Inorganic polyphosphates (polyPs) are linear polymers composed of repeated phosphate (PO4 3-) units linked together by multiple high-energy phosphoanhydride bonds. In addition to being a source of energy, polyPs have cytoprotective and antiviral activities. Here, we investigated the antiviral activities of long-chain polyPs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In molecular docking analyses, polyPs interacted with several conserved amino acid residues in angiotensin-converting enzyme 2 (ACE2), the host receptor that facilitates virus entry, and in viral RNA-dependent RNA polymerase (RdRp). ELISA and limited proteolysis assays using nano- LC-MS/MS mapped polyP120 binding to ACE2, and site-directed mutagenesis confirmed interactions between ACE2 and SARS-CoV-2 RdRp and identified the specific amino acid residues involved. PolyP120 enhanced the proteasomal degradation of both ACE2 and RdRp, thus impairing replication of the British B.1.1.7 SARS-CoV-2 variant. We thus tested polyPs for functional interactions with the virus in SARS-CoV-2-infected Vero E6 and Caco2 cells and in primary human nasal epithelial cells. Delivery of a nebulized form of polyP120 reduced the amounts of viral positive-sense genomic and subgenomic RNAs, of RNA transcripts encoding proinflammatory cytokines, and of viral structural proteins, thereby presenting SARS-CoV-2 infection in cells in vitro.

Update of medRxiv. 2020 Sep 08;:

We report the clinical and molecular phenotype of three siblings from one family, who presented with short stature and immunodeficiency and carried uncharacterized variants in RGS10 (c.489_491del:p.E163del and c.G511T:p.A171S). This gene encodes regulator of G protein signaling 10 (RGS10), a member of a large family of GTPase-activating proteins (GAPs) that targets heterotrimeric G proteins to constrain the activity of G protein-coupled receptors, including receptors for chemoattractants. The affected individuals exhibited systemic abnormalities directly related to the RGS10 mutations, including recurrent infections, hypergammaglobulinemia, profoundly reduced lymphocyte chemotaxis, abnormal lymph node architecture, and short stature due to growth hormone deficiency. Although the GAP activity of each RGS10 variant was intact, each protein exhibited aberrant patterns of PKA-mediated phosphorylation and increased cytosolic and cell membrane localization and activity compared to the wild-type protein. We propose that the RGS10 p.E163del and p.A171S mutations lead to mislocalization of the RGS10 protein in the cytosol, thereby resulting in attenuated chemokine signaling. This study suggests that RGS10 is critical for both immune competence and normal hormonal metabolism in humans and that rare RGS10 variants may contribute to distinct systemic genetic disorders.

Mutations in the kinase LRRK2 and impaired endocytic trafficking are both implicated in the pathogenesis of Parkinson's disease (PD). Expression of the PD-associated LRRK2 mutant in mouse dopaminergic neurons was shown to disrupt clathrin-mediated endocytic trafficking. Here, we explored the molecular mechanism linking LRRK2 to endocytosis and found that LRRK2 bound to and phosphorylated the μ2 subunit of the adaptor protein AP2 (AP2M1), a core component of the clathrin-mediated endocytic machinery. Analysis of human SH-SY5Y cells and mouse neurons and tissues revealed that loss of LRRK2 abundance or kinase function resulted in decreased phosphorylation of AP2M1, which is required for the initial formation of clathrin-coated vesicles (CCVs). In contrast, overexpression of LRRK2 or expression of a Parkinson's disease-associated gain-of-function mutant LRRK2 (G2019S) inhibited the uncoating of AP2M1 from CCVs at later stages and prevented new cycles of CCV formation. Thus, the abundance and activity of LRRK2 must be calibrated to ensure proper endocytosis. Dysregulated phosphorylation of AP2M1 from the brain but not thyroid tissues of LRRK2 knockout and G2019S-knockin mice suggests a tissue-specific regulatory mechanism of endocytosis. Furthermore, we found that LRRK2-dependent phosphorylation of AP2M1 mediated dopaminergic neurodegeneration in a Drosophila model of PD. Together, our findings provide a mechanistic link between LRRK2, AP2, and endocytosis in the pathogenesis of PD.

