2'-N-Alkylaminocarbonyl-2'-amino-LNA: Synthesis, duplex stability, nuclease resistance, and in vitro anti-microRNA activity.

2'-Amino-LNA has the potential to acquire various functions through chemical modification at the 2'-nitrogen atom. This study focused on 2'-N-alkylaminocarbonyl 2'-amino-LNA, which is a derivative of 2'-amino-LNA. We evaluated its practical usefulness as a chemical modification of anti-miRNA oligonucleotide. The synthesis of phosphoramidites of 2'-N-alkylaminocarbonyl substituted 2'-amino-LNA bearing thymine and 5-methylcytosine proceeded in good yields. Incorporating the 2'-N-alkylaminocarbonyl-2'-amino-LNA monomers into oligonucleotides improved the duplex stability for complementary RNA strands and robust nuclease resistance. Moreover, 2'-N-alkylaminocarbonyl-2'-amino-LNA is a promising scaffold that significantly increases the potency of anti-miRNA oligonucleotides.

Parallel synthesis of oligonucleotides containing N-acyl amino-LNA and their therapeutic effects as anti-microRNAs.

2'-Amino-locked nucleic acid (ALNA), maintains excellent duplex stability, and the nitrogen at the 2'-position is an attractive scaffold for functionalization. Herein, a facile and efficient method for the synthesis of various 2'-N-acyl amino-LNA derivatives by direct acylation of the 2'-amino moiety contained in the synthesized oligonucleotides and its fundamental properties are described. The introduction of the acylated amino-LNA enhances the potency of the molecules as therapeutic anti-microRNA oligonucleotides.

Structure-Activity Relationships of Anti-microRNA Oligonucleotides Containing Cationic Guanidine-Modified Nucleic Acids.

Anti-microRNA oligonucleotides (AMOs) are valuable tools for the treatment of diseases caused by the dysregulation of microRNA expression. However, the correlation between chemical modifications in AMO sequences and the microRNA-inhibitory activity has not been fully elucidated. In this study, we synthesized a series of AMOs containing cationic guanidine-bridged nucleic acids (GuNA) and evaluated their activities using a dual luciferase assay. We also optimized the site of GuNA substitution and found an effective design for the inhibition of microRNA-21, which was partially different from that of conventional nucleic acid derivatives. This study showed that GuNA-substituted AMOs are effective in inhibiting the function of microRNA.

Mitochondrial reactive zones in antiviral innate immunity.

Mitochondria are multi-functioning organelles that participate in a wide range of biologic processes from energy metabolism to cellular suicide. Mitochondria are also involved in the cellular innate immune response against microorganisms or environmental irritants, particularly in mammals. Mitochondrial-mediated innate immunity is achieved by the activation of two discrete signaling pathways, the NLR family pyrin domain-containing 3 inflammasomes and the retinoic acid-inducible gene I-like receptor pathway. In both pathways, a mitochondrial outer membrane adaptor protein, called mitochondrial antiviral signaling MAVS, and mitochondria-derived components play a key role in signal transduction. In this review, we discuss current insights regarding the fundamental phenomena of mitochondrial-related innate immune responses, and review the specific roles of various mitochondrial subcompartments in fine-tuning innate immune signaling events. We propose that specific targeting of mitochondrial functions is a potential therapeutic approach for the management of infectious diseases and autoinflammatory disorders with an excessive immune response.

The microRNAs miR-302b and miR-372 regulate mitochondrial metabolism via the SLC25A12 transporter, which controls MAVS-mediated antiviral innate immunity.

MicroRNAs (miRNAs) are small noncoding RNAs that suppress the expression of multiple genes and are involved in numerous biologic functions and disorders, including human diseases. Here, we report that two miRNAs, miR-302b and miR-372, target mitochondrial-mediated antiviral innate immunity by regulating mitochondrial dynamics and metabolic demand. Using human cell lines transfected with the synthetic analog of viral dsRNA, poly(I-C), or challenged with Sendai virus, we found that both miRNAs are up-regulated in the cells late after viral infection and ultimately terminate the production of type I interferons and inflammatory cytokines. We found that miR-302b and miR-372 are involved in dynamin-related protein 1 (DRP1)-dependent mitochondrial fragmentation and disrupt mitochondrial metabolism by attenuating solute carrier family 25 member 12 (SLC25A12), a member of the SLC25 family. Neutralizing the effects of the two miRNAs through specific inhibitors re-established the mitochondrial dynamics and the antiviral responses. We found that SLC25A12 contributes to regulating the antiviral response by inducing mitochondrial-related metabolite changes in the organelle. Structure-function analysis indicated that SLC25A12, as part of a prohibitin complex, associates with the mitochondrial antiviral-signaling protein in mitochondria, providing structural insight into the regulation of the mitochondrial-mediated antiviral response. Our results contribute to the understanding of how miRNAs modulate the innate immune response by altering mitochondrial dynamics and metabolic demand. Manipulating the activities of miR-302b and miR-372 may be a potential therapeutic approach to target RNA viruses.

