RetroCHMP3 blocks budding of enveloped viruses without blocking cytokinesis.

Many enveloped viruses require the endosomal sorting complexes required for transport (ESCRT) pathway to exit infected cells. This highly conserved pathway mediates essential cellular membrane fission events, which restricts the acquisition of adaptive mutations to counteract viral co-option. Here, we describe duplicated and truncated copies of the ESCRT-III factor CHMP3 that block ESCRT-dependent virus budding and arose independently in New World monkeys and mice. When expressed in human cells, these retroCHMP3 proteins potently inhibit release of retroviruses, paramyxoviruses, and filoviruses. Remarkably, retroCHMP3 proteins have evolved to reduce interactions with other ESCRT-III factors and have little effect on cellular ESCRT processes, revealing routes for decoupling cellular ESCRT functions from viral exploitation. The repurposing of duplicated ESCRT-III proteins thus provides a mechanism to generate broad-spectrum viral budding inhibitors without blocking highly conserved essential cellular ESCRT functions.

Viral houseguests undertake interior redesign.

As part of their life cycle some single-stranded RNA viruses remodel host cytoplasmic membranes into specialized organelles. In this issue, Hsu et al. (2010) demonstrate how the viruses selectively co-opt host machinery to make this unique organelle, which has a lipid composition favorable to viral replication.

GalNAc-conjugated siRNA targeting the DNAJB1-PRKACA fusion junction in fibrolamellar hepatocellular carcinoma.

Fibrolamellar hepatocellular carcinoma (FLC) is a rare liver cancer caused by a dominant recurrent fusion of the heat shock protein (DNAJB1) and the catalytic subunit of protein kinase A (PRKACA). Current therapies such as chemotherapy and radiation have limited efficacy, and new treatment options are needed urgently. We have previously shown that FLC tumors are dependent on the fusion kinase DNAJB1::PRKACA, making the oncokinase an ideal drug target. mRNA degrading modalities such as antisense oligonucleotides or small interfering RNAs (siRNAs) provide an opportunity to specifically target the fusion junction. Here, we identify a potent and specific siRNA that inhibits DNAJB1::PRKACA expression. We found expression of the asialoglycoprotein receptor in FLC to be maintained at sufficient levels to effectively deliver siRNA conjugated to the GalNAc ligand. We observe productive uptake and siRNA activity in FLC patient-derived xenografts (PDX) models in vitro and in vivo. Knockdown of DNAJB1::PRKACA results in durable growth inhibition of FLC PDX in vivo with no detectable toxicities. Our results suggest that this approach could be a treatment option for FLC patients.

Exocytosis of post-Golgi vesicles is regulated by components of the endocytic machinery.

Post-Golgi vesicles target and deliver most biosynthetic cargoes to the cell surface. However, the molecules and mechanisms involved in fusion of these vesicles are not well understood. We have employed a system to simultaneously monitor release of luminal and membrane biosynthetic cargoes from individual post-Golgi vesicles. Exocytosis of these vesicles is not calcium triggered. Release of luminal cargo can be accompanied by complete, partial, or no release of membrane cargo. Partial and no release of membrane cargo of a fusing vesicle are fates associated with kiss-and-run exocytosis, and we find that these are the predominant mode of post-Golgi vesicle exocytosis. Partial cargo release by post-Golgi vesicles occurs because of premature closure of the fusion pore and is modulated by the activity of clathrin, actin, and dynamin. Our results demonstrate that these components of the endocytic machinery modulate the nature and extent of biosynthetic cargo delivery by post-Golgi vesicles at the cell membrane.

Single molecule microscopy reveals diverse actions of substrate sequences that impair ClpX AAA+ ATPase function.

