Gary S. McDowell

Cell cycle-regulated multi-site phosphorylation of Neurogenin 2 coordinates cell cycling with differentiation during neurogenesis

During development of the central nervous system, the transition from progenitor maintenance to differentiation is directly triggered by a lengthening of the cell cycle that occurs as development progresses. However, the mechanistic basis of this regulation is unknown. The proneural transcription factor Neurogenin 2 (Ngn2) acts as a master regulator of neuronal differentiation. Here, we demonstrate that Ngn2 is phosphorylated on multiple serine-proline sites in response to rising cyclin-dependent kinase (cdk) levels. This multi-site phosphorylation results in quantitative inhibition of the ability of Ngn2 to induce neurogenesis in vivo and in vitro. Mechanistically, multi-site phosphorylation inhibits binding of Ngn2 to E box DNA, and inhibition of DNA binding depends on the number of phosphorylation sites available, quantitatively controlling promoter occupancy in a rheostat-like manner. Neuronal differentiation driven by a mutant of Ngn2 that cannot be phosphorylated by cdks is no longer inhibited by elevated cdk kinase levels. Additionally, phosphomutant Ngn2-driven neuronal differentiation shows a reduced requirement for the presence of cdk inhibitors. From these results, we propose a model whereby multi-site cdk-dependent phosphorylation of Ngn2 interprets cdk levels to control neuronal differentiation in response to cell cycle lengthening during development.

Post-translational modification of Ngn2 differentially affects transcription of distinct targets to regulate the balance between progenitor maintenance and differentiation

Neurogenin 2 (Ngn2) controls neuronal differentiation cell-autonomously by transcriptional activation of targets such as NeuroD, while simultaneously controlling progenitor maintenance non-cell-autonomously by upregulating Delta expression and Notch signalling. Reduction in Cdk-dependent multisite phosphorylation of Ngn2 enhances its promoter binding affinity. This leads specifically to an increase in neuronal differentiation without an apparent increase in progenitor maintenance via Delta-Notch signalling, although the mechanism underlying this imbalance remains unclear. Here we show in Xenopus embryos and mouse P19 cells that the NeuroD promoter is substantially more sensitive to the phosphorylation status of Ngn2 than the Delta promoter, and that this can be attributed to differences in the ease of promoter activation. In addition, we also show that the phosphorylation status of Ngn2 regulates sensitivity to Notch signalling. These observations explain how Ngn2 post-translational modification in response to changes in the cell cycle kinase environment results in enhanced neuronal differentiation upon cell cycle lengthening.

Ubiquitylation on canonical and non-canonical sites targets the transcription factor neurogenin for ubiquitin-mediated proteolysis

Polyubiquitylation targets multiple proteins for degradation by the proteasome. Typically, the first ubiquitin is linked to lysine residues in the substrate for degradation via an isopeptide bond, although rarely ubiquitin linkage to the N-terminal residue has also been observed. We have recently shown that Neurogenin (NGN), a basic helix-loop-helix transcription factor that plays a central role in regulating neuronal differentiation, is degraded by ubiquitin-mediated proteolysis. We have taken a biochemical and mutagenesis approach to investigate sites of ubiquitylation of NGN, initially using extracts of eggs from the frog Xenopus laevis as a source of ubiquitylation and degradation components. NGN can be targeted for destruction by ubiquitylation via lysines or the N terminus. However, we see that a modified NGN, where canonical lysine ubiquitylation and N-terminally linked ubiquitylation are prevented, is nevertheless ubiquitylated and degraded by the proteasome. We show that polyubiquitin chains covalently attach to non-canonical cysteine residues in NGN, and these non-canonical linkages alone are capable of targeting NGN protein for destruction. Importantly, canonical and non-canonical ubiquitylation occurs simultaneously in the native protein and may differ in importance for driving degradation in interphase and mitosis. We conclude that native NGN is ubiquitylated on multiple canonical and non-canonical sites by cellular ubiquitin ligases, and all types of linkage can contribute to protein turnover.

Formin Is Associated with Left-Right Asymmetry in the Pond Snail and the Frog

Summary While components of the pathway that establishes left-right asymmetry have been identified in diverse animals, from vertebrates to flies, it is striking that the genes involved in the first symmetry-breaking step remain wholly unknown in the most obviously chiral animals, the gastropod snails. Previously, research on snails was used to show that left-right signaling of Nodal, downstream of symmetry breaking, may be an ancestral feature of the Bilateria [1, 2]. Here, we report that a disabling mutation in one copy of a tandemly duplicated, diaphanous-related formin is perfectly associated with symmetry breaking in the pond snail. This is supported by the observation that an anti-formin drug treatment converts dextral snail embryos to a sinistral phenocopy, and in frogs, drug inhibition or overexpression by microinjection of formin has a chirality-randomizing effect in early (pre-cilia) embryos. Contrary to expectations based on existing models [3, 4, 5], we discovered asymmetric gene expression in 2- and 4-cell snail embryos, preceding morphological asymmetry. As the formin-actin filament has been shown to be part of an asymmetry-breaking switch in vitro [6, 7], together these results are consistent with the view that animals with diverse body plans may derive their asymmetries from the same intracellular chiral elements [8].

