Cohesin cleavage by separase is enhanced by a substrate motif distinct from the cleavage site.

Chromosome segregation begins when the cysteine protease, separase, cleaves the Scc1 subunit of cohesin at the metaphase-to-anaphase transition. Separase is inhibited prior to metaphase by the tightly bound securin protein, which contains a pseudosubstrate motif that blocks the separase active site. To investigate separase substrate specificity and regulation, here we develop a system for producing recombinant, securin-free human separase. Using this enzyme, we identify an LPE motif on the Scc1 substrate that is distinct from the cleavage site and is required for rapid and specific substrate cleavage. Securin also contains a conserved LPE motif, and we provide evidence that this sequence blocks separase engagement of the Scc1 LPE motif. Our results suggest that rapid cohesin cleavage by separase requires a substrate docking interaction outside the active site. This interaction is blocked by securin, providing a second mechanism by which securin inhibits cohesin cleavage

An engineered gastrointestinally stable microbial leucine decarboxylase for potential treatment of maple syrup urine disease

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MECHANISMS OF CELL CYCLE CONTROL

Ordered phosphorylation of cyclin-dependent kinase (CDK) substrates leads to the sequential transcriptional activation and inhibition of hundreds of cell cycle-regulated genes. We find that Ndd1, an activator of genes required for mitotic progression, is both positively and negatively regulated by CDK activity. CDK activity initially stimulates Ndd1-dependent transcription as cells enter mitosis, but prolonged high CDK activity in a mitotic arrest inhibits transcription. The result is a time-delayed negative feedback circuit that generates a pulse of mitotic gene expression. Our results suggest that high CDK activity catalyzes the formation of multiple weak phosphodegrons on Ndd1, leading to its destabilization. Cyclin specificity and phosphorylation kinetics contribute to the timing of Ndd1 destruction. Failure to degrade Ndd1 in a mitotic arrest leads to elevated mitotic gene expression. We conclude that a combination of positive and negative Ndd1 regulation by CDKs governs the timing and magnitude of the mitotic transcriptional program

Lipid droplet formation in <i>Mycobacterium tuberculosis</i> infected macrophages requires IFN-γ/HIF-1α signaling and supports host defense

<div><p>Lipid droplet (LD) formation occurs during infection of macrophages with numerous intracellular pathogens, including <i>Mycobacterium tuberculosis</i>. It is believed that <i>M</i>. <i>tuberculosis</i> and other bacteria specifically provoke LD formation as a pathogenic strategy in order to create a depot of host lipids for use as a carbon source to fuel intracellular growth. Here we show that LD formation is not a bacterially driven process during <i>M</i>. <i>tuberculosis</i> infection, but rather occurs as a result of immune activation of macrophages as part of a host defense mechanism. We show that an IFN-γ driven, HIF-1α dependent signaling pathway, previously implicated in host defense, redistributes macrophage lipids into LDs. Furthermore, we show that <i>M</i>. <i>tuberculosis</i> is able to acquire host lipids in the absence of LDs, but not in the presence of IFN-γ induced LDs. This result uncouples macrophage LD formation from bacterial acquisition of host lipids. In addition, we show that IFN-γ driven LD formation supports the production of host protective eicosanoids including PGE₂ and LXB₄. Finally, we demonstrate that HIF-1α and its target gene <i>Hig2</i> are required for the majority of LD formation in the lungs of mice infected with <i>M</i>. <i>tuberculosis</i>, thus demonstrating that immune activation provides the primary stimulus for LD formation <i>in vivo</i>. Taken together our data demonstrate that macrophage LD formation is a host-driven component of the adaptive immune response to <i>M</i>. <i>tuberculosis</i>, and suggest that macrophage LDs are not an important source of nutrients for <i>M</i>. <i>tuberculosis</i>.</p></div

Multisite phosphorylation by Cdk1 initiates delayed negative feedback to control mitotic transcription