Murine γδ17 cells, which are T cells that bear the γδ T cell receptor (TCRγδ) and secrete interleukin-17A (IL-17A), are generated in the thymus and are critical for various immune responses. Although strong TCRγδ signals are required for the development of interferon-γ (IFN-γ)-secreting γδ cells (γδIFN cells), the generation of γδ17 cells requires weaker TCRγδ signaling. Here, we demonstrated that constrained activation of the kinase Syk downstream of TCRγδ was required for the thymic development of γδ17 cells. Increasing or decreasing Syk activity by stimulating TCRγδ or inhibiting Syk, respectively, substantially reduced γδ17 cell numbers. This delimited Syk activity optimally engaged the phosphoinositide 3-kinase (PI3K)-Akt signaling pathway, which maintained the expression of master regulators of the IL-17 program, RORγt and c-Maf. Inhibition of PI3K not only abrogated γδ17 cell development but also augmented the development of a distinct, previously undescribed subset of γδ T cells. These CD8+Ly6a+ γδ T cells had a type-I IFN gene expression signature and expanded in response to stimulation with IFN-β. Collectively, these studies elucidate how weaker TCRγδ signaling engages distinct signaling pathways to specify the γδ17 cell fate and identifies a role for type-I IFNs in γδ T cell development.

Cellular adaptation to low-oxygen environments is mediated in part by the hypoxia-inducible factors (HIFs). Like other transcription factors, the stability and transcriptional activity of HIFs-and consequently, the hypoxic response-are regulated by post-translational modifications (PTMs) and changes in protein-protein interactions. Our current understanding of PTM-mediated regulation of HIFs is primarily based on in vitro protein fragment-based studies typically validated in fragment-expressing cells treated with hypoxia-mimicking compounds. Here, we used immunoprecipitation-based mass spectrometry to characterize the PTMs and binding partners for full-length HIF-1α and HIF-2α under normoxic (21% oxygen) and hypoxic (1% oxygen) conditions. Hypoxia substantially altered the complexity and composition of the HIFα protein interaction networks, particularly for HIF-2α, with the hypoxic networks of both isoforms being enriched for mitochondrial proteins. Moreover, both HIFα isoforms were heavily covalently modified. We identified ~40 PTM sites composed of 13 different types of modification on both HIFα isoforms, including multiple cysteine modifications and an unusual phosphocysteine. More than 80% of the PTMs identified were not previously known and about half exhibited oxygen dependency. We further characterized an evolutionarily conserved phosphorylation of Ser31 in HIF-1α as a regulator of its transcriptional function, and we propose functional roles for Thr406, Thr528, and Ser581 in HIF-2α. These data will help to delineate the different physiological roles of these closely related isoforms in fine-tuning the hypoxic response.

The type I interferon (IFN) response is an important component of the innate immune response to viral infection. Precise control of IFN responses is critical because insufficient expression of IFN-stimulated genes (ISGs) can lead to a failure to restrict viral spread, whereas excessive ISG activation can result in IFN-related pathologies. Although both positive and negative regulatory factors control the magnitude and duration of IFN signaling, it is also appreciated that several ISGs regulate aspects of the IFN response themselves. In this study, we performed a CRISPR activation screen to identify previously unknown regulators of the type I IFN response. We identified the strongly induced ISG encoding ETS variant transcription factor 7 (ETV7) as a negative regulator of the type I IFN response. However, ETV7 did not uniformly suppress ISG transcription. Instead, ETV7 preferentially targeted a subset of antiviral ISGs that were particularly important for IFN-mediated control of influenza viruses. Together, our data assign a function for ETV7 as an IFN response regulator and also identify ETV7 as a potential therapeutic target to increase innate antiviral responses and enhance IFN-based antiviral therapies.

Synaptic plasticity involves structural modifications in dendritic spines that are modulated by local protein synthesis and actin remodeling. Here, we investigated the molecular mechanisms that connect synaptic stimulation to these processes. We found that the phosphorylation of isoform-specific sites in eEF1A2-an essential translation elongation factor in neurons-is a key modulator of structural plasticity in dendritic spines. Expression of a nonphosphorylatable eEF1A2 mutant stimulated mRNA translation but reduced actin dynamics and spine density. By contrast, a phosphomimetic eEF1A2 mutant exhibited decreased association with F-actin and was inactive as a translation elongation factor. Activation of metabotropic glutamate receptor signaling triggered transient dissociation of eEF1A2 from its regulatory guanine exchange factor (GEF) protein in dendritic spines in a phosphorylation-dependent manner. We propose that eEF1A2 establishes a cross-talk mechanism that coordinates translation and actin dynamics during spine remodeling.