Recent Publications

Distinct roles of UVRAG and EGFR signaling in skeletal muscle homeostasis

Mol Metab. 2021 Feb 6;47:101185. doi: 10.1016/j.molmet.2021.101185. Online ahead of print.


OBJECTIVE: Autophagy is a physiological self-eating process that can promote cell survival or activate cell death in eukaryotic cells. In skeletal muscle, it is important for maintaining muscle mass and function that is critical to sustain mobility and regulate metabolism. The UV radiation resistance-associated gene (UVRAG) regulates the early stages of autophagy and autophagosome maturation and plays a key role in endosomal trafficking. This study investigated the essential in vivo role of UVRAG in skeletal muscle biology.

METHODS: To determine the role of UVRAG in skeletal muscle in vivo, we generated muscle-specific UVRAG knockout mice using the Cre-loxP system driven by Myf6 promoter that is exclusively expressed in skeletal muscle. Myf6-Cre+ UVRAGfl/fl (M-UVRAG-/-) mice were compared to littermate Myf6-Cre+ UVRAG+/+ (M-UVRAG+/+) controls under basal conditions on a normal chow diet. Body composition, muscle function, and mitochondria morphology were assessed in muscles of the WT and KO mice at 24 weeks of age.

RESULTS: M-UVRAG-/- mice developed accelerated sarcopenia and impaired muscle function compared to M-UVRAG+/+ littermates at 24 weeks of age. Interestingly, these mice displayed improved glucose tolerance and increased energy expenditure likely related to upregulated Fgf21, a marker of muscle dysfunction. Skeletal muscle of the M-UVRAG-/- mice showed altered mitochondrial morphology with increased mitochondrial fission and EGFR accumulation reflecting defects in endosomal trafficking. To determine whether increased EGFR signaling had a causal role in muscle dysfunction, the mice were treated with an EGFR inhibitor, gefitinib, which partially restored markers of muscle and mitochondrial deregulation. Conversely, constitutively active EGFR transgenic expression in UVRAG-deficient muscle led to further detrimental effects with non-overlapping distinct defects in muscle function, with EGFR activation affecting the muscle fiber type whereas UVRAG deficiency impaired mitochondrial homeostasis.

CONCLUSIONS: Our results show that both UVRAG and EGFR signaling are critical for maintaining muscle mass and function with distinct mechanisms in the differentiation pathway.

PMID:33561544 | DOI:10.1016/j.molmet.2021.101185

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Diverse Eukaryotic CGG Binding Proteins Produced by Independent Domestications of hAT Transposons

Mol Biol Evol. 2021 Feb 9:msab007. doi: 10.1093/molbev/msab007. Online ahead of print.


The human transcription factor (TF) CGGBP1 ("CGG Binding Protein") is conserved only in amniotes, and is believed to derive from the zf-BED and Hermes transposase DNA-binding domains (DBDs) of a hAT DNA transposon. Here, we show that sequence-specific DNA binding proteins with this bipartite domain structure have resulted from dozens of independent hAT domestications in different eukaryotic lineages. CGGBPs display a wide range of sequence specificity, usually including preferences for CGG or CGC trinucleotides, while some bind AT-rich motifs. The CGGBPs are almost entirely non-syntenic, and their protein sequences, DNA binding motifs, and patterns of presence or absence in genomes are uncharacteristic of ancestry via speciation. At least eight CGGBPs in the coelacanth Latimeria chalumnae bind distinct motifs, and the expression of the corresponding genes varies considerably across tissues, suggesting tissue-restricted function.

PMID:33561217 | DOI:10.1093/molbev/msab007

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Crystal structure of the BRPF2 PWWP domain in complex with DNA reveals a different binding mode than the HDGF family of PWWP domains

Biochim Biophys Acta Gene Regul Mech. 2021 Mar;1864(3):194688. doi: 10.1016/j.bbagrm.2021.194688. Epub 2021 Feb 6.


