Shuobing Chen*, Jiayi Wu*, Ying Lu*, Yong-Bei Ma, Byung-hoon Lee, Zhou Yu, Qi Ouyang, Daniel J. Finley, Marc W. Kirschner, and Youdong Mao (* equal contribution). 10/21/2016. “Structural Basis for Dynamic Regulation of The Human 26S Proteasome.” PNAS , 113, 46, Pp. 12991-12996. Publisher's Version
Nicholas G. Brown, Ryan VanderLinden, Edmond R. Watson, Florian Weissmann, Alban Ordureau, Kuen-Phon Wu, Wei Zhang, Shanshan Yu, Joseph S. Harrison, Renping Q. Coudevylle, Iain F. Davidson, Ying Lu, Prakash Dube, Michael R. Brunner, Christy R. R. Grace, Darcie J. Miller, Marc A. Jarvis, Masaya Yamaguchi, Peter Y. Mercredi, Sachdev S. Sidhu, Brian Kuhlman, Marc W. Kirschner, J. Wade Harper, Jan-Michael Peters, Holger Stark, and Brenda A. Schulman. 6/2/2016. “Dual RING E3 Architectures for Substrate Polyubiquitination Visualized for Anaphase Promoting Complex/Cyclosome.” Cell, 165, 6, Pp. 1440–1453. Publisher's VersionAbstract

Protein ubiquitination involves E1, E2, and E3 trienzyme cascades. E2 and RING E3 enzymes often collaborate to first prime a substrate with a single ubiquitin (UB) and then achieve different forms of polyubiquitination: multiubiquitination of several sites and elongation of linkage-specific UB chains. Here, cryo-EM and biochemistry show that the human E3 anaphase-promoting complex/cyclosome (APC/C) and its two partner E2s, UBE2C (aka UBCH10) and UBE2S, adopt specialized catalytic architectures for these two distinct forms of polyubiquitination. The APC/C RING constrains UBE2C proximal to a substrate and simultaneously binds a substrate-linked UB to drive processive multiubiquitination. Alternatively, during UB chain elongation, the RING does not bind UBE2S but rather lures an evolving substrate-linked UB to UBE2S positioned through a cullin interaction to generate a Lys11-linked chain. Our findings define mechanisms of APC/C regulation, and establish principles by which specialized E3–E2–substrate-UB architectures control different forms of polyubiquitination.

Byung-hoon Lee, Ying Lu, Miguel A. Prado, Yuan Shi, Shuangwu Sun, Suzanne Elsasser, Marc W. Kirschner, Steven P. Gygi, Randall W. King, and Daniel Finley. 4/21/2016. “USP14 Deubiquitinates Proteasome-bound Substrates that are Ubiquitinated at Multiple Sites.” Nature, 532, Pp. 398–401. Publisher's VersionAbstract

USP14 is a major regulator of the proteasome and one of three proteasome-associated deubiquitinating enzymes1, 2, 3, 4, 5, 6, 7, 8, 9. Its effects on protein turnover are substrate-specific, for unknown reasons. We report that USP14 shows a marked preference for ubiquitin–cyclin B conjugates that carry more than one ubiquitin modification or chain. This specificity is conserved from yeast to humans and is independent of chain linkage type. USP14 has been thought to cleave single ubiquitin groups from the distal tip of a chain, but we find that it removes chains from cyclin B en bloc, proceeding until a single chain remains. The suppression of degradation by USP14’s catalytic activity reflects its capacity to act on a millisecond time scale, before the proteasome can initiate degradation of the substrate. In addition, single-molecule studies showed that the dwell time of ubiquitin conjugates at the proteasome was reduced by USP14-dependent deubiquitination. In summary, the specificity of the proteasome can be regulated by rapid ubiquitin chain removal, which resolves substrates based on a novel aspect of ubiquitin conjugate architecture.

