenetically more conserved than the known fraction and predominantly taxonomically restricted at the species level. From the 71M genes identified to be of unknown function, we compiled a collection of 283,874 lineage-specific genes of unknown function for Cand. Patescibacteria (also known as Candidate Phyla Radiation, CPR), which provides a significant resource to expand our understanding of their unusual biology. Finally, by identifying a target gene of unknown function for antibiotic resistance, we demonstrate how we can enable the generation of hypotheses that can be used to augment experimental data. © 2022, eLife Sciences Publications Ltd. All rights reserved.
    • Universal and taxon-specific trends in protein sequences as a function of age

      James, Jennifer E; Willis, Sara M; Nelson, Paul G; Weibel, Catherine; Kosinski, Luke J; Masel, Joanna; Department of Ecology and Evolutionary Biology, University of Arizona; Department of Physics, University of Arizona; Department of Mathematics, University of Arizona; Department of Molecular and Cellular Biology, University of Arizona (eLife Sciences Publications Ltd, 2021-01-08)
      Extant protein-coding sequences span a huge range of ages, from those that emerged only recently to those present in the last universal common ancestor. Because evolution has had less time to act on young sequences, there might be ‘phylostratigraphy’ trends in any properties that evolve slowly with age. A long-term reduction in hydrophobicity and hydrophobic clustering was found in previous, taxonomically restricted studies. Here we perform integrated phylostratigraphy across 435 fully sequenced species, using sensitive HMM methods to detect protein domain homology. We find that the reduction in hydrophobic clustering is universal across lineages. However, only young animal domains have a tendency to have higher structural disorder. Among ancient domains, trends in amino acid composition reflect the order of recruitment into the genetic code, suggesting that the composition of the contemporary descendants of ancient sequences reflects amino acid availability during the earliest stages of life, when these sequences first emerged. © James et al.