Amir Sapir

Viral and Developmental Cell Fusion Mechanisms: Conservation and Divergence

Membrane fusion is a fundamental requirement in numerous developmental, physiological, and pathological processes in eukaryotes. So far, only a limited number of viral and cellular fusogens, proteins that fuse membranes, have been isolated and characterized. Despite the diversity in structures and functions of known fusogens, some common principles of action apply to all fusion reactions. These can serve as guidelines in the search for new fusogens, and may allow the formulation of a cross-species, unified theory to explain divergent and convergent evolutionary principles of membrane fusion.

Conserved eukaryotic fusogens can fuse viral envelopes to cells.

A Caenorhabditis elegans cell-surface fusion protein can promote viral fusion with mammalian cells. Caenorhabditis elegans proteins AFF-1 and EFF-1 [C. elegans fusion family (CeFF) proteins] are essential for developmental cell-to-cell fusion and can merge insect cells. To study the structure and function of AFF-1, we constructed vesicular stomatitis virus (VSV) displaying AFF-1 on the viral envelope, substituting the native fusogen VSV glycoprotein. Electron microscopy and tomography revealed that AFF-1 formed distinct supercomplexes resembling pentameric and hexameric “flowers” on pseudoviruses. Viruses carrying AFF-1 infected mammalian cells only when CeFFs were on the target cell surface. Furthermore, we identified fusion family (FF) proteins within and beyond nematodes, and divergent members from the human parasitic nematode Trichinella spiralis and the chordate Branchiostoma floridae could also fuse mammalian cells. Thus, FF proteins are part of an ancient family of cellular fusogens that can promote fusion when expressed on a viral particle.

Unidirectional Notch signaling depends on continuous cleavage of Delta

Unidirectional signaling from cells expressing Delta (Dl) to cells expressing Notch is a key feature of many developmental processes. We demonstrate that the Drosophila ADAM metalloprotease Kuzbanian-like (Kul) plays a key role in promoting this asymmetry. Kul cleaves Dl efficiently both in cell culture and in flies, and has previously been shown not to be necessary for Notch processing during signaling. In the absence of Kul in the developing wing, the level of Dl in cells that normally receive the signal is elevated, and subsequent alterations in the directionality of Notch signaling lead to prominent phenotypic defects. Proteolytic cleavage of Dl by Kul represents a general mechanism for refining and maintaining the asymmetric distribution of Dl, in cases where transcriptional repression of Dl expression does not suffice to eliminate Dl protein.

Overexpression of mouse Mdm2 induces developmental phenotypes in Drosophila

The Mdm2 proto-oncogene is amplified and over-expressed in a variety of tumors. One of the major functions of Mdm2 described to date is its ability to modulate the levels and activity of the tumor suppressor protein p53. Mdm2 binds to the N-terminus of p53 and, through its action as an E3 ubiquitin ligase, targets p53 for rapid proteasomal degradation. Mdm2 can also bind to other cellular proteins such as hNumb, E2F1, Rb and Akt; however, the biological significance of these interactions is less clear. To gain insight into the function of Mdm2 in vivo, we have generated a transgenic Drosophila strain bearing the mouse Mdm2 gene. Ectopic expression of Mdm2, using the UAS/GAL4 system, causes eye and wing phenotypes in the fly. Analysis of wing imaginal discs from third instar larvae showed that expression of Mdm2 induces apoptosis. Crosses did not reveal genetic interactions between Mdm2 and the Drosophila homolog of E2F, Numb and Akt. These transgenic flies may provide a unique experimental model for exploring the molecular interactions of Mdm2 in a developmental context.

Controlled sumoylation of the mevalonate pathway enzyme HMGS-1 regulates metabolism during aging

Significance The mevalonate pathway plays a critical role in cholesterol homeostasis and cancer development, two major challenges in modern medicine. Consequently, cholesterol-reducing medications (statins) that target this pathway are the best-selling pharmaceutical drugs in history. Beyond regulation of the enzyme HMG-CoA reductase, little is known about additional posttranslational regulatory nodes in the mevalonate pathway or how this cascade is controlled with age. We have discovered a regulatory circuit that controls HMGS-1, the first enzyme of the mevalonate pathway, during aging. HMGS-1 is regulated by posttranslational ubiquitination and age-dependent sumoylation. Sumoylation is reversed by the spatiotemporally controlled activity of a specific small ubiquitin-like modifier protease. This conserved molecular circuit could serve as a handle for targeting the mevalonate pathway in future therapeutics. Many metabolic pathways are critically regulated during development and aging but little is known about the molecular mechanisms underlying this regulation. One key metabolic cascade in eukaryotes is the mevalonate pathway. It catalyzes the synthesis of sterol and nonsterol isoprenoids, such as cholesterol and ubiquinone, as well as other metabolites. In humans, an age-dependent decrease in ubiquinone levels and changes in cholesterol homeostasis suggest that mevalonate pathway activity changes with age. However, our knowledge of the mechanistic basis of these changes remains rudimentary. We have identified a regulatory circuit controlling the sumoylation state of Caenorhabditis elegans HMG-CoA synthase (HMGS-1). This protein is the ortholog of human HMGCS1 enzyme, which mediates the first committed step of the mevalonate pathway. In vivo, HMGS-1 undergoes an age-dependent sumoylation that is balanced by the activity of ULP-4 small ubiquitin-like modifier protease. ULP-4 exhibits an age-regulated expression pattern and a dynamic cytoplasm-to-mitochondria translocation. Thus, spatiotemporal ULP-4 activity controls the HMGS-1 sumoylation state in a mechanism that orchestrates mevalonate pathway activity with the age of the organism. To expand the HMGS-1 regulatory network, we combined proteomic analyses with knockout studies and found that the HMGS-1 level is also governed by the ubiquitin–proteasome pathway. We propose that these conserved molecular circuits have evolved to govern the level of mevalonate pathway flux during aging, a flux whose dysregulation is associated with numerous age-dependent cardiovascular and cancer pathologies.