AAA+ (ATPases Associated with diverse cellular Activities) proteases unfold substrate proteins by pulling the substrate polypeptide through a narrow pore. To overcome the barrier to unfolding, substrates may require extended association with the ATPase. Failed unfolding attempts can lead to a slip of grip, which may result in substrate dissociation, but how substrate sequence affects slippage is unresolved. Here, we measured single molecule dwell time using total internal reflection fluorescence microscopy, scoring time-dependent dissociation of engaged substrates from bacterial AAA+ ATPase unfoldase/translocase ClpX. Substrates comprising a stable domain resistant to unfolding and a C-terminal unstructured tail, tagged with a degron for initiating translocase insertion, were used to determine dwell time in relation to tail length and composition. We found greater tail length promoted substrate retention during futile unfolding. Additionally, we tested two tail compositions known to frustrate unfolding. A poly-glycine tract (polyG) promoted release, but only when adjacent to the folded domain, whereas glycine-alanine repeats (GAr) did not promote release. A high complexity motif containing polar and charged residues also promoted release. We further investigated the impact of these and related motifs on substrate degradation rates and ATP consumption, using the unfoldase-protease complex ClpXP. Here, substrate domain stability modulates the effects of substrate tail sequences. polyG and GAr are both inhibitory for unfolding, but act in different ways. GAr motifs only negatively affected degradation of highly stable substrates, which is accompanied by reduced ClpXP ATPase activity. Together, our results specify substrate characteristics that affect unfolding and degradation by ClpXP.

Structural analyses of the PKA RIIß holoenzyme containing the oncogenic DnaJB1-PKAc fusion protein reveal protomer asymmetry and fusion-induced allosteric perturbations in fibrolamellar hepatocellular carcinoma.

When the J-domain of the heat shock protein DnaJB1 is fused to the catalytic (C) subunit of cAMP-dependent protein kinase (PKA), replacing exon 1, this fusion protein, J-C subunit (J-C), becomes the driver of fibrolamellar hepatocellular carcinoma (FL-HCC). Here, we use cryo-electron microscopy (cryo-EM) to characterize J-C bound to RIIß, the major PKA regulatory (R) subunit in liver, thus reporting the first cryo-EM structure of any PKA holoenzyme. We report several differences in both structure and dynamics that could not be captured by the conventional crystallography approaches used to obtain prior structures. Most striking is the asymmetry caused by the absence of the second cyclic nucleotide binding (CNB) domain and the J-domain in one of the RIIß:J-C protomers. Using molecular dynamics (MD) simulations, we discovered that this asymmetry is already present in the wild-type (WT) RIIß2C2 but had been masked in the previous crystal structure. This asymmetry may link to the intrinsic allosteric regulation of all PKA holoenzymes and could also explain why most disease mutations in PKA regulatory subunits are dominant negative. The cryo-EM structure, combined with small-angle X-ray scattering (SAXS), also allowed us to predict the general position of the Dimerization/Docking (D/D) domain, which is essential for localization and interacting with membrane-anchored A-Kinase-Anchoring Proteins (AKAPs). This position provides a multivalent mechanism for interaction of the RIIß holoenzyme with membranes and would be perturbed in the oncogenic fusion protein. The J-domain also alters several biochemical properties of the RIIß holoenzyme: It is easier to activate with cAMP, and the cooperativity is reduced. These results provide new insights into how the finely tuned allosteric PKA signaling network is disrupted by the oncogenic J-C subunit, ultimately leading to the development of FL-HCC.

Golgi governance: the third way.

It is a subject of intense debate whether proteins are transported by vesicles through the membranous stacks of the Golgi or whether the stacks mature, carrying the cargo along. In this issue, Patterson et al. (2008) present evidence for a third model in which the Golgi stacks are a continuous structure and proteins rapidly equilibrate between the layers.

Novel Chemically Modified Curcumin (CMC) Analogs Exhibit Anti-Melanogenic Activity in Primary Human Melanocytes.