The PWWP domain was first identified in the HDGF protein family and named after the conserved Proline-Tryptophan-Tryptophan-Proline motif in WHSC1. The PWWP domain-containing proteins play important roles in different biological processes, such as DNA replication, transcription, DNA repair, pre-mRNA processing by recognizing methylated histone and dsDNA simultaneously. Recently, how the HDGF family of PWWP domains recognize histone H3K36me3-modified nucleosome has been reported. In order to better understand the interactions between the PWWP domain and dsDNA, we carried out family-wide characterization of dsDNA binding abilities of human PWWP domains. Our binding assays confirmed that PWWP domains bind to dsDNA without sequence selectivity. Our crystal structure of the BRPF2 PWWP domain in complex with a 12-mer dsDNA reveals that the PWWP domain interacts with dsDNA by binding to its major groove, instead of the minor groove observed in the HDGF family of PWWP domains. Our study indicates that PWWP domains could bind to dsDNA in different modes.

PMID:33556623 | DOI:10.1016/j.bbagrm.2021.194688

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Angiomotin Counteracts the Negative Regulatory Effect of Host WWOX on Viral PPxY-Mediated Egress

J Virol. 2021 Feb 3:JVI.00121-21. doi: 10.1128/JVI.00121-21. Online ahead of print.


Filoviridae family members Ebola (EBOV) and Marburg (MARV) viruses and Arenaviridae family member Lassa virus (LASV) are emerging pathogens that can cause hemorrhagic fever and high rates of mortality in humans. A better understanding of the interplay between these viruses and the host will inform about the biology of these pathogens, and may lead to the identification of new targets for therapeutic development. Notably, expression of the filovirus VP40 and LASV Z matrix proteins alone drives assembly and egress of virus-like particles (VLPs). The conserved PPxY Late (L) domain motifs in the filovirus VP40 and LASV Z proteins play a key role in the budding process by mediating interactions with select host WW-domain containing proteins that then regulate virus egress and spread. To identify the full complement of host WW-domain interactors, we utilized WT and PPxY mutant peptides from EBOV and MARV VP40 and LASV Z proteins to screen an array of GST-WW-domain fusion proteins. We identified WW domain-containing oxidoreductase (WWOX) as a novel PPxY-dependent interactor, and we went on to show that full-length WWOX physically interacts with eVP40, mVP40 and LASV Z to negatively regulate egress of VLPs and of a live VSV/Ebola recombinant virus (M40). Interestingly, WWOX is a versatile host protein that regulates multiple signaling pathways and cellular processes via modular interactions between its WW-domains and PPxY motifs of select interacting partners, including host angiomotin (AMOT). Notably, we demonstrated recently that expression of endogenous AMOT not only positively regulates egress of VLPs, but also promotes egress and spread of live EBOV and MARV. Toward the mechanism of action, we show that the competitive and modular interplay among WWOX-AMOT-VP40/Z regulates VLP and M40 virus egress. Thus, WWOX is the newest member of an emerging group of host WW-domain interactors (e.g. BAG3; YAP/TAZ) that negatively regulate viral egress. These findings further highlight the complex interplay of virus-host PPxY/WW-domain interactions and their potential impact on the biology of both the virus and the host during infection.Author Summary Filoviruses (Ebola [EBOV] and Marburg [MARV]) and arenavirus (Lassa virus; LASV) are zoonotic, emerging pathogens that cause outbreaks of severe hemorrhagic fever in humans. A fundamental understanding of the virus-host interface is critical for understanding the biology of these viruses and for developing future strategies for therapeutic intervention. Here, we identified host WW-domain containing protein WWOX as a novel interactor with VP40 and Z, and showed that WWOX inhibited budding of VP40/Z virus-like particles (VLPs) and live virus in a PPxY/WW-domain dependent manner. Our findings are important to the field as they expand the repertoire of host interactors found to regulate PPxY-mediated budding of RNA viruses, and further highlight the competitive interplay and modular virus-host interactions that impact both the virus lifecycle and the host cell.

