SUMO modification of nuclear receptors, including the constitutively active receptor steroidogenic factor 1 (SF-1; NR5A1), is proposed to repress their transcriptional activity. We examined the functional and structural consequences of SF-1 sumoylation at two conserved lysines (Lys119 and Lys194) that reside adjacent to the DNA-binding domain (DBD) and ligand-binding domain (LBD), respectively. Surprisingly, while previous loss-of-function studies predicted that sumoylation at Lys194 would greatly impact SF-1 function, the conformation and coregulator recruitment of fully sumoylated SF-1 LBD protein was either unchanged or modestly impaired. Sumoylation at Lys194 also modestly reduced Ser203 phosphorylation. In contrast to these findings, sumoylation of the DBD at Lys119 resulted in a marked and selective loss of DNA binding to noncanonical SF-1 targets, such as inhibin␣; this binding deficit was extended to all sites when the sumoylated human mutant (R92Q) protein, which exhibits lower activity, was used. Consistent with this result, the K119R mutant, compared to wild-type SF-1, was selectively recruited to a "SUMO-sensitive" site in the endogenous inhibin␣ promoter, leading to increased transcription. DNA binding and sumoylation of Lys119 appeared to be mutually exclusive, suggesting that once SF-1 is bound to DNA, sumoylation may be less important in regulating SF-1 activity. We propose that sumoylation of nuclear receptors imposes an active posttranslational mark that dampens recognition of SUMO-sensitive target genes to restrain their expression.Posttranslational modification with ubiquitin-like proteins has emerged as an important regulatory mechanism in many aspects of cellular function (29). Indeed, the small ubiquitinlike modifier (SUMO) conjugate modifies many transcription factors and results in marked transcriptional repression (27). Sumoylation occurs on lysines within consensus KxE sites through an enzymatic mechanism analogous to ubiquitination (6). While the requirement for an obligate SUMO E3 ligase is still debated, several proteins exhibit SUMO E3 ligase activity in cells, with the largest group belonging to the protein inhibitors of activated STATs (PIAS) family (47). Sumoylation is easily reversible by the action of SUMO isopeptidases (SUSPs or SENPs), with seven members identified in humans thus far (40).While recent structural studies have helped to elucidate the enzymatic details of SUMO conjugation and substrate recognition (44, 45), the mechanisms underlying transcriptional repression by sumoylation are less clear. Identification of a SUMO-interacting motif found in the PML protein and transcriptional repressors such as Daxx has helped to establish how the SUMO conjugate might function as a passive molecular mark to attenuate gene expression (28,39). However, it is also plausible that the SUMO conjugate functions as an active mark to modify either protein-protein or protein-DNA interactions. Evidence for this latter hypothesis remains controversial. For instance, sumoylation of thymine-...
Monoclonal antibodies (mAbs) and remdesivir, a small-molecule antiviral, are promising monotherapies for many viruses, including members of the genera Marburgvirus and Ebolavirus (family Filoviridae), and more recently, SARS-CoV-2. One of the major challenges of acute viral infections is the treatment of advanced disease. Thus, extending the window of therapeutic intervention is critical. Here, we explore the benefit of combination therapy with a mAb and remdesivir in a non-human primate model of Marburg virus (MARV) disease. While rhesus monkeys are protected against lethal infection when treatment with either a human mAb (MR186-YTE; 100%), or remdesivir (80%), is initiated 5 days post-inoculation (dpi) with MARV, no animals survive when either treatment is initiated alone beginning 6 dpi. However, by combining MR186-YTE with remdesivir beginning 6 dpi, significant protection (80%) is achieved, thereby extending the therapeutic window. These results suggest value in exploring combination therapy in patients presenting with advanced filovirus disease.
Poor drug delivery and penetration of antibody-mediated therapies pose significant obstacles to effective treatment of solid tumors. This study explored the role of pharmacokinetics, valency, and molecular weight in maximizing drug delivery. Biodistribution of a fibroblast growth factor receptor 4 (FGFR4) targeting CovX-body (an FGFR4-binding peptide covalently linked to a nontargeting IgG scaffold; 150 kDa) and enzymatically generated FGFR4 targeting F(ab)2 (100 kDa) and Fab (50 kDa) fragments was measured. Peak tumor levels were achieved in 1 to 2 hours for Fab and F(ab)2 versus 8 hours for IgG, and the percentage injected dose in tumors was 0.45%, 0.5%, and 2.5%, respectively, compared to 0.3%, 2%, and 6% of their nontargeting controls. To explore the contribution of multivalent binding, homodimeric peptides were conjugated to the different sized scaffolds, creating FGFR4 targeting IgG and F(ab)2 with four peptides and Fab with two peptides. Increased valency resulted in an increase in cell surface binding of the bivalent constructs. There was an inverse relationship between valency and intratumoral drug concentration, consistent with targeted consumption. Immunohistochemical analysis demonstrated increased size and increased cell binding decreased tumor penetration. The binding site barrier hypothesis suggests that limited tumor penetration, as a result of high-affinity binding, could result in decreased efficacy. In our studies, increased target binding translated into superior efficacy of the IgG instead, because of superior inhibition of FGFR4 proliferation pathways and dosing through the binding site barrier. Increasing valency is therefore an effective way to increase the efficacy of antibody-based drugs.
Inhalation of ricin toxin (RT), a Category B biothreat agent, provokes an acute respiratory distress syndrome marked by proinflammatory cytokine and chemokine production, neutrophilic exudate, and pulmonary edema. The severity of RT exposure is attributed to the tropism of the toxin's B subunit (RTB) for alveolar macrophages and airway epithelial cells, coupled with the extraordinarily potent ribosome-inactivating properties of the toxin's enzymatic subunit (RTA). While there are currently no vaccines or treatments approved to prevent RT intoxication, we recently described a humanized anti-RTA IgG 1 MAb, huPB10, that was able to rescue non-human primates (NHPs) from lethal dose RT aerosol challenge if administered by intravenous (IV) infusion within hours of toxin exposure. We have now engineered an extended serum half-life variant of that MAb, huPB10-LS, and evaluated it as a pre-exposure prophylactic. Five Rhesus macaques that received a single intravenous infusion (25 mg/kg) of huPB10-LS survived a lethal dose aerosol RT challenge 28 days later, whereas three control animals succumbed to RT intoxication within 48 h. The huPB10-LS treated animals remained clinically normal in the hours and days following toxin insult, suggesting that pre-existing antibody levels were sufficient to neutralize RT locally. Moreover, pro-inflammatory markers in sera and BAL fluids collected 24 h following RT challenge were significantly dampened in huPB10-LS treated animals, as compared to controls. Finally, we found that all five surviving animals, within days after RT exposure, had anti-RT serum IgG titers against epitopes other than huPB10-LS, indicative of active immunization by residual RT and/or RT-immune complexes.
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