Hyperpigmentation is a dermatological condition characterized by the overaccumulation and/or oversecretion of melanin pigment. The efficacy of curcumin as an anti-melanogenic therapeutic has been recognized, but the poor stability and solubility that have limited its use have inspired the synthesis of novel curcumin analogs. We have previously reported on comparisons of the anti-melanogenic activity of four novel chemically modified curcumin (CMC) analogs, CMC2.14, CMC2.5, CMC2.23 and CMC2.24, with that of parent curcumin (PC), using a B16F10 mouse melanoma cell model, and we have investigated mechanisms of inhibition. In the current study, we have extended our findings using normal human melanocytes from a darkly pigmented donor (HEMn-DP) and we have begun to study aspects of melanosome export to human keratinocytes. Our results showed that all the CMCs downregulated the protein levels of melanogenic paracrine mediators, endothelin-1 (ET-1) and adrenomedullin (ADM) in HaCaT cells and suppressed the phagocytosis of FluoSphere beads that are considered to be melanosome mimics. All the three CMCs were similarly potent (except CMC2.14, which was highly cytotoxic) in inhibiting melanin production; furthermore, they suppressed dendricity in HEMn-DP cells. CMC2.24 and CMC2.23 robustly suppressed cellular tyrosinase activity but did not alter tyrosinase protein levels, while CMC2.5 did not suppress tyrosinase activity but significantly downregulated tyrosinase protein levels, indicative of a distinctive mode of action for the two structurally related CMCs. Moreover, HEMn-DP cells treated with CMC2.24 or CMC2.23 partially recovered their suppressed tyrosinase activity after cessation of the treatment. All the three CMCs were nontoxic to human dermal fibroblasts while PC was highly cytotoxic. Our results provide a proof-of-principle for the novel use of the CMCs for skin depigmentation, since at low concentrations, ranging from 5 to 25 µM, the CMCs (CMC2.24, CMC2.23 and CMC2.5) were more potent anti-melanogenic agents than PC and tetrahydrocurcumin (THC), both of which were ineffective at melanogenesis at similar doses, as tested in HEMn-DP cells (with PC being highly toxic in dermal fibroblasts and keratinocytes). Further studies to evaluate the efficacy of CMCs in human skin tissue and in vivo studies are warranted.

Recruitment of 7SL RNA to assembling HIV-1 virus-like particles.

Retroviruses incorporate specific host cell RNAs into virions. In particular, the host noncoding 7SL RNA is highly abundant in all examined retroviruses compared with its cellular levels or relative to common mRNAs such as actin. Using live cell imaging techniques, we have determined that the 7SL RNA does not arrive with the HIV-1 RNA genome. Instead, it is recruited contemporaneously with assembly of the protein HIV-1 Gag at the plasma membrane. Further, we demonstrate that complexes of 7SL RNA and Gag can be immunoprecipitated from both cytosolic and plasma membrane fractions. This indicates that 7SL RNAs likely interact with Gag prior to high-order Gag multimerization at the plasma membrane. Thus, the interactions between Gag and the host RNA 7SL occur independent of the interactions between Gag and the host endosomal sorting complex required for transport (ESCRT) proteins, which are recruited temporarily at late stages of assembly. The interactions of 7SL and Gag are also independent of interactions of Gag and the HIV-1 genome which are seen on the plasma membrane prior to assembly of Gag.

DNAJB1-PRKACA fusion kinase interacts with ß-catenin and the liver regenerative response to drive fibrolamellar hepatocellular carcinoma.

A segmental deletion resulting in DNAJB1-PRKACA gene fusion is now recognized as the signature genetic event of fibrolamellar hepatocellular carcinoma (FL-HCC), a rare but lethal liver cancer that primarily affects adolescents and young adults. Here we implement CRISPR-Cas9 genome editing and transposon-mediated somatic gene transfer to demonstrate that expression of either the endogenous fusion protein or a chimeric cDNA leads to the formation of indolent liver tumors in mice that closely resemble human FL-HCC. Notably, overexpression of the wild-type PRKACA was unable to fully recapitulate the oncogenic activity of DNAJB1-PRKACA, implying that FL-HCC does not simply result from enhanced PRKACA expression. Tumorigenesis was significantly enhanced by genetic activation of ß-catenin, an observation supported by evidence of recurrent Wnt pathway mutations in human FL-HCC, as well as treatment with the hepatotoxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine, which causes tissue injury, inflammation, and fibrosis. Our study validates the DNAJB1-PRKACA fusion kinase as an oncogenic driver and candidate drug target for FL-HCC, and establishes a practical model for preclinical studies to identify strategies to treat this disease.

Asoprisnil, a Selective Progesterone Receptor Modulator (SPRM), Inhibits Melanosome Export in B16F10 Cells and HEMn-DP Melanocytes.

