Octyl itaconate enhances VSVΔ51 oncolytic virotherapy by multitarget inhibition of antiviral and inflammatory pathways.

The presence of heterogeneity in responses to oncolytic virotherapy poses a barrier to clinical effectiveness, as resistance to this treatment can occur through the inhibition of viral spread within the tumor, potentially leading to treatment failures. Here we show that 4-octyl itaconate (4-OI), a chemical derivative of the Krebs cycle-derived metabolite itaconate, enhances oncolytic virotherapy with VSVΔ51 in various models including human and murine resistant cancer cell lines, three-dimensional (3D) patient-derived colon tumoroids and organotypic brain tumor slices. Furthermore, 4-OI in combination with VSVΔ51 improves therapeutic outcomes in a resistant murine colon tumor model. Mechanistically, we find that 4-OI suppresses antiviral immunity in cancer cells through the modification of cysteine residues in MAVS and IKKß independently of the NRF2/KEAP1 axis. We propose that the combination of a metabolite-derived drug with an oncolytic virus agent can greatly improve anticancer therapeutic outcomes by direct interference with the type I IFN and NF-κB-mediated antiviral responses.

SAM68 directs STING signaling to apoptosis in macrophages.

DNA is a danger signal sensed by cGAS to engage signaling through STING to activate innate immune functions. The best-studied downstream responses to STING activation include expression of type I interferon and inflammatory genes, but STING also activates other pathways, including apoptosis. Here, we report that STING-dependent induction of apoptosis in macrophages occurs through the intrinsic mitochondrial pathway and is mediated via IRF3 but acts independently of gene transcription. By intersecting four mass spectrometry datasets, we identify SAM68 as crucial for the induction of apoptosis downstream of STING activation. SAM68 is essential for the full activation of apoptosis. Still, it is not required for STING-mediated activation of IFN expression or activation of NF-κB. Mechanistic studies reveal that protein trafficking is required and involves SAM68 recruitment to STING upon activation, with the two proteins associating at the Golgi or a post-Golgi compartment. Collectively, our work identifies SAM68 as a STING-interacting protein enabling induction of apoptosis through this DNA-activated innate immune pathway.

Novel homozygous CD46 variant with C-isoform expression affects C3b inactivation in atypical hemolytic uremic syndrome.

Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy that may lead to organ failure. Dysregulation of the complement system can cause aHUS, and various disease-related variants in the complement regulatory protein CD46 are described. We here report a pediatric patient with aHUS carrying a hitherto unreported homozygous variant in CD46 (NM_172359.3:c.602C>T p.(Ser201Leu)). In our functional analyses, this variant caused complement dysregulation through three separate mechanisms. First, CD46 surface expression on the patient's blood cells was significantly reduced. Second, stably expressing CD46(Ser201Leu) cells bound markedly less to patterns of C3b than CD46 WT cells. Third, the patient predominantly expressed the rare isoforms of CD46 (C dominated) instead of the more common isoforms (BC dominated). Using BC1 and C1 expressing cell lines, we found that the C1 isoform bound markedly less C3b than the BC1 isoform. These results highlight the coexistence of multiple mechanisms that may act synergistically to disrupt CD46 function during aHUS development.

CD46 Isoforms Influence the Mode of Entry by Human Herpesvirus 6A/B in T Cells.

CD46 is a receptor for human herpesvirus 6A (HHV-6A) and is in some cells also important for infection with HHV-6B. CD46 has several isoforms of which the most commonly expressed can be distinguished by expression of a BC domain or a C domain in a serine-threonine-proline-rich (STP) extracellular region. Using a SupT1 CD46 CRISPR-Cas9 knockout model system reconstituted with specific CD46 isoforms, we demonstrated that HHV-6A infection was more efficient when BC isoforms were expressed as opposed to C isoforms, measured by higher levels of intracellular viral transcripts and recovery of more progeny virus. Although the B domain contains several O-glycosylations, mutations of Ser and Thr residues did not prevent infection with HHV-6A. The HHV-6A infection was blocked by inhibitors of clathrin-mediated endocytosis. In contrast, infection with HHV-6B was preferentially promoted by C isoforms mediating fusion-from-without, and this infection was less affected by inhibitors of clathrin-mediated endocytosis. Taken together, HHV-6A preferred BC isoforms, mediating endocytosis, whereas HHV-6B preferred C isoforms, mediating fusion-from-without. This demonstrates that the STP region of CD46 is important for regulating the mode of infection in SupT1 cells and suggests an epigenetic regulation of the host susceptibility to HHV-6A and HHV-6B infection. IMPORTANCE CD46 is the receptor used by human herpesvirus 6A (HHV-6A) during infection of T cells, but it is also involved in infection of certain T cells by HHV-6B. The gene for CD46 allows expression of several variants of CD46, known as isoforms, but whether the isoforms matter for infection of T cells is unknown. We used a genetic approach to delete CD46 from T cells and reconstituted them with separate isoforms to study them individually. We expressed the isoforms known as BC and C, which are distinguished by the potential inclusion of a B domain in the CD46 molecule. We demonstrate that HHV-6A prefers the BC isoform to infect T cells, and this occurs predominantly by clathrin-mediated endocytosis. In contrast, HHV-6B prefers the C isoform and infects predominantly by fusion-from-without. Thus, CD46 isoforms may affect susceptibility of T cells to infection with HHV-6A and HHV-6B.

