Emerging Connections between Nuclear Pore Complex Homeostasis and ALS.

Developing effective treatments for neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) requires understanding of the underlying pathomechanisms that contribute to the motor neuron loss that defines the disease. As it causes the largest fraction of familial ALS cases, considerable effort has focused on hexanucleotide repeat expansions in the C9ORF72 gene, which encode toxic repeat RNA and dipeptide repeat (DPR) proteins. Both the repeat RNA and DPRs interact with and perturb multiple elements of the nuclear transport machinery, including shuttling nuclear transport receptors, the Ran GTPase and the nucleoporin proteins (nups) that build the nuclear pore complex (NPC). Here, we consider recent work that describes changes to the molecular composition of the NPC in C9ORF72 model and patient neurons in the context of quality control mechanisms that function at the nuclear envelope (NE). For example, changes to NPC structure may be caused by the dysregulation of a conserved NE surveillance pathway mediated by the endosomal sorting complexes required for the transport protein, CHMP7. Thus, these studies are introducing NE and NPC quality control pathways as key elements in a pathological cascade that leads to C9ORF72 ALS, opening entirely new experimental avenues and possibilities for targeted therapeutic intervention.

Atg39 selectively captures inner nuclear membrane into lumenal vesicles for delivery to the autophagosome.

Mechanisms that turn over components of the nucleus and inner nuclear membrane (INM) remain to be fully defined. We explore how components of the INM are selected by a cytosolic autophagy apparatus through a transmembrane nuclear envelope-localized cargo adaptor, Atg39. A split-GFP reporter showed that Atg39 localizes to the outer nuclear membrane (ONM) and thus targets the INM across the nuclear envelope lumen. Consistent with this, sequence elements that confer both nuclear envelope localization and a membrane remodeling activity are mapped to the Atg39 lumenal domain; these lumenal motifs are required for the autophagy-mediated degradation of integral INM proteins. Interestingly, correlative light and electron microscopy shows that the overexpression of Atg39 leads to the expansion of the ONM and the enclosure of a network of INM-derived vesicles in the nuclear envelope lumen. Thus, we propose an outside-in model of nucleophagy where INM is delivered into vesicles in the nuclear envelope lumen, which can be targeted by the autophagosome.

Ectopic expression of 35 kDa and knocking down of 78 kDa SG2NAs induce cytoskeletal reorganization, alter membrane sialylation, and modulate the markers of EMT.

SG2NA is a protein of the striatin family that organizes STRIPAK complexes. It has splice variants expressing differentially in tissues. Its 78 kDa isoform regulates cell cycle, maintains homeostasis in the endoplasmic reticulum, and prevents oxidative injuries. The 35 kDa variant is devoid of the signature WD-40 repeats in the carboxy terminal, and its function is unknown. We expressed it in NIH 3T3 cells that otherwise express 78 kDa variant only. These cells (35 EE) have altered morphology, faster rate of migration, and enhanced growth as measured by the MTT assay. Similar phenotypes were also seen in cells where the endogenous 78 kDa isoform was downregulated by siRNA (78 KD). Proteomic analyses showed that several cancer-associated proteins are modulated in both 35 EE and 78 KD cells. The 35 EE cells have diffused actin fibers, distinctive ultrastructure, reduced sialylation, and increased expression of MMP2 & 9. The 78 KD cells also had diffused actin fibers and an upregulated expression of MMP2. In both cells, markers epithelial to mesenchymal transition (EMT) viz, E- & N-cadherins, ß-catenin, slug, vimentin, and ZO-1 were modulated partially in tune with the EMT process. Since NIH 3T3 cells are mesenchymal, we also expressed 35 kDa SG2NA in MCF-7 cells of epithelial origin. In these cells (MCF-7-35), the actin fibers were also diffused and the modulation of the markers was more in tune with the EMT process. However, unlike in 35 EE cells, in MCF-7-35 cells, membrane sialylation rather increased. We infer that ectopic expression of 35 kDa and downregulation of 78 kDa SG2NAs partially induce transformed phenotypes.

Site-specific phosphorylation of villin remodels the actin cytoskeleton to regulate Sendai viral glycoprotein-mediated membrane fusion.

Connivance of cellular factors during virus-host cell membrane fusion is poorly understood. We have recently shown that cellular villin plays an important role during membrane fusion of reconstituted Sendai virosomes with hepatocytes. Here, we employed villin-null Chinese Hamster Ovary (CHO) cells, where villin expression led to an increased fusion with virosomes, which was further enhanced due to tyrosine phosphorylation in the presence of c-src. However, the villin RRI mutant, lacking actin-severing function, failed to augment membrane fusion. Furthermore, quantitative mass spectrometry and detailed analysis revealed Tyr499 to be the key phosphorylation site of villin responsible for the enhancement of virosome-CHO cell fusion. Overall, our results demonstrate a critical role for villin and its cell-type dependent phosphorylation in regulating membrane fusion.