Previous studies have reported that estrogen hormone promotes melanogenesis while progesterone inhibits it. A selective estrogen receptor modulator (SERM), tamoxifen, has been shown to promote melanogenesis; however, to date, there have been no reports on the effects of a selective progesterone receptor modulator (SPRM) on melanogenesis. In the present study, we hypothesized that asoprisnil (AP), a SPRM, inhibits melanogenesis. AP was tested for cytotoxicity to B16F10 mouse melanoma cells for screening the nontoxic concentrations using MTS cytotoxicity assay. Extracellular and intracellular melanin levels were estimated at nontoxic concentrations of AP. To evaluate the direct effect of AP on tyrosinase enzyme, tyrosinase activity and copper chelating activities were measured. Next, the effects of AP on melanogenesis were tested in normal human melanocytes, neonatal, darkly pigmented (HEMn-DP). Our results demonstrate that AP was nontoxic at a concentration range of 10-50 µM in B16F10 cells; AP at 50 µM significantly suppressed extracellular melanin levels comparable to kojic acid at 500 µM, with no significant effect on intracellular melanin levels. The mechanism of melanogenesis inhibition was studied to assess if AP downregulated tyrosinase activity in cell lysates or in a cell-free system. However, AP was found to increase intracellular tyrosinase activity without any effect on tyrosinase enzyme activity or copper chelating activity in a cell-free system, indicating that AP inhibits melanogenesis by mechanisms other than direct effects on tyrosinase enzyme activity. The capacity of AP to inhibit melanosome export was further validated in HEMn-DP cells; AP significantly suppressed dendricity at concentrations of 20 and 30 µM in the absence of effects on melanin synthesis or intracellular tyrosinase activity. In addition, AP was nontoxic to human keratinocytes (HaCaT) at these concentrations, validating its safety for topical use. Taken together, our preliminary results demonstrate that AP might be repurposed as a candidate therapeutic for treatment of hyperpigmentation disorders via a unique mechanism, which encompasses a selective inhibition of melanosome export.

Green fluorescent protein-tagged apolipoprotein E: A useful marker for the study of hepatic lipoprotein egress.

Apolipoprotein E (ApoE), a component of very-low-density and high-density lipoproteins, participates in many aspects of lipid transport in the bloodstream. Underscoring its important functions, ApoE isoforms have been associated with metabolic and circulatory disease. ApoE is also incorporated into hepatitis C virus (HCV) particles, and promotes their production and infectivity. Live cell imaging analysis of ApoE behavior during secretion from producing cells thus has the potential to reveal important details regarding lipoprotein and HCV particle biogenesis and secretion from cells. However, this approach requires expression of fluorescently tagged ApoE constructs that need to faithfully reproduce known ApoE behaviors. Herein, we evaluate the usefulness of using an ApoE-GFP fusion protein in studying hepatocyte-derived, ApoE-containing lipoproteins and HCV particles. We show that while ApoE-GFP alone is not sufficient to support infectious HCV production, it nonetheless colocalizes intracellularly and associates with secreted untagged lipoprotein components. Furthermore, its rate of secretion from hepatic cells is indistinguishable from that of untagged ApoE. ApoE-GFP thus represents a useful marker for ApoE-containing hepatic lipoproteins.

Fibrolamellar carcinoma in the Carney complex: PRKAR1A loss instead of the classic DNAJB1-PRKACA fusion.

Fibrolamellar carcinomas are characterized by activation of protein kinase A, a kinase composed of catalytic and regulatory subunits. PRKACA encodes a catalytic subunit of protein kinase A, and almost all fibrolamellar carcinomas have a heterozygous 400-kb deletion that leads to the fusion of DNAJB1 and PRKACA. The resulting DNAJB1-PRKACA fusion transcript is believed to activate protein kinase A by dysregulation of the catalytic portion of the protein. In contrast, PRKAR1A encodes one of the regulatory subunits of protein kinase A. We hypothesized that loss of function of this regulatory unit could also lead to protein kinase A activation and thus to fibrolamellar carcinoma. Because PRKAR1A mutations underlie the Carney complex, we searched for liver tumors in individuals with the Carney complex. We identified 3 individuals with fibrolamellar carcinomas and a personal history of the Carney complex. All three tumors displayed the typical morphology of fibrolamellar carcinoma and were positive for arginase, cytokeratin 7, and cluster of differentiation 68. Fluorescence in situ hybridization was negative for PRKACA rearrangements. However, PRKAR1A sequencing identified pathogenic mutations in two of two cases with successful sequencing. In addition, all three cases were negative for PRKAR1A protein expression, consistent with inactivation of this key regulatory unit of protein kinase A. We also identified one additional fibrolamellar carcinoma in an individual without a documented history of the Carney complex who was negative for PRKACA rearrangements but had loss of PRKAR1A protein expression as well as PRKAR1A mutations. CONCLUSION: Fibrolamellar carcinoma can be part of the Carney complex; in this setting, fibrolamellar carcinomas have inactivating PRKAR1A mutations instead of the DNAJB1-PRKACA fusion gene found in sporadic fibrolamellar carcinomas, providing an alternate means for activation of protein kinase A. (Hepatology 2017).