PMID:33536174 | DOI:10.1128/JVI.00121-21

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TGFβ signaling curbs cell fusion and muscle regeneration

Nat Commun. 2021 Feb 2;12(1):750. doi: 10.1038/s41467-020-20289-8.


Muscle cell fusion is a multistep process involving cell migration, adhesion, membrane remodeling and actin-nucleation pathways to generate multinucleated myotubes. However, molecular brakes restraining cell-cell fusion events have remained elusive. Here we show that transforming growth factor beta (TGFβ) pathway is active in adult muscle cells throughout fusion. We find TGFβ signaling reduces cell fusion, regardless of the cells' ability to move and establish cell-cell contacts. In contrast, inhibition of TGFβ signaling enhances cell fusion and promotes branching between myotubes in mouse and human. Exogenous addition of TGFβ protein in vivo during muscle regeneration results in a loss of muscle function while inhibition of TGFβR2 induces the formation of giant myofibers. Transcriptome analyses and functional assays reveal that TGFβ controls the expression of actin-related genes to reduce cell spreading. TGFβ signaling is therefore requisite to limit mammalian myoblast fusion, determining myonuclei numbers and myofiber size.

PMID:33531466 | PMC:PMC7854756 | DOI:10.1038/s41467-020-20289-8

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Niche-dependent inhibition of neural stem cell proliferation and oligodendrogenesis is mediated by the presence of myelin basic protein

Stem Cells. 2021 Feb 2. doi: 10.1002/stem.3344. Online ahead of print.


Neural stem and progenitor cells (collectively termed neural precursor cells [NPCs]) are found along the ventricular neuraxis extending from the spinal cord to the forebrain in regionally distinct niches comprised of different cell types, architecture, and cell-cell interactions. An understanding of the factors that regulate NPC behavior is critical for developing therapeutics to repair the injured central nervous system. Herein, we demonstrate that myelin basic protein (MBP), the major cytoplasmic protein constituent of the myelin sheath in oligodendrocytes, can regulate NPC behavior. Under physiological conditions, NPCs are not in contact with intracellular MBP; however, upon injury, MBP is released into the neural parenchyma. We reveal that MBP presented in a spinal cord niche is inhibitory to NPC proliferation. This inhibitory effect is regionally distinct as spinal cord NPCs, but not forebrain-derived NPCs, are inhibited by MBP. We performed coculture and conditioned media experiments that reveal the stem cell niche is a key regulator of MBP's inhibitory actions on NPCs. The inhibition is mediated by a heat-labile protein released by spinal cord niche cells, but not forebrain niche cells. However, forebrain NPCs are also inhibited by the spinal cord derived factor as revealed following in vivo infusion of the spinal cord niche-derived conditioned media. Moreover, we show that MBP inhibits oligodendrogenesis from NPCs. Together, these findings highlight the role of MBP and the regionally distinct microenvironment in regulating NPC behavior which has important implications for stem cell-based regenerative strategies.

PMID:33529418 | DOI:10.1002/stem.3344

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RNF168 regulates R-loop resolution and genomic stability in BRCA1/2-deficient tumors

J Clin Invest. 2021 Feb 1;131(3):e140105. doi: 10.1172/JCI140105.


Germline mutations in BRCA1 and BRCA2 (BRCA1/2) genes considerably increase breast and ovarian cancer risk. Given that tumors with these mutations have elevated genomic instability, they exhibit relative vulnerability to certain chemotherapies and targeted treatments based on poly (ADP-ribose) polymerase (PARP) inhibition. However, the molecular mechanisms that influence cancer risk and therapeutic benefit or resistance remain only partially understood. BRCA1 and BRCA2 have also been implicated in the suppression of R-loops, triple-stranded nucleic acid structures composed of a DNA:RNA hybrid and a displaced ssDNA strand. Here, we report that loss of RNF168, an E3 ubiquitin ligase and DNA double-strand break (DSB) responder, remarkably protected Brca1-mutant mice against mammary tumorigenesis. We demonstrate that RNF168 deficiency resulted in accumulation of R-loops in BRCA1/2-mutant breast and ovarian cancer cells, leading to DSBs, senescence, and subsequent cell death. Using interactome assays, we identified RNF168 interaction with DHX9, a helicase involved in the resolution and removal of R-loops. Mechanistically, RNF168 directly ubiquitylated DHX9 to facilitate its recruitment to R-loop-prone genomic loci. Consequently, loss of RNF168 impaired DHX9 recruitment to R-loops, thereby abrogating its ability to resolve R-loops. The data presented in this study highlight a dependence of BRCA1/2-defective tumors on factors that suppress R-loops and reveal a fundamental RNF168-mediated molecular mechanism that governs cancer development and vulnerability.