Shaping the Future of Research: a perspective from junior scientists

The landscape of scientific research and funding is in flux as a result of tight budgets, evolving models of both publishing and evaluation, and questions about training and workforce stability. As future leaders, junior scientists are uniquely poised to shape the culture and practice of science in response to these challenges. A group of postdocs in the Boston area who are invested in improving the scientific endeavor, planned a symposium held on October 2 nd and 3 rd, 2014, as a way to join the discussion about the future of US biomedical research. Here we present a report of the proceedings of participant-driven workshops and the organizers’ synthesis of the outcomes.

iFASP: Combining Isobaric Mass Tagging with Filter-Aided Sample Preparation

Careful, clean and controlled preparation of samples for mass spectrometry proteomics is crucial to obtain reproducible and reliable data. This is especially important when carrying out quantitative proteomics by chemical isobaric labeling (aka tandem mass tagging), since the differentially labeled samples are combined quite late during the sample processing. Addressing this need for robust and reliable sample processing for quantitative proteomics, we describe here iFASP, a simple protocol for combining isobaric mass tagging with the recently introduced filter-aided sample preparation (FASP) method. iFASP provides a quick, simple and effective method for obtaining clean samples, ensuring efficient digestion and providing excellent labeling yields for quantitative proteomics experiments. We have carried out our iFASP protocol using several highly complex Xenopus laevis egg and embryo lysates and compared the labeling yields and number of high-confidence peptide identifications to a standard in-solution digestion and labeling protocol. Although the labeling efficiency with both techniques is in the 99+% range, the number of peptides identified with a 1% false discovery rate and the corresponding number of quantified peptide spectral matches are as much as doubled with iFASP compared to the corresponding non-FASP-based method.

Topology is the Principal Determinant in the Folding of a Complex All-alpha Greek Key Death Domain from Human FADD

In order to elucidate the relative importance of secondary structure and topology in determining folding mechanism, we have carried out a phi-value analysis of the death domain (DD) from human FADD. FADD DD is a 100 amino acid domain consisting of six anti-parallel alpha helices arranged in a Greek key structure. We asked how does the folding of this domain compare with that of (a) other all-alpha-helical proteins and (b) other Greek key proteins? Is the folding pathway determined mainly by secondary structure or is topology the principal determinant? Our Φ-value analysis reveals a striking resemblance to the all-beta Greek key immunoglobulin-like domains. Both fold via diffuse transition states and, importantly, long-range interactions between the four central elements of secondary structure are established in the transition state. The elements of secondary structure that are less tightly associated with the central core are less well packed in both cases. Topology appears to be the dominant factor in determining the pathway of folding in all Greek key domains.

Non-canonical ubiquitylation of the proneural protein Ngn2 occurs in both Xenopus embryos and mammalian cells

Poly-ubiquitin chains targeting proteins for 26S proteasomal degradation are classically anchored on internal lysines of substrates via iso-peptide linkages. However recently, linkage of ubiquitin moieties to non-canonical nucleophilic residues, such as cysteines, serines and threonines, has been demonstrated in a small number of cases. Non-canonical ubiquitylation of the proneural protein Ngn2 has previously been seen in Xenopus egg extract, but it was not clear whether such highly unusual modes of ubiquitylation were restricted to the environment of egg cytoplasm. Here we show that Ngn2 is, indeed, ubiquitylated on non-canonical sites in extracts from neurula stage Xenopus embryos, when Ngn2 is usually active. Moreover, in the P19 mammalian embryonal carcinoma cell line capable of differentiating into neurons, xNgn2 is ubiquitylated on both canonical and non-canonical sites. We see that mutation of cysteines alone results stabilisation of the protein in P19 cells, indicating that non-canonical ubiquitylation on these residues normally contributes to the fast turnover of xNgn2 in mammalian cells.

A call for transparency in tracking student and postdoc career outcomes

There is a common misconception that the United States is suffering from a “STEM shortage,” a dearth of graduates with scientific, technological, engineering, and mathematical backgrounds. In biomedical science, however, we are likely suffering from the opposite problem and could certainly better tailor training to actual career outcomes. At the Future of Research Symposium, various workshops identified this as a key issue in a pipeline traditionally geared toward academia. Proposals for reform all ultimately come up against the same problem: there is a shocking lack of data at institutional and national levels on the size, shape, and successful careers of participants in the research workforce. In this paper, we call for improved institutional reporting of the number of graduate students and postdocs and their training and career outcomes.

SweetSEQer: simple de novo filtering and annotation of glycoconjugate mass spectra

The past 15 years have seen significant progress in LC-MS/MS peptide sequencing, including the advent of successful de novo and database search methods; however, analysis of glycopeptide and, more generally, glycoconjugate spectra remains a much more open problem, and much annotation is still performed manually. This is partly because glycans, unlike peptides, need not be linear chains and are instead described by trees. In this study, we introduce SweetSEQer, an extremely simple open source tool for identifying potential glycopeptide MS/MS spectra. We evaluate SweetSEQer on manually curated glycoconjugate spectra and on negative controls, and we demonstrate high quality filtering that can be easily improved for specific applications. We also demonstrate a high overlap between peaks annotated by experts and peaks annotated by SweetSEQer, as well as demonstrate inferred glycan graphs consistent with canonical glycan tree motifs. This study presents a novel tool for annotating spectra and producing glycan graphs from LC-MS/MS spectra. The tool is evaluated and shown to perform similarly to an expert on manually curated data.