Comparison of electrostatic and mechanical cell sorting with limited starting material.

Isolation of multiple cell populations from limited starting material and with minimal influence on cell homeostasis and viability are common requirements in both basic and clinical research. Fluorescence-activated cell sorting (FACS) is the most commonly applied sorting methodology with the majority of instruments being based on high pressure and electrostatic deflection. A more recent technology is based on a mechanical valve, operating at low pressure. In the present work we compared the two technologies by parallel sorting of small amounts of peripheral blood and umbilical cord blood on a BD FACSAria™ III and Miltenyi MACSQuant® Tyto® instrument. Concurrent manually performed magnetic-based cell sorting served as reference. Sorting metrics, including purity and viability, were compared. Expression of the heat-shock protein HSPA1A immediately post sorting and the proliferation potential of sorted T-cells in vitro was assessed. In general, there was little to distinguish the two fluorescence-activated technologies with regard to sorting metrics and HSPA1A expression. Variation, however, with respect to recovery and viability, was much smaller among Tyto sorted samples. The proliferation potential of Tyto-sorted T-cells was significantly higher compared to Aria-sorted T-cells, indicating that T-cells of the Tyto instrument are less perturbed. In summary, cell types of blood origin including CD34+ cells could effectively be isolated from small input amounts with either fluorescence-activated technology with little immediate effect on viability. The mechanical valve-based sorting by the Tyto instrument; however, appeared to perturb the cells to a lesser extent as judged by their proliferation potential.

The tetraspanin protein CD9 modulates infection with human herpesvirus 6A and 6B in a CD46-dependent manner.

Tetraspanins are four-span transmembrane proteins that organize the membrane by forming tetraspanin-enriched microdomains. These have been shown to be important for virus entry. The human herpesvirus (HHV)-6A receptor CD46 is known to form complexes with the tetraspanin CD9 and ß1-integrins, however the significance of this for HHV-6A infection remains unexplored. Using a genetic approach, we demonstrate that knock out of CD46 abolishes binding to and infection of SupT1 cells by both HHV-6A and HHV-6B, establishing CD46 as a necessary receptor for productive infection of these cells. Knock out of CD9 in SupT1 cells had no effect on binding of either virus to the cell surface, but it reduced expression of immediate early transcripts to between 25-60% compared with the wild type cells. Although HHV-6B required CD46 for infection of SupT1, infection of Molt3 cells was independent of CD46 expression. Conversely, the absence of CD9 expression promoted infection of Molt3 cells with HHV-6B, indicating a negative role of CD9 for CD46-independent infection. Taken together, these data demonstrate that CD9 modulates infection with HHV-6A/B by promoting CD46-dependent infection and impairing CD46-independent infection. This also suggests that HHV-6A is strictly dependent on CD46 for entry, although other proteins, like CD9, may enhance the infection, whereas HHV-6B is more promiscuous and may use CD134, as demonstrated by others, CD46 in SupT1, and a novel yet unidentified receptor in Molt3 cells.Importance The mechanisms of entry of human herpesvirus (HHV)-6A and HHV-6B into host cells are of significance in order to develop novel drugs that may inhibit infection. To elucidate the contribution of the membrane proteins CD9 and CD46, we employed a genetic approach that eliminated these molecules from the host cell. This demonstrated that CD46 is critical for infection by HHV-6A, whereas infection by HHV-6B appeared to be more promiscuous. The infection of a T-cell line in the absence of CD46 and CD134 strongly suggest that an additional receptor for HHV-6B entry exists. Moreover, elimination of CD9 and subsequent reconstitution experiments demonstrated that CD9 promoted infection with HHV-6A and HHV-6B mediated by CD46, but inhibited infection with HHV-6B that occurred independent of CD46. Together, this demonstrated a CD46-dependent role of CD9 during infection with HHV-6A and HHV-6B and emphasized that HHV-6B may employ different entry mechanisms in various cells.

Functional consequences of the CAPOS mutation E818K of Na+,K+-ATPase.

