The structurally related somatic and germinal isoforms of angiotensin-converting enzyme (ACE) contain the same catalytic active center and are encoded by the same gene, whose disruption causes renal atrophy, hypotension, and male sterility. The reason for the evolutionary conservation of both isozymes is an enigma, because, in vitro, they have very similar enzymatic properties. Despite the common enzymatic properties, discrete expression of both isoforms is maintained in alternate cell types. We have previously shown that sperm-specific expression of transgenic germinal ACE in Ace ؊/؊ male mice restores fertility without curing their other abnormalities (Ramaraj, P., Kessler, S. P., Colmenares, C. & Sen, G. C. (1998) J. Clin. Invest. 102, 371-378). In this report we tested the biological equivalence of somatic ACE and germinal ACE utilizing an in vivo isozymic substitution approach. Here we report that restoration of male fertility was not achieved by the transgenic expression of enzymatically active, somatic ACE in the sperm of Ace ؊/؊ mice. Therefore, the requisite physiological functions of the two tissue-specific isozymes of ACE are not interchangeable.

SUMMARY The intracellular microbial nucleic acid sensors, TLR3 and STING, recognize pathogen molecules and signal to activate the interferon pathway. The TIR-domain containing protein TRIF is the sole adaptor of TLR3. Here we report an essential role for TRIF in STING signaling: various activators of STING could not induce genes in the absence of TRIF. TRIF and STING interacted directly, through their carboxyl terminal domains, to promote STING dimerization, intermembrane translocation and signaling. Herpes simplex virus (HSV), which triggers the STING signaling pathway and is controlled by it, replicated more efficiently in the absence of TRIF and HSV-infected TRIF−/− mice displayed pronounced pathology. Our results indicate that defective STING signaling may be responsible for the observed genetic association between TRIF mutations and Herpes Simplex Encephalitis in patients.

RNase L and RNA-dependent protein kinase (PKR) are effectors of the interferon antiviral response that share homology in their pseudokinase and protein kinase domains, respectively. Sunitinib is an orally available, ATP-competitive inhibitor of VEGF and PDGF receptors used clinically to suppress angiogenesis and tumor growth. Sunitinib also impacts IRE1, an endoplasmic reticulum protein involved in the unfolded protein response that is closely related to RNase L. Here, we report that sunitinib is a potent inhibitor of both RNase L and PKR with IC 50 values of 1.4 and 0.3 M, respectively. In addition, flavonol activators of IRE1 inhibited RNase L. Sunitinib treatment of wild type (WT) mouse embryonic fibroblasts resulted in about a 12-fold increase in encephalomyocarditis virus titers. However, sunitinib had no effect on encephalomyocarditis virus growth in cells lacking both PKR and RNase L. Furthermore, oral delivery of sunitinib in WT mice resulted in 10-fold higher viral titers in heart tissues while suppressing by about 2-fold the IFN-␤ levels. In contrast, sunitinib had no effect on viral titers in mice deficient in both RNase L and PKR. Also, sunitinib reduced mean survival times from 12 to 6 days in virus-infected WT mice while having no effect on survival of mice lacking both RNase L and PKR. Results indicate that sunitinib treatments prevent antiviral innate immune responses mediated by RNase L and PKR.RNase L and PKR 2 are host enzymes of higher vertebrates that participate in innate immunity against viral infections (1-4). Activation of both RNase L and PKR is triggered by the viral pathogen-associated molecular pattern, double-stranded RNA (dsRNA). However, whereas dsRNA directly binds to and activates PKR, in the case of RNase L activation is indirect. Interferon (IFN) treatment of cells induces PKR that, upon binding to dsRNA, phosphorylates first itself and then EIF2␣ thus blocking protein synthesis among other effects. RNase L degrades single-stranded RNA resulting in pleiotropic antiviral effects (5). Short 5Ј-triphosphorylated, 2Ј,5Ј-oligoadenylates (2-5A) are produced from ATP when viral dsRNA stimulates IFN-inducible oligoadenylate synthetases. 2-5A binds ankyrin repeats 2 and 4 in the N-terminal region of RNase L causing its dimerization and activation (6). RNase L is also pseudokinase with amino acid sequence homology to the PKR kinase domains (7).IRE1, a kinase and endoribonuclease involved in the unfolded protein response, is another relative of RNase L (8). IRE1 spans the endoplasmic reticulum (ER) membrane. The intralumenal domains of IRE1 directly or indirectly sense unfolded proteins in the ER leading to autophosphorylation and ribonuclease activities in the cytoplasmic domains. IRE1 excises an intron from pre-mRNA for a transcription factor (HAC1 in yeast and XBP1 in mammals) leading to splicing and translation (9). HAC1/XBP1 drives expression of ER chaperones and protein folding enzymes that re-establish ER function. The kinase-extension-nuclease (KEN) domains of RNase L and IRE1 ar...

STING (STimulator of INterferon Genes) mediates protective cellular response to microbial infection and tissue damage, but its aberrant activation can lead to autoinflammatory diseases. Upon ligand stimulation, the endoplasmic reticulum (ER) protein STING translocates to endosomes for induction of interferon production, while an alternate trafficking route delivers it directly to the autophagosomes. Here, we report that phosphorylation of a specific tyrosine residue in STING by the epidermal growth factor receptor (EGFR) is required for directing STING to endosomes, where it interacts with its downstream effector IRF3. In the absence of EGFR-mediated phosphorylation, STING rapidly transits into autophagosomes, and IRF3 activation, interferon production, and antiviral activity are compromised in cell cultures and mice, while autophagic activity is enhanced. Our observations illuminate a new connection between the tyrosine kinase activity of EGFR and innate immune functions of STING and suggest new experimental and therapeutic approaches for selective regulation of STING functions.

Anaplastic histology and metastasis are each associated with higher relapse and mortality rates in Wilms tumor patients. However, not all anaplastic tumors relapse and some nonanaplastic tumors relapse unexpectedly. To identify more accurate early prognostic indicators, we analyzed expression of 4,900 cancer-related genes in 26 primary Wilms tumors. This analysis revealed that expression of a set of four genes predicts future relapse of primary Wilms tumors with high accuracy, independent of anaplasia. Random permutation testing of this prognostic gene expression signature yielded P = = = 0.003. Real-time reverse transcription-PCR analysis of the four genes in an independent primary tumor set resulted in correct prediction of future relapse with an accuracy of 92%. One of the four genes in the prognostic signature, CCAAT/ /enhancer binding protein h h (C/ /EBPB), is expressed at higher levels in both primary relapsing tumors and metastatic tumors than in primary nonrelapsing tumors. Short interfering RNA-mediated down-regulation of C/ / /EBPB expression in WiT49, a cell line derived from a metastatic Wilms tumor, resulted in spontaneous apoptosis. These findings suggest that C/ / /EBPB is a critical survival factor for Wilms tumor cells and that its expression contributes to the prognosis of Wilms tumor patients. (Cancer Res 2005; 65(7): 2592-601)