Ying Lu, Weiping Wang, and Marc W. Kirschner. 4/10/2015. “Specificity of the Anaphase-Promoting Complex: A Single-Molecule Study.” Science, 348, 6231. Publisher's VersionAbstract

Biological processes require specific enzymatic reactions, paradoxically involving short recognition sequences. As an example, cell-cycle timing depends on a sequence of ubiquitylation events mediated by the anaphase-promoting complex (APC) based on short redundant motifs. To understand the origin of specificity, we designed single-molecule fluorescence assays that capture transient ubiquitylation reactions. We find that the APC-mediated ubiquitylation involves a highly processive initial reaction on the substrate, followed by multiple encounters and reactions at a slower rate. The initial ubiquitylation greatly enhances the substrate’s binding affinity in subsequent reactions, by both increasing the on-rate and decreasing the off-rate. We postulate that these cycles of positive feedback enable high specificity for substrates with short recognition motifs in a complex cellular environment.

Ying Lu, Byung-hoon Lee, Randall King, Daniel Finley, and Marc W. Kirschner. 4/10/2015. “Substrate Degradation by the Proteasome: A Single-Molecule Kinetic Analysis.” Science, 348, 6231, Pp. 1250834. Publisher's VersionAbstract

To address how the configuration of conjugated ubiquitins determines the recognition of substrates by the proteasome, we analyzed the degradation kinetics of substrates with chemically defined ubiquitin configurations. Contrary to the view that a tetraubiquitin chain is the minimal signal for efficient degradation, we find that distributing the ubiquitins as diubiquitin chains provides a more efficient signal. To understand how the proteasome actually discriminates among ubiquitin configurations, we developed single-molecule assays that distinguished intermediate steps of degradation kinetically. The level of ubiquitin on a substrate drives proteasome-substrate interaction, whereas the chain structure of ubiquitin affects translocation into the axial channel on the proteasome. Together these two features largely determine the susceptibility of substrates for proteasomal degradation.

Hidefumi* Fukushima, Lixin* Wan, Hiroyuki* Inuzuka, Ying Lu*, Daming Gao, Victor Li, Pengda Liu, Alan W Lau, Tao Wu, Marc W. Kirschner, and Wenyi (* equal contribution) Wei. 8/29/2013. “SCF-mediated Cdh1 Destruction Defines a Negative Feedback Regulation to Coordinate Cell Cycle Progression.” Cell Reports, 4, 4, Pp. 803–816. Publisher's VersionAbstract

Proper cell-cycle transitions are driven by waves of ubiquitin-dependent degradation of key regulators by the anaphase-promoting complex (APC) and Skp1-Cullin1-F-box (SCF) E3 ubiquitin ligase complexes. But precisely how APC and SCF activities are coordinated to regulate cell-cycle progression remains largely unclear. We previously showed that APC/Cdh1 earmarks the SCF component Skp2 for degradation. Here, we continue to report that SCFβ-TRCP reciprocally controls APC/Cdh1 activity by governing Cdh1 ubiquitination and subsequent degradation. Furthermore, we define both cyclin A and Plk1, two well-known Cdh1 substrates, as upstream modifying enzymes that promote Cdh1 phosphorylation to trigger Cdh1 ubiquitination and subsequent degradation by SCFβ-TRCP. Thus, our work reveals a negative repression mechanism for SCF to control APC, thereby illustrating an elegant dual repression system between these two E3 ligase complexes to create the ordered cascade of APC and SCF activities governing timely cell-cycle transitions.

Ying Lu and Frederick Cross. 4/16/2010. “Periodic Cyclin-Cdk Activity Entrains an Autonomous Cdc14 Release Oscillator.” Cell, 141, 2, Pp. 224-226. Publisher's VersionAbstract

One oscillation of Cyclin-dependent kinase (Cdk) activity, largely driven by periodic synthesis and destruction of cyclins, is tightly coupled to a single complete eukaryotic cell division cycle. Tight linkage of different steps in diverse cell-cycle processes to Cdk activity has been proposed to explain this coupling. Here, we demonstrate an intrinsically oscillatory module controlling nucleolar release and resequestration of the Cdc14 phosphatase, which is essential for mitotic exit in budding yeast. We find that this Cdc14 release oscillator functions at constant and physiological cyclin-Cdk levels, and is therefore independent of the Cdk oscillator. However, the frequency of the release oscillator is regulated by cyclin-Cdk activity. This observation together with its mechanism suggests that the intrinsically autonomous Cdc14 release cycles are locked at once-per-cell-cycle through entrainment by the Cdk oscillator in wild-type cells. This concept may have broad implications for the structure and evolution of eukaryotic cell-cycle control.