The cerebellar ataxia, areflexia, pes cavus, optic atrophy, and sensorineural hearing loss (CAPOS) syndrome is caused by the single mutation E818K of the α3-isoform of Na+,K+-ATPase. Here, using biochemical and electrophysiological approaches, we examined the functional characteristics of E818K, as well as of E818Q and E818A mutants. We found that these amino acid substitutions reduce the apparent Na+ affinity at the cytoplasmic-facing sites of the pump protein and that this effect is more pronounced for the lysine and glutamine substitutions (3-4-fold) than for the alanine substitution. The electrophysiological measurements indicated a more conspicuous, ∼30-fold reduction of apparent Na+ affinity for the extracellular-facing sites in the CAPOS mutant, which was related to an accelerated transition between the phosphoenzyme intermediates E1P and E2P. The apparent affinity for K+ activation of the ATPase activity was unaffected by these substitutions, suggesting that primarily the Na+-specific site III is affected. Furthermore, the apparent affinities for ATP and vanadate were WT-like in E818K, indicating a normal E1-E2 equilibrium of the dephosphoenzyme. Proton-leak currents were not increased in E818K. However, the CAPOS mutation caused a weaker voltage dependence of the pumping rate and a stronger inhibition by cytoplasmic K+ than the WT enzyme, which together with the reduced Na+ affinity of the cytoplasmic-facing sites precluded proper pump activation under physiological conditions. The functional deficiencies could be traced to the participation of Glu-818 in an intricate hydrogen-bonding/salt-bridge network, connecting it to key residues involved in Na+ interaction at site III.

Neurological disease mutations of α3 Na+,K+-ATPase: Structural and functional perspectives and rescue of compromised function.

Na+,K+-ATPase creates transmembrane ion gradients crucial to the function of the central nervous system. The α-subunit of Na+,K+-ATPase exists as four isoforms (α1-α4). Several neurological phenotypes derive from α3 mutations. The effects of some of these mutations on Na+,K+-ATPase function have been studied in vitro. Here we discuss the α3 disease mutations as well as information derived from studies of corresponding mutations of α1 in the light of the high-resolution crystal structures of the Na+,K+-ATPase. A high proportion of the α3 disease mutations occur in the transmembrane sector and nearby regions essential to Na+ and K+ binding. In several cases the compromised function can be traced to disturbance of the Na+ specific binding site III. Recently, a secondary mutation was found to rescue the defective Na+ binding caused by a disease mutation. A perspective is that it may be possible to develop an efficient pharmaceutical mimicking the rescuing effect.

Somatic ATP1A1, ATP2B3, and KCNJ5 mutations in aldosterone-producing adenomas.

Aldosterone-producing adenomas (APAs) cause a sporadic form of primary aldosteronism and somatic mutations in the KCNJ5 gene, which encodes the G-protein-activated inward rectifier K(+) channel 4, GIRK4, account for ≈40% of APAs. Additional somatic APA mutations were identified recently in 2 other genes, ATP1A1 and ATP2B3, encoding Na(+)/K(+)-ATPase 1 and Ca(2+)-ATPase 3, respectively, at a combined prevalence of 6.8%. We have screened 112 APAs for mutations in known hotspots for genetic alterations associated with primary aldosteronism. Somatic mutations in ATP1A1, ATP2B3, and KCNJ5 were present in 6.3%, 0.9%, and 39.3% of APAs, respectively, and included 2 novel mutations (Na(+)/K(+)-ATPase p.Gly99Arg and GIRK4 p.Trp126Arg). CYP11B2 gene expression was higher in APAs harboring ATP1A1 and ATP2B3 mutations compared with those without these or KCNJ5 mutations. Overexpression of Na(+)/K(+)-ATPase p.Gly99Arg and GIRK4 p.Trp126Arg in HAC15 adrenal cells resulted in upregulation of CYP11B2 gene expression and its transcriptional regulator NR4A2. Structural modeling of the Na(+)/K(+)-ATPase showed that the Gly99Arg substitution most likely interferes with the gateway to the ion binding pocket. In vitro functional assays demonstrated that Gly99Arg displays severely impaired ATPase activity, a reduced apparent affinity for Na(+) activation of phosphorylation and K(+) inhibition of phosphorylation that indicate decreased Na(+) and K(+) binding, respectively. Moreover, whole cell patch-clamp studies established that overexpression of Na(+)/K(+)-ATPase Gly99Arg causes membrane voltage depolarization. In conclusion, somatic mutations are common in APAs that result in an increase in CYP11B2 gene expression and may account for the dysregulated aldosterone production in a subset of patients with sporadic primary aldosteronism.

Relationship between intracellular Na+ concentration and reduced Na+ affinity in Na+,K+-ATPase mutants causing neurological disease.

