A potent, selective and cell-active allosteric inhibitor of protein arginine methyltransferase†3 (PRMT3).

PRMT3 catalyzes the asymmetric dimethylation of arginine residues of various proteins. It is essential for maturation of ribosomes, may have a role in lipogenesis, and is implicated in several diseases. A potent, selective, and cell-active PRMT3 inhibitor would be a valuable tool for further investigating PRMT3 biology. Here we report the discovery of the first PRMT3 chemical probe, SGC707, by structure-based optimization of the allosteric PRMT3 inhibitors we reported previously, and thorough characterization of this probe in biochemical, biophysical, and cellular assays. SGC707 is a potent PRMT3 inhibitor (IC50 =31±2 nM, KD =53±2 nM) with outstanding selectivity (selective against 31 other methyltransferases and more than 250 non-epigenetic targets). The mechanism of action studies and crystal structure of the PRMT3-SGC707 complex confirm the allosteric inhibition mode. Importantly, SGC707 engages PRMT3 and potently inhibits its methyltransferase activity in cells. It is also bioavailable and suitable for animal studies. This well-characterized chemical probe is an excellent tool to further study the role of PRMT3 in health and disease.

Discovery of Potent and Selective Allosteric Inhibitors of Protein Arginine Methyltransferase 3 (PRMT3).

PRMT3 catalyzes the asymmetric dimethylation of arginine residues of various proteins. It is crucial for maturation of ribosomes and has been implicated in several diseases. We recently disclosed a highly potent, selective, and cell-active allosteric inhibitor of PRMT3, compound 4. Here, we report comprehensive structure-activity relationship studies that target the allosteric binding site of PRMT3. We conducted design, synthesis, and evaluation of novel compounds in biochemical, selectivity, and cellular assays that culminated in the discovery of 4 and other highly potent (IC50 values: ∼10-36 nM), selective, and cell-active allosteric inhibitors of PRMT3 (compounds 29, 30, 36, and 37). In addition, we generated compounds that are very close analogs of these potent inhibitors but displayed drastically reduced potency as negative controls (compounds 49-51). These inhibitors and negative controls are valuable chemical tools for the biomedical community to further investigate biological functions and disease associations of PRMT3.

Retina-specific protein fascin 2 is an actin cross-linker associated with actin bundles in photoreceptor inner segments and calycal processes.

PURPOSE: Fascin 2 is a retinal-specific member of the fascin family of actin filament-bundling proteins. Fascin 2 mutation in humans results in autosomal dominant retinitis pigmentosa or macular degeneration. To investigate the role of fascin 2 in photoreceptor survival, the authors examined its localization in photoreceptors and characterized its interactions with actin filaments in vitro. METHODS: Fascin 2 localization was determined by immunohistochemistry and transgenic expression of green fluorescent protein (GFP)-tagged fascin 2 in Xenopus laevis rods. Fascin 2 actin-binding and actin-bundling activity were examined in sedimentation assays using bacterially expressed fusion proteins and polymerized actin. To assess the role of phosphorylation of a conserved serine (amino acid 39) in fascin 2 on subcellular localization and actin-binding, effects of serine mutants were also examined in transgenic Xenopus and in in vitro assays. RESULTS: Fascin 2 is localized to actin filament bundles of the photoreceptor inner segment and calycal processes. Like fascin 1, fascin 2 binds and cross-links actin filaments. Mutation of serine 39 to an aspartic acid reduced fascin 2 binding of actin filaments and abolished fascin 2 bundling of actin filaments in vitro but produced no detectable effect on GFP-tagged fascin 2 localization in transgenic Xenopus. CONCLUSIONS: These observations suggest that fascin 2 plays a role in the assembly or stabilization of inner segment and calycal process actin filament bundles in photoreceptors and that serine 39 phosphorylation reduces actin-binding and cross-linking activity and, thus, is likely to regulate the inner segment actin cytoskeleton.

Myo3A, one of two class III myosin genes expressed in vertebrate retina, is localized to the calycal processes of rod and cone photoreceptors and is expressed in the sacculus.

The striped bass has two retina-expressed class III myosin genes, each composed of a kinase, motor, and tail domain. We report the cloning, sequence analysis, and expression patterns of the long (Myo3A) and short (Myo3B) class III myosins, as well as cellular localization and biochemical characterization of the long isoform, Myo3A. Myo3A (209 kDa) is expressed in the retina, brain, testis, and sacculus, and Myo3B (155 kDa) is expressed in the retina, intestine, and testis. The tails of these two isoforms contain two highly conserved domains, 3THDI and 3THDII. Whereas Myo3B has three IQ motifs, Myo3A has nine IQ motifs, four in its neck and five in its tail domain. Myo3A localizes to actin filament bundles of photoreceptors and is concentrated in the calycal processes. An anti-Myo3A antibody decorates the actin cytoskeleton of rod inner/outer segments, and this labeling is reduced by the presence of ATP. The ATP-sensitive actin association is a feature characteristic of myosin motors. The numerous IQ motifs may play a structural or signaling role in the Myo3A, and its localization to calycal processes indicates that this myosin mediates a local function at this site in vertebrate photoreceptors.

Disruption of kinesin II function using a dominant negative-acting transgene in Xenopus laevis rods results in photoreceptor degeneration.

