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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.

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.

RNA interference (RNAi) is a useful technique for knocking down a protein of interest, allowing for the study of the function of a gene product. However, RNAi techniques are prone to off-target effects, such as non-specific knockdown of genes besides the protein of interest. An important control and companion to RNAi knockdown experiments is the rescue experiment, wherein gene function is restored by expression of an RNAi-resistant construct of the protein of interest. Generating an RNAi-resistant construct of the protein of interest involves generating silent mutations within the coding sequence of the protein so that the resulting amino acid product is the same, but the protein mRNA is no longer a target for the RNAi. Here, Synonymous Mutation Generator, a Python-based web tool that takes an input DNA coding sequence and outputs a synonymous DNA coding sequence that is RNAi-resistant, is described. This web tool should be a useful resource for researchers cloning RNAi-resistant constructs. Synonymous Mutation Generator is easy to use and can be found at jong2.pythonanywhere.com, and the source code is available on GitHub.

Ubiquitination plays many critical roles in protein function and regulation. Consequently, mutation and aberrant expression of E3 ubiquitin ligases can drive cancer progression. Identifying key ligase-substrate relationships is crucial to understanding the molecular basis and pathways behind cancer and toward identifying novel targets for cancer therapeutics. Here, we review the importance of E3 ligases in the regulating the hallmarks of cancer, discuss some of the key and novel E3 ubiquitin ligases that drive tumor formation and angiogenesis, and review the clinical development of inhibitors that antagonize their function. We conclude with perspectives on the field and future directions toward understanding ubiquitination and cancer progression.

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) Omethyltransferase (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 domaincontaining 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.

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 181,420 drug-like 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.

Cell processes like growth and division are tightly regulated. One such mechanism of regulation is ubiquitination. Ubiquitination can change a protein's localization or activity, or it can mark the protein for degradation by the ubiquitin proteasome system. The final step of ubiquitination, the transfer of ubiquitin to the target protein, is mediated by E3 ubiquitin ligases and their substrate adaptors, proteins that allow E3 ligases to be selective in choosing their targets. Understanding the targets of E3 ligases and substrate adaptors, then, is crucial to understanding cell regulation and disease mechanisms linked to misregulation of protein levels and activity. SPOP is a Cul3 E3 ubiquitin ligase substrate adaptor whose targets, such as c‐Myc, PD‐L1, and ERG, are crucial for cell cycle progression and cancer proliferation. Through a mass spectrometry screen, we identified SPOP as a potential regulator of NupJ, a nuclear pore protein. Nuclear pore proteins play canonical roles in transport across the nuclear envelope and emerging roles in nuclear envelope morphology and cell division. SPOP and NupJ both co‐localize at the nuclear envelope via immunofluorescence microscopy, and co‐immunoprecipitation experiments demonstrate that SPOP and NupJ bind to each other in vitro and from cell lysates. Similar to overexpression of NupJ, siRNA against SPOP leads to an increase in the number of nuclear envelope defects. Moreoever, overexpressed NupJ leads to defects in cell division. Our results suggest that SPOP regulates NupJ activity and perhaps protein stability of NupJ.Support or Funding InformationJYO is supported by Edwin W. Pauley Fellowship, NSF Graduate Research Fellowship DGE‐1650604, and Ruth L. Kirschstein National Research Service Award GM007185. This work was funded by NSF MCB1243645 (to JZT).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.