The neurological disorders familial hemiplegic migraine type 2 (FHM2), alternating hemiplegia of childhood (AHC), and rapid-onset dystonia parkinsonism (RDP) are caused by mutations of Na(+),K(+)-ATPase α2 and α3 isoforms, expressed in glial and neuronal cells, respectively. Although these disorders are distinct, they overlap in phenotypical presentation. Two Na(+),K(+)-ATPase mutations, extending the C terminus by either 28 residues ("+28" mutation) or an extra tyrosine ("+Y"), are associated with FHM2 and RDP, respectively. We describe here functional consequences of these and other neurological disease mutations as well as an extension of the C terminus only by a single alanine. The dependence of the mutational effects on the specific α isoform in which the mutation is introduced was furthermore studied. At the cellular level we have characterized the C-terminal extension mutants and other mutants, addressing the question to what extent they cause a change of the intracellular Na(+) and K(+) concentrations ([Na(+)]i and [K(+)]i) in COS cells. C-terminal extension mutants generally showed dramatically reduced Na(+) affinity without disturbance of K(+) binding, as did other RDP mutants. No phosphorylation from ATP was observed for the +28 mutation of α2 despite a high expression level. A significant rise of [Na(+)]i and reduction of [K(+)]i was detected in cells expressing mutants with reduced Na(+) affinity and did not require a concomitant reduction of the maximal catalytic turnover rate or expression level. Moreover, two mutations that increase Na(+) affinity were found to reduce [Na(+)]i. It is concluded that the Na(+) affinity of the Na(+),K(+)-ATPase is an important determinant of [Na(+)]i.

IGF dependent modulation of IGF binding protein (IGFBP) proteolysis by pregnancy-associated plasma protein-A (PAPP-A): multiple PAPP-A-IGFBP interaction sites.

BACKGROUND: Pregnancy-associated plasma protein-A (PAPP-A) is a local regulator of insulin-like growth factor (IGF) bioavailability in physiological systems, but many structural and functional aspects of the metzincin metalloproteinase remain to be elucidated. PAPP-A cleaves IGF binding protein (IGFBP)-4 and IGFBP-5. Cleavage of IGFBP-4, but not IGFBP-5, depends on the binding of IGF before proteolysis by PAPP-A can occur. The paralogue PAPP-A2 has two substrates among the six IGFBPs: IGFBP-3 and IGFBP-5. METHODS: Sets of chimeric proteins between IGFBP-4 and -5, and IGFBP-3 and -5 were constructed to investigate the structural requirements for IGF modulation. At the proteinase level, we investigated the importance of individual acidic amino acids positioned in the proteolytic domain of PAPP-A for proteolytic activity against IGFBP-4 and -5. Interaction between PAPP-A and its substrates was analyzed by surface plasmon resonance. RESULTS AND CONCLUSION: We provide data suggesting that the C-terminal domain of the IGFBPs is responsible for IGF-dependent modulation of access to the scissile bond. Loss or reduction of IGFBP proteolysis by PAPP-A was observed upon mutation of residues positioned in the unique 63-residue stretch separating the zinc and Met-turn motifs, and in the short sequence following the Met-turn methionine. A model of the proteolytic domain of PAPP-A suggests the presence of structural calcium ions in the C-terminal subdomain, implicated in IGFBP substrate interactions. GENERAL SIGNIFICANCE: Detailed knowledge of interactions between PAPP-A and its substrates is required to understand the modulatory role of PAPP-A on IGF receptor stimulation.

Somatic mutations in ATP1A1 and ATP2B3 lead to aldosterone-producing adenomas and secondary hypertension.

Primary aldosteronism is the most prevalent form of secondary hypertension. To explore molecular mechanisms of autonomous aldosterone secretion, we performed exome sequencing of aldosterone-producing adenomas (APAs). We identified somatic hotspot mutations in the ATP1A1 (encoding an Na(+)/K(+) ATPase α subunit) and ATP2B3 (encoding a Ca(2+) ATPase) genes in three and two of the nine APAs, respectively. These ATPases are expressed in adrenal cells and control sodium, potassium and calcium ion homeostasis. Functional in vitro studies of ATP1A1 mutants showed loss of pump activity and strongly reduced affinity for potassium. Electrophysiological ex vivo studies on primary adrenal adenoma cells provided further evidence for inappropriate depolarization of cells with ATPase alterations. In a collection of 308 APAs, we found 16 (5.2%) somatic mutations in ATP1A1 and 5 (1.6%) in ATP2B3. Mutation-positive cases showed male dominance, increased plasma aldosterone concentrations and lower potassium concentrations compared with mutation-negative cases. In summary, dominant somatic alterations in two members of the ATPase gene family result in autonomous aldosterone secretion.