PURPOSE: Kinesin II is a motor protein that moves on microtubules and whose importance in ciliary and flagellar transport has been well documented. In the current study, the role of kinesin II in rod photoreceptors was examined by expressing a dominant negative-acting transgene that disrupts kinesin II function in Xenopus laevis rods of transgenic tadpoles. METHODS: A previously characterized dominant negative-acting kinesin II transgene tagged with enhanced green fluorescent protein (EGFP) driven by the Xenopus rod opsin promoter was used to make Xenopus transgenic tadpoles to disrupt kinesin II function specifically in rod photoreceptors. Transgenic tadpole retinas were examined to ascertain transgene expression pattern and morphologic phenotype. Rod-to-cone ratios were determined in experimental and control retinas. RESULTS: Visualized by its EGFP tag, the kinesin II transgene was expressed in rods in a mosaic pattern in the retina. Subcellular localization of transgenic kinesin II was similar to that of endogenous kinesin II subunit photoreceptor expression-that is, it was localized to the connecting cilium, inner segment, and synapse. However, in kinesin II transgene-expressing animals, fluorescence was transient. Ocular fluorescence was lost 6 days after its first detection. The disappearance of fluorescence was due to degeneration of rods expressing the transgene. Retinas of 7- to 9-day old kinesin II transgenic tadpoles had significantly fewer rods than did control retinas. CONCLUSIONS: The observation that rod degeneration is produced by expression of a dominant negative-acting kinesin II transgene in Xenopus rods is consistent with previous studies in mice, suggesting that kinesin II function is required for photoreceptor survival.

Fascin 2b is a component of stereocilia that lengthens actin-based protrusions.

Stereocilia are actin-filled protrusions that permit mechanotransduction in the internal ear. To identify proteins that organize the cytoskeleton of stereocilia, we scrutinized the hair-cell transcriptome of zebrafish. One promising candidate encodes fascin 2b, a filamentous actin-bundling protein found in retinal photoreceptors. Immunolabeling of zebrafish hair cells and the use of transgenic zebrafish that expressed fascin 2b fused to green fluorescent protein demonstrated that fascin 2b localized to stereocilia specifically. When filamentous actin and recombinant fusion protein containing fascin 2b were combined in vitro to determine their dissociation constant, a K(d)≈0.37 µM was observed. Electron microscopy showed that fascin 2b-actin filament complexes formed parallel actin bundles in vitro. We demonstrated that expression of fascin 2b or espin, another actin-bundling protein, in COS-7 cells induced the formation of long filopodia. Coexpression showed synergism between these proteins through the formation of extra-long protrusions. Using phosphomutant fascin 2b proteins, which mimicked either a phosphorylated or a nonphosphorylated state, in COS-7 cells and in transgenic hair cells, we showed that both formation of long filopodia and localization of fascin 2b to stereocilia were dependent on serine 38. Overexpression of wild-type fascin 2b in hair cells was correlated with increased stereociliary length relative to controls. These findings indicate that fascin 2b plays a key role in shaping stereocilia.

Identification and localization of myosin superfamily members in fish retina and retinal pigmented epithelium.

Myosins are cytoskeletal motors critical for generating the forces necessary for establishing cell structure and mediating actin-dependent cell motility. In each cell type a multitude of myosins are expressed, each myosin contributing to aspects of morphogenesis, transport, or motility occurring in that cell type. To examine the roles of myosins in individual retinal cell types, we first used polymerase chain reaction (PCR) screening to identify myosins expressed in retina and retinal pigmented epithelium (RPE), followed by immunohistochemistry to examine the cellular and subcellular localizations of seven of these expressed myosins. In the myosin PCR screen of cDNA from striped bass retina and striped bass RPE, we amplified 17 distinct myosins from eight myosin classes from retinal cDNA and 11 distinct myosins from seven myosin classes from RPE cDNA. By using antibodies specific for myosins IIA, IIB, IIIA, IIIB, VI, VIIA, and IXB, we examined the localization patterns of these myosins in retinas and RPE of fish, and in isolated inner/outer segment fragments of green sunfish photoreceptors. Each of the myosins exhibited unique expression patterns in fish retina. Individual cell types expressed multiple myosin family members, some of which colocalized within a particular cell type. Because much is known about the functions and properties of these myosins from studies in other systems, their cellular and subcellular localization patterns in the retina help us understand which roles they might play in the vertebrate retina and RPE.

Myosin 3A transgene expression produces abnormal actin filament bundles in transgenic Xenopus laevis rod photoreceptors.

Myo3A, a class III myosin, localizes to the distal (plus) ends of inner segment actin filament bundles that form the core of microvillus-like calycal processes encircling the base of the photoreceptor outer segment. To investigate Myo3A localization and function, we expressed green fluorescent protein-tagged bass Myo3A and related constructs in transgenic Xenopus rods using a modified opsin promoter. Tagged intact Myo3A localized to rod calycal processes, as previously reported for native bass Myo3A. Transgenic rods developed abnormally large calycal processes and subsequently degenerated. Modified Myo3A expression constructs demonstrated that calycal process localization required an active motor domain and the tail domain. Expressed tail domain alone localized to actin bundles along the entire inner segment length, rather than to the distal end. This tail domain localization required the conserved C-terminal domain (3THDII) previously shown to possess an actin-binding motif. Our findings suggest that Myo3A plays a role in the morphogenesis and maintenance of calycal processes of vertebrate photoreceptors.