Interaction between Viral Proteins and Mitochondria.

Recent advances reveal that mitochondria are not limited to functioning only as the cellular powerhouse and in apoptosis, but that they act as central hubs for multiple signal transductions. Studies over the last decade indicate that mitochondria in vertebrates are involved in the front line of host defense, especially against RNA viruses. Mitochondrial-mediated antiviral innate immunity depends on activation of the retinoic acid-inducible gene I (RIG-I)-like receptors signal transduction pathway, and the mitochondrial surface acts as a platform for the assembly of signaling molecules, including mitochondrial antiviral signaling (MAVS) during the process. Some viral encoded proteins target to the mitochondria post-infection, however, thereby evading the cellular immune response. Here we review specific interactions between mitochondria and viral proteins and discuss their physiologic effects on the host cells.

Mitochondrial membrane potential is required for MAVS-mediated antiviral signaling.

Mitochondria, dynamic organelles that undergo cycles of fusion and fission, are the powerhouses of eukaryotic cells and are also involved in cellular innate antiviral immunity in mammals. Mitochondrial antiviral immunity depends on activation of the cytoplasmic retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) signaling pathway and the participation of a mitochondrial outer membrane adaptor protein called MAVS (mitochondrial antiviral signaling). We found that cells that lack the ability to undergo mitochondrial fusion as a result of targeted deletion of both mitofusin 1 (Mfn1) and mitofusin 2 (Mfn2) exhibited impaired induction of interferons and proinflammatory cytokines in response to viral infection, resulting in increased viral replication. In contrast, cells with null mutations in either Mfn1 or Mfn2 retained their RLR-induced antiviral responses. We also found that a reduced mitochondrial membrane potential (ΔΨ(m)) correlated with the reduced antiviral response. The dissipation in ΔΨ(m) did not affect the activation of the transcription factor interferon regulatory factor 3 downstream of MAVS, which suggests that ΔΨ(m) and MAVS are coupled at the same stage in the RLR signaling pathway. Our results provide evidence that the physiological function of mitochondria plays a key role in innate antiviral immunity.

Structure-function analysis of the yeast mitochondrial Rho GTPase, Gem1p: implications for mitochondrial inheritance.

Mitochondria undergo continuous cycles of homotypic fusion and fission, which play an important role in controlling organelle morphology, copy number, and mitochondrial DNA maintenance. Because mitochondria cannot be generated de novo, the motility and distribution of these organelles are essential for their inheritance by daughter cells during division. Mitochondrial Rho (Miro) GTPases are outer mitochondrial membrane proteins with two GTPase domains and two EF-hand motifs, which act as receptors to regulate mitochondrial motility and inheritance. Here we report that although all of these domains are biochemically active, only the GTPase domains are required for the mitochondrial inheritance function of Gem1p (the yeast Miro ortholog). Mutations in either of the Gem1p GTPase domains completely abrogated mitochondrial inheritance, although the mutant proteins retained half the GTPase activity of the wild-type protein. Although mitochondrial inheritance was not dependent upon Ca(2+) binding by the two EF-hands of Gem1p, a functional N-terminal EF-hand I motif was critical for stable expression of Gem1p in vivo. Our results suggest that basic features of Miro protein function are conserved from yeast to humans, despite differences in the cellular machinery mediating mitochondrial distribution in these organisms.

Mitofusin 2 inhibits mitochondrial antiviral signaling.

The innate immune response to viral infection involves the activation of multiple signaling steps that culminate in the production of type I interferons (IFNs). Mitochondrial antiviral signaling (MAVS), a mitochondrial outer membrane adaptor protein, plays an important role in this process. Here, we report that mitofusin 2 (Mfn2), a mediator of mitochondrial fusion, interacts with MAVS to modulate antiviral immunity. Overexpression of Mfn2 resulted in the inhibition of retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5), two cytosolic sensors of viral RNA, as well as of MAVS-mediated activation of the transcription factors interferon regulatory factor 3 (IRF-3) and nuclear factor kappaB (NF-kappaB). In contrast, loss of endogenous Mfn2 enhanced virus-induced production of IFN-beta and thereby decreased viral replication. Structure-function analysis revealed that Mfn2 interacted with the carboxyl-terminal region of MAVS through a heptad repeat region, providing a structural perspective on the regulation of the mitochondrial antiviral response. Our results suggest that Mfn2 acts as an inhibitor of antiviral signaling, a function that may be distinct from its role in mitochondrial dynamics.