Cell-cycle progression is driven by the phosphorylation of cyclin-dependent kinase (Cdk) substrates.1-3 The order of substrate phosphorylation depends in part on the general rise in Cdk activity during the cell cycle,4-7 together with variations in substrate docking to sites on associated cyclin and Cks subunits.3,6,8-10 Many substrates are modified at multiple sites to provide more complex regulation.10-14 Here, we describe an elegant regulatory circuit based on multisite phosphorylation of Ndd1, a transcriptional co-activator of budding yeast genes required for mitotic progression.11,12 As cells enter mitosis, Ndd1 phosphorylation by Cdk1 is known to promote mitotic cyclin (CLB2) gene transcription, resulting in positive feedback.13-16 Consistent with these findings, we show that low Cdk1 activity promotes CLB2 expression at mitotic entry. We also find, however, that when high Cdk1 activity accumulates in a mitotic arrest, CLB2 expression is inhibited. Inhibition is accompanied by Ndd1 degradation, and we present evidence that degradation is triggered by multisite Ndd1 phosphorylation by high mitotic Cdk1-Clb2 activity. Complete Ndd1 phosphorylation by Clb2-Cdk1-Cks1 requires the phosphothreonine-binding site of Cks1, as well as a recently identified phosphate-binding pocket on the cyclin Clb2.17 We therefore propose that initial phosphorylation by Cdk1 primes Ndd1 for delayed secondary phosphorylation at suboptimal sites that promote degradation. Together, our results suggest that rising levels of mitotic Cdk1 activity act at multiple phosphorylation sites on Ndd1, first triggering rapid positive feedback and then promoting delayed negative feedback, resulting in a pulse of mitotic gene expression

HIF-1α target gene <i>Hig2</i> is required for LD maintenance during <i>M</i>. <i>tuberculosis</i> infection.

<p>(A) RNA-seq data for transcript levels of <i>Hig2</i> in wildtype BMDM either unstimulated [U], IFN-γ activated [G], <i>M</i>. <i>tuberculosis</i> infected [TB], or IFN-γ activated and <i>M</i>. <i>tuberculosis</i> infected [TB/G]. Samples were collected 1 day post-infection in 3 independent experiments. (B-E) IFN-γ activated wildtype and <i>Hig2</i>^-/- BMDM were infected with <i>M</i>. <i>tuberculosis</i> 635-Turbo. BODIPY 493/503 staining and confocal microscopy was performed 1 day post-infection (B,D) and 3 days post-infection (C,E). CellProfiler was used to quantify (F) the percentage of BMDM with LDs, (G) the number of LDs per BMDM, and (H) LD size. Each data point is quantified from ~500 BMDM. Figures are representative of three experiments, error bars are standard deviation, *p<0.05, **p<0.01, ***p < .001, ****p<0.0001 by unpaired t-test.</p

LDs support host immunity in <i>M</i>. <i>tuberculosis</i> infected and IFN-γ activated macrophages.

<p>(A) Resting and IFN-γ activated BMDM were infected with TB-Lux <i>M</i>. <i>tuberculosis</i> at MOI = 5, and T863 was added after the 4 hour phagocytosis. Lux readings were taken at day 0 (after the 4 hour phagocytosis), and days 1, 2, and 3 post-infection. Data is normalized to the day 0 read. (B) Wildtype and <i>Hig2</i>^-/- BMDM were infected as in (A) and bacterial numbers were enumerated by plating for CFU at the indicated time points. (C) LC-MS/MS measurement of PGE₂ in supernatants from wildtype BMDM either unstimulated [U], IFN-γ activated [G], <i>M</i>. <i>tuberculosis</i> infected [TB], or IFN-γ activated and <i>M</i>. <i>tuberculosis</i> infected [TB/G]. Supernatant samples were taken in quadruplicate at both 48 hours post-infection and again at 72 hours post-infection from the same cells. (D,E) LC-MS/MS measurement of PGE₂ (D) and LXB₄ (E) from supernatants of BMDM infected with <i>M</i>. <i>tuberculosis</i>, 48 hours post-infection. Samples are from wildtype [WT], <i>Hif1a</i>^-/- [<i>Hif</i>] and <i>Hig2</i>^-/- [<i>Hig</i>] BMDM, with IFN-γ and T863 treatment as indicated. (F,G) Wildtype and <i>Hif1a</i>^-/- BMDM were activated with IFN-γ and infected with <i>M</i>. <i>tuberculosis</i> 635-Turbo and stained with BODIPY 493/503 at 3 days post-infection. (H) Quantification of bacterial area colocalizing with lipid staining at 3 days post-infection in wildtype, <i>Cd36</i>^<i>-/-</i>, <i>Hif1a</i>^<i>-/-</i>, and <i>Hig2</i>^<i>-/-</i> BMDM activated with IFN-γ and infected with <i>M</i>. <i>tuberculosis</i>. Figures are representative of a minimum of three experiments, except for LC-MS/MS eicosanoid profiling which was performed once in quadruplicate. Error bars are standard deviation. *p<0.05, ***p<0.001 by unpaired t-test.</p

IFN-γ signaling is required for LD formation during <i>M</i>. <i>tuberculosis</i> infection <i>in vivo</i>.

<p><b>(A-J)</b> Mice were infected with ~200 CFU of <i>M</i>. <i>tuberculosis</i> via the aerosol route and lungs were collected for histological analysis of LD formation by Oil Red O (ORO) staining. (A-D) ORO stained lung sections from wildtype and <i>Ifng</i>^-/- mice 21 days post-infection. Sections were counter-stained with hematoxylin. (E-J) ORO stained lung sections from wildtype (E,F), <i>LysMcre</i>^<i>+/+</i><i>; Hif1a</i>^<i>fl/fl</i> (G,H), and <i>Hig2</i>^-/- mice (I,J) at 28 days post-infection. (K) ORO signal as a percentage of lesion area was quantified in sections from wildtype, <i>LysMcre</i>^<i>+/+</i><i>; Hif1a</i>^<i>fl/fl</i>, and <i>Hig2</i>^-/- mice. (L) Bacterial burden in the lungs of wildtype and <i>Hig2</i>^-/- mice was enumerated by plating for CFU at 1, 18 and 28 days post-infection. Images and figures are representative of at least 3 independent experiments. Error bars are standard deviation, *p<0.05, **p<0.01 by unpaired t-test.</p

Macrophage LD formation during <i>M</i>. <i>tuberculosis</i> infection requires IFN-γ.

<p>(A) Unstimulated and (B) IFN-γ activated BMDM were infected with fluorescent <i>M</i>. <i>tuberculosis</i> 635-Turbo at MOI = 5 and imaged by confocal microscopy 3 days post-infection. Nuclei were visualized with DAPI staining and neutral lipids were visualized with BODIPY 493/503 staining. (C,D) Images from 1 and 3 days post-infection were quantified using CellProfiler for (C) the average number of LDs per BMDM and (D) the percentage of BMDM containing LDs. Each data point is quantified from ~500 BMDM. (E,F) TEM was performed on IFN-γ activated BMDMs infected with <i>M</i>. <i>tuberculosis</i> 3 days post-infection. LDs (arrowheads) and <i>M</i>. <i>tuberculosis</i> (asterisk) are indicated. (E) is at 890x magnification and (F) is at 9300x magnification. (G) IFN-γ activated BMDM were infected with fluorescent <i>M</i>. <i>tuberculosis</i> 635-Turbo. Immunofluorescence microscopy was performed 1 day post-infection, and localization of PLIN2 to BODIPY-stained structures was observed. (H,I) IFN-γ activated <i>Plin2</i>^<i>-/-</i> BMDM were infected with <i>M</i>. <i>tuberculosis</i> and LD formation was evaluated using BODIPY 493/503 staining 1 day post-infection (H) and 3 days post-infection (I). (J) Unstimulated and (K) IFN-γ activated primary human monocyte derived macrophages were infected with <i>M</i>. <i>tuberculosis</i> 635-Turbo at MOI = 3 and imaged by confocal microscopy 1 day post-infection. Nuclei were visualized by Hoechst 33342 and neutral lipids with BODIPY 493/503. (L) The average number of LDs per cell was quantified using CellProfiler in uninfected [U], IFN-γ activated [G], <i>M</i>. <i>tuberculosis</i> infected [TB], or IFN-γ activated and <i>M</i>. <i>tuberculosis</i> infected [TB/G] human macrophages at 1 day post-infection. Each data point is quantified from ~200 cells. All figures are representative of a minimum of three independent experiments with the exception of electron microscopy which was performed once in triplicate and human macrophages infections which were performed in duplicate. Error bars are standard deviation, **p<0.01, ****p<0.0001 by unpaired t-test.</p