Readily Accessible Multiplane Microscopy: 3D Tracking the HIV-1 Genome in Living Cells.

Human immunodeficiency virus (HIV)-1 infection and the associated disease AIDS are a major cause of human death worldwide with no vaccine or cure available. The trafficking of HIV-1 RNAs from sites of synthesis in the nucleus, through the cytoplasm, to sites of assembly at the plasma membrane are critical steps in HIV-1 viral replication, but are not well characterized. Here we present a broadly accessible microscopy method that captures multiple focal planes simultaneously, which allows us to image the trafficking of HIV-1 genomic RNAs with high precision. This method utilizes a customization of a commercial multichannel emission splitter that enables high-resolution 3D imaging with single-macromolecule sensitivity. We show with high temporal and spatial resolution that HIV-1 genomic RNAs are most mobile in the cytosol, and undergo confined mobility at sites along the nuclear envelope and in the nucleus and nucleolus. These provide important insights regarding the mechanism by which the HIV-1 RNA genome is transported to the sites of assembly of nascent virions.

Fibrolamellar Carcinoma: Recent Advances and Unresolved Questions on the Molecular Mechanisms.

Fibrolamellar hepatocellular carcinoma (FLC) is a rare form of primary liver cancer that affects adolescents and young adults without underlying liver disease. Surgery remains the mainstay of therapy; however, most patients are either not surgical candidates or suffer from recurrence. There is no approved systemic therapy and the overall survival remains poor. Historically classified as a subtype of hepatocellular carcinoma (HCC), FLC has a unique clinical, histological, and molecular presentation. At the genomic level, FLC contains a single 400kB deletion in chromosome 19, leading to a functional DNAJB1-PRKACA fusion protein. In this review, we detail the recent advances in our understanding of the molecular underpinnings of FLC and outline the current knowledge gaps.

Intracranial metastasis in fibrolamellar hepatocellular carcinoma.

Fibrolamellar hepatocellular carcinoma (FLHCC) is a rare liver malignancy in adolescents and young adults. Surgery is the mainstay of therapy for primary and metastatic disease. Most patients relapse, with development of both local and distant metastases. Brain metastases from solid tumors are rare in the pediatric and young adult population. Here, we document three patients with brain metastases from FLHCC, confirmed by histology and molecular characterization of the chimeric fusion DNAJB1-PRKACA, each necessitating neurosurgical intervention. These observations highlight the ability of FLHCC to metastasize to the brain and suggest the need for surveillance neuroimaging for patients with advanced-stage disease.

Transcriptomic characterization of fibrolamellar hepatocellular carcinoma.

Fibrolamellar hepatocellular carcinoma (FLHCC) tumors all carry a deletion of ∼ 400 kb in chromosome 19, resulting in a fusion of the genes for the heat shock protein, DNAJ (Hsp40) homolog, subfamily B, member 1, DNAJB1, and the catalytic subunit of protein kinase A, PRKACA. The resulting chimeric transcript produces a fusion protein that retains kinase activity. No other recurrent genomic alterations have been identified. Here we characterize the molecular pathogenesis of FLHCC with transcriptome sequencing (RNA sequencing). Differential expression (tumor vs. adjacent normal tissue) was detected for more than 3,500 genes (log2 fold change ≥ 1, false discovery rate ≤ 0.01), many of which were distinct from those found in hepatocellular carcinoma. Expression of several known oncogenes, such as ErbB2 and Aurora Kinase A, was increased in tumor samples. These and other dysregulated genes may serve as potential targets for therapeutic intervention.