A combinatorial approach for robust transgene delivery and targeted expression in mammary gland for generating biotherapeutics in milk, bypassing germline gene integration.

Protein expression in the milk of transgenic farmed animals offers a cost-effective system for producing therapeutics. However, transgenesis in farmed animals is not only cumbersome but also involves risk of potential hazard by germline gene integration, due to interruptions caused by the transgene in the native genome. Avoiding germline gene integration, we have delivered buffalo ß-casein promoter-driven transgene construct entrapped in virosomes directly in the milk gland through intraductal perfusion delivery. Virosomes were generated from purified Sendai viral membrane, containing hemagglutinin-neuraminidase (HN) and fusion factor (F) proteins on surface (HNF-Virosomes) which initiate membrane fusion, devoid of any viral nucleic acids. Intraductal delivery of HNF-Virosomes predominantly transfected luminal epithelial cells lining the milk duct and buffalo ß-casein promoter of the construct ensured mammary luminal epithelial cell specific expression of the transgene. Mammary epithelial cells expressed EGFP at lactation when egfp was used as a transgene. Similarly, human interferon-γ (hIFN-γ) was expressed in the mammary gland as well as in the milk when hIFN-γ was used as a transgene. This combinatorial approach of using Sendai viral membrane-derived virosomes for entrapment and delivery of the transgene and using buffalo ß-casein promoter for mammary gland specific gene expression provided a better option for generating therapeutic proteins in milk, bypassing germline gene integration avoiding risks associated with animal bioreactor generated through germline gene integration.

Sendai virus recruits cellular villin to remodel actin cytoskeleton during fusion with hepatocytes.

Reconstituted Sendai viral envelopes (virosomes) are well recognized for their promising potential in membrane fusion-mediated delivery of bioactive molecules to liver cells. Despite the known function of viral envelope glycoproteins in catalyzing fusion with cellular membrane, the role of host cell proteins remains elusive. Here, we used two-dimensional differential in-gel electrophoresis to analyze hepatic cells in early response to virosome-induced membrane fusion. Quantitative mass spectrometry together with biochemical analysis revealed that villin, an actin-modifying protein, is differentially up-regulated and phosphorylated at threonine 206-an early molecular event during membrane fusion. We found that villin influences actin dynamics and that this influence, in turn, promotes membrane mixing through active participation of Sendai viral envelope glycoproteins. Modulation of villin in host cells also resulted in a discernible effect on the entry and egress of progeny Sendai virus. Taken together, these results suggest a novel mechanism of regulated viral entry in animal cells mediated by host factor villin.

Phosphorylation of Nonmuscle myosin II-A regulatory light chain resists Sendai virus fusion with host cells.

Enveloped viruses enter host cells through membrane fusion and the cells in turn alter their shape to accommodate components of the virus. However, the role of nonmuscle myosin II of the actomyosin complex of host cells in membrane fusion is yet to be understood. Herein, we show that both (-) blebbistatin, a specific inhibitor of nonmuscle myosin II (NMII) and small interfering RNA markedly augment fusion of Sendai virus (SeV), with chinese hamster ovary cells and human hepatocarcinoma cells. Inhibition of RLC phosphorylation using inhibitors against ROCK, but not PKC and MRCK, or overexpression of phospho-dead mutant of RLC enhances membrane fusion. SeV infection increases cellular stiffness and myosin light chain phosphorylation at two hour post infection. Taken together, the present investigation strongly indicates that Rho-ROCK-NMII contractility signaling pathway may provide a physical barrier to host cells against viral fusion.

Glycobiology of the Leishmania parasite and emerging targets for antileishmanial drug discovery.

IMPORTANCE OF THE FIELD: Parasitic diseases that pose a threat to human life include leishmaniasis - caused by protozoa of Leishmania species. Existing drugs have limitations due to deleterious side effects like teratogenicity and factors like cost and drug resistance, thus furthering the need to develop this area of research. AREAS COVERED IN THIS REVIEW: We came across drug targets, very recently characterised, cloned and validated by genomics and bioinformatics. We bring these promising drug targets into focus so that they can be explored to their fullest. WHAT THE READER WILL GAIN: In an effort to bridge the gaps between existing knowledge and future prospects of drug discovery, we found interesting studies validating drug targets and paving the way for better experiments to be designed. In a few cases, novel pathways have been characterized, while in others, well established pathways when probed further, led to the discovery of new drug targets. TAKE HOME MESSAGE: The review constitutes a comprehensive report on upcoming drug targets, with emphasis on glycosylphosphatidylinositol (GPI)-anchored glycoconjugates along with related biochemistry of enolase, glycosome and purine salvage pathways, as we strive to bring ourselves a step closer to being able to combat this deadly disease.