Benjamin J. Drapkin, Ying Lu, Andrea L. Procko, Benjamin L. Timney, and Frederick R. Cross. 11/17/2009. “1. Analysis of The Mitotic Exit Control System Using Locked Levels of Stable Mitotic Cyclin.” Molecular System Biology, 5, Pp. 328. Publisher's VersionAbstract

Cyclin‐dependent kinase (Cdk) both promotes mitotic entry (spindle assembly and anaphase) and inhibits mitotic exit (spindle disassembly and cytokinesis), leading to an elegant quantitative hypothesis that a single cyclin oscillation can function as a ratchet to order these events. This ratchet is at the core of a published ODE model for the yeast cell cycle. However, the ratchet model requires appropriate cyclin dose–response thresholds. Here, we test the inhibition of mitotic exit in budding yeast using graded levels of stable mitotic cyclin (Clb2). In opposition to the ratchet model, stable levels of Clb2 introduced dose‐dependent delays, rather than hard thresholds, that varied by mitotic exit event. The ensuing cell cycle was highly abnormal, suggesting a novel reason for cyclin degradation. Cdc14 phosphatase antagonizes Clb2–Cdk, and Cdc14 is released from inhibitory nucleolar sequestration independently of stable Clb2. Thus, Cdc14/Clb2 balance may be the appropriate variable for mitotic regulation. Although our results are inconsistent with the aforementioned ODE model, revision of the model to allow Cdc14/Clb2 balance to control mitotic exit corrects these discrepancies, providing theoretical support for our conclusions.

Ying Lu and Frederick Cross. 3/1/2009. “Mitotic Exit in the Absence of Separase Activity.” Mol Biol Cell, 20, 5, Pp. 1576-91. Publisher's VersionAbstract

In budding yeast, three interdigitated pathways regulate mitotic exit (ME): mitotic cyclin–cyclin-dependent kinase (Cdk) inactivation; the Cdc14 early anaphase release (FEAR) network, including a nonproteolytic function of separase (Esp1); and the mitotic exit network (MEN) driven by interaction between the spindle pole body and the bud cortex. Here, we evaluate the contributions of these pathways to ME kinetics. Reducing Cdk activity is critical for ME, and the MEN contributes strongly to ME efficiency. Esp1 contributes to ME kinetics mainly through cohesin cleavage: the Esp1 requirement can be largely bypassed if cells are provided Esp1-independent means of separating sister chromatids. In the absence of Esp1 activity, we observed only a minor ME delay consistent with a FEAR defect. Esp1 overexpression drives ME in Cdc20-depleted cells arrested in metaphase. We have found that this activity of overexpressed Esp1 depended on spindle integrity and the MEN. We defined the first quantitative measure for Cdc14 release based on colocalization with the Net1 nucleolar anchor. This measure indicates efficient Cdc14 release upon MEN activation; release driven by Esp1 in the absence of microtubules was inefficient and incapable of driving ME. We also found a novel role for the MEN: activating Cdc14 nuclear export, even in the absence of Net1.

Xiaonan Wang, Ying Lu, Minxi Jiang, and Qi Ouyang. 5/27/2004. “Attraction of spiral waves by localized inhomogeneities with small-world connections in excitable media.” Physical Review E, 69, 5, Pp. 056223. Publisher's VersionAbstract

Trapping and untrapping of spiral tips in a two-dimensional homogeneous excitable medium with local small-world connections are studied by numerical simulation. In a homogeneous medium which can be simulated with a lattice of regular neighborhood connections, the spiral wave is in the meandering regime. When changing the topology of a small region from regular connections to small-world connections, the tip of the spiral waves is attracted by the small-world region, where the average path length declines with the introduction of long distant connections. The “trapped” phenomenon also occurs in regular lattices where the diffusion coefficient of the small region is increased. The above results can be explained by the eikonal equation, the Luther equation, and the relation between the core radius and the diffusion coefficient.

Fangting Li*, Tao Long*, Ying Lu*, Chao Tang, and Qi Ouyang (*equal contribution). 4/6/2004. “The Yeast Cell-Cycle Network is Robustly Designed.” PNAS, 101, 14, Pp. 4781-6. Publisher's VersionAbstract

The interactions between proteins, DNA, and RNA in living cells constitute molecular networks that govern various cellular functions. To investigate the global dynamical properties and stabilities of such networks, we studied the cell-cycle regulatory network of the budding