PMID:33529165 | PMC:PMC7843228 | DOI:10.1172/JCI140105

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Glucocorticoid agonists enhance retinal stem cell self-renewal and proliferation

Stem Cell Res Ther. 2021 Jan 25;12(1):83. doi: 10.1186/s13287-021-02136-9.


BACKGROUND: Adult mammalian retinal stem cells (RSCs) readily proliferate, self-renew, and generate progeny that differentiate into all retinal cell types in vitro. RSC-derived progeny can be induced to differentiate into photoreceptors, making them a potential source for retinal cell transplant therapies. Despite their proliferative propensity in vitro, RSCs in the adult mammalian eye do not proliferate and do not have a regenerative response to injury. Thus, identifying and modulating the mechanisms that regulate RSC proliferation may enhance the capacity to produce RSC-derived progeny in vitro and enable RSC activation in vivo.

METHODS: Here, we used medium-throughput screening to identify small molecules that can expand the number of RSCs and their progeny in culture. In vitro differentiation assays were used to assess the effects of synthetic glucocorticoid agonist dexamethasone on RSC-derived progenitor cell fate. Intravitreal injections of dexamethasone into adult mouse eyes were used to investigate the effects on endogenous RSCs.

RESULTS: We discovered that high-affinity synthetic glucocorticoid agonists increase RSC self-renewal and increase retinal progenitor proliferation up to 6-fold without influencing their differentiation in vitro. Intravitreal injection of synthetic glucocorticoid agonist dexamethasone induced in vivo proliferation in the ciliary epithelium-the niche in which adult RSCs reside.

CONCLUSIONS: Together, our results identify glucocorticoids as novel regulators of retinal stem and progenitor cell proliferation in culture and provide evidence that GCs may activate endogenous RSCs.

PMID:33494791 | PMC:PMC7831262 | DOI:10.1186/s13287-021-02136-9

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Nanotechnology for Modern Medicine: Next Steps Towards Clinical Translation

J Intern Med. 2021 Jan 22. doi: 10.1111/joim.13254. Online ahead of print.


The field of nanotechnology has been a significant research focus in the last thirty years. This emphasis is due to the unique optical, electrical, magnetic, chemical and biological properties of materials approximately ten thousand times smaller than the diameter of a hair strand. Researchers have developed methods to synthesize and characterize large libraries of nanomaterials and have demonstrated their preclinical utility. We have entered a new phase of nanomedicine development, where the focus is to translate these technologies to benefit patients. This review article provides an overview of nanomedicine's unique properties, the current state of the field, and discusses the challenge of clinical translation. Finally, we discuss the need to build and strengthen partnerships between engineers and clinicians to create a feedback loop between the bench and bedside. This partnership will guide fundamental studies on the nanoparticle-biological interactions, address clinical challenges, and change the development and evaluation of new drug delivery systems, sensors, imaging agents, and therapeutic systems.

PMID:33480120 | DOI:10.1111/joim.13254

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The promise of machine learning to inform the management of juvenile idiopathic arthritis

Expert Rev Clin Immunol. 2021 Jan;17(1):1-3. doi: 10.1080/1744666X.2020.1850268. Epub 2021 Jan 26.


PMID:33475006 | DOI:10.1080/1744666X.2020.1850268

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