Characterizing Glycosylation of Adeno-Associated Virus Serotype 9 Capsid Proteins Generated from HEK293 Cells through Glycopeptide Mapping and Released Glycan Analysis.

Recombinant adeno-associated viral (AAV) vectors have emerged as prominent gene delivery vehicles for gene therapy. AAV capsid proteins determine tissue specificity and immunogenicity and play important roles in receptor binding, the escape of the virus from the endosome, and the transport of the viral DNA to the nuclei of target cells. Therefore, the comprehensive characterization of AAV capsid proteins is necessary for a better understanding of the vector assembly, stability, and transduction efficiency of AAV gene therapies. Glycosylation is one of the most common post-translational modifications (PTMs) and may affect the tissue tropism of AAV gene therapy. However, there are few studies on the characterization of the N- and O-glycosylation of AAV capsid proteins. In this study, we identified the N- and O-glycosylation sites and forms of AAV9 capsid proteins generated from HEK293 cells using liquid chromatography-tandem mass spectrometry (LC-MS)-based glycopeptide mapping and identified free N-glycans released from AAV9 capsid proteins by PNGase F using hydrophilic interaction (HILIC) LC-MS and HILIC LC-fluorescence detection (FLD) methods. This study demonstrates that AAV9 capsids are sprinkled with sugars, including N- and O-glycans, albeit at low levels. It may provide valuable information for a better understanding of AAV capsids in supporting AAV-based gene therapy development.

Cul3 substrate adaptor SPOP targets Nup153 for degradation.

SPOP is a Cul3 substrate adaptor responsible for degradation of many proteins related to cell growth and proliferation. Because mutation or misregulation of SPOP drives cancer progression, understanding the suite of SPOP substrates is important to understanding regulation of cell proliferation. Here, we identify Nup153, a component of the nuclear basket of the nuclear pore complex, as a novel substrate of SPOP. SPOP and Nup153 bind to each other and colocalize at the nuclear envelope and some nuclear foci in cells. The binding interaction between SPOP and Nup153 is complex and multivalent. Nup153 is ubiquitylated and degraded upon expression of SPOPWT but not its substrate binding-deficient mutant SPOPF102C. Depletion of SPOP via RNAi leads to Nup153 stabilization. Upon loss of SPOP, the nuclear envelope localization of spindle assembly checkpoint protein Mad1, which is tethered to the nuclear envelope by Nup153, is stronger. Altogether, our results demonstrate SPOP regulates Nup153 levels and expands our understanding of the role of SPOP in protein and cellular homeostasis.

Human Protein-l-isoaspartate O-Methyltransferase Domain-Containing Protein 1 (PCMTD1) Associates with Cullin-RING Ligase Proteins.

The spontaneous l-isoaspartate protein modification has been observed to negatively affect protein function. However, this modification can be reversed in many proteins in reactions initiated by the protein-l-isoaspartyl (d-aspartyl) O-methyltransferase (PCMT1). It has been hypothesized that an additional mechanism exists in which l-isoaspartate-damaged proteins are recognized and proteolytically degraded. Herein, we describe the protein-l-isoaspartate O-methyltransferase domain-containing protein 1 (PCMTD1) as a putative E3 ubiquitin ligase substrate adaptor protein. The N-terminal domain of PCMTD1 contains l-isoaspartate and S-adenosylmethionine (AdoMet) binding motifs similar to those in PCMT1. This protein also has a C-terminal domain containing suppressor of cytokine signaling (SOCS) box ubiquitin ligase recruitment motifs found in substrate receptor proteins of the Cullin-RING E3 ubiquitin ligases. We demonstrate specific PCMTD1 binding to the canonical methyltransferase cofactor S-adenosylmethionine (AdoMet). Strikingly, while PCMTD1 is able to bind AdoMet, it does not demonstrate any l-isoaspartyl methyltransferase activity under the conditions tested here. However, this protein is able to associate with the Cullin-RING proteins Elongins B and C and Cul5 in vitro and in human cells. The previously uncharacterized PCMTD1 protein may therefore provide an alternate maintenance pathway for modified proteins in mammalian cells by acting as an E3 ubiquitin ligase adaptor protein.

Yeast grown in continuous culture systems can detect mutagens with improved sensitivity relative to the Ames test.

Continuous culture systems allow for the controlled growth of microorganisms over a long period of time. Here, we develop a novel test for mutagenicity that involves growing yeast in continuous culture systems exposed to low levels of mutagen for a period of approximately 20 days. In contrast, most microorganism-based tests for mutagenicity expose the potential mutagen to the biological reporter at a high concentration of mutagen for a short period of time. Our test improves upon the sensitivity of the well-established Ames test by at least 20-fold for each of two mutagens that act by different mechanisms (the intercalator ethidium bromide and alkylating agent methyl methanesulfonate). To conduct the tests, cultures were grown in small, inexpensive continuous culture systems in media containing (potential) mutagen, and the resulting mutagenicity of the added compound was assessed via two methods: a canavanine-based plate assay and whole genome sequencing. In the canavanine-based plate assay, we were able to detect a clear relationship between the amount of mutagen and the number of canavanine-resistant mutant colonies over a period of one to three weeks of exposure. Whole genome sequencing of yeast grown in continuous culture systems exposed to methyl methanesulfonate demonstrated that quantification of mutations is possible by identifying the number of unique variants across each strain. However, this method had lower sensitivity than the plate-based assay and failed to distinguish the different concentrations of mutagen. In conclusion, we propose that yeast grown in continuous culture systems can provide an improved and more sensitive test for mutagenicity.

Phospho-regulation of mitotic spindle assembly.

The assembly of the bipolar mitotic spindle requires the careful orchestration of a myriad of enzyme activities like protein posttranslational modifications. Among these, phosphorylation has arisen as the principle mode for spatially and temporally activating the proteins involved in early mitotic spindle assembly processes. Here, we review key kinases, phosphatases, and phosphorylation events that regulate critical aspects of these processes. We highlight key phosphorylation substrates that are important for ensuring the fidelity of centriole duplication, centrosome maturation, and the establishment of the bipolar spindle. We also highlight techniques used to understand kinase-substrate relationships and to study phosphorylation events. We conclude with perspectives on the field of posttranslational modifications in early mitotic spindle assembly.

Phase Separation in Cell Division.

Cell division requires the assembly and organization of a microtubule spindle for the proper separation of chromosomes in mitosis and meiosis. Phase separation is an emerging paradigm for understanding spatial and temporal regulation of a variety of cellular processes, including cell division. Phase-separated condensates have been recently discovered at many structures during cell division as a possible mechanism for properly localizing, organizing, and activating proteins involved in cell division. Here, we review how these condensates play roles in regulating microtubule density and organization and spindle assembly and function and in activating some of the key players in cell division. We conclude with perspectives on areas of future research for this exciting and rapidly advancing field.

Dissecting the mechanisms of cell division.

Cell division is a highly regulated and carefully orchestrated process. Understanding the mechanisms that promote proper cell division is an important step toward unraveling important questions in cell biology and human health. Early studies seeking to dissect the mechanisms of cell division used classical genetics approaches to identify genes involved in mitosis and deployed biochemical approaches to isolate and identify proteins critical for cell division. These studies underscored that post-translational modifications and cyclin-kinase complexes play roles at the heart of the cell division program. Modern approaches for examining the mechanisms of cell division, including the use of high-throughput methods to study the effects of RNAi, cDNA, and chemical libraries, have evolved to encompass a larger biological and chemical space. Here, we outline some of the classical studies that established a foundation for the field and provide an overview of recent approaches that have advanced the study of cell division.

Leukemia Cell Cycle Chemical Profiling Identifies the G2-Phase Leukemia Specific Inhibitor Leusin-1.

Targeting the leukemia proliferation cycle has been a successful approach to developing antileukemic therapies. However, drug screening efforts to identify novel antileukemic agents have been hampered by the lack of a suitable high-throughput screening platform for suspension cells that does not rely on flow-cytometry analyses. We report the development of a novel leukemia cell-based high-throughput chemical screening platform for the discovery of cell cycle phase specific inhibitors that utilizes chemical cell cycle profiling. We have used this approach to analyze the cell cycle response of acute lymphoblastic leukemia CCRF-CEM cells to each of 181420 druglike compounds. This approach yielded cell cycle phase specific inhibitors of leukemia cell proliferation. Further analyses of the top G2-phase and M-phase inhibitors identified the leukemia specific inhibitor 1 (Leusin-1). Leusin-1 arrests cells in G2 phase and triggers an apoptotic cell death. Most importantly, Leusin-1 was more active in acute lymphoblastic leukemia cells than other types of leukemias, non-blood cancers, or normal cells and represents a lead molecule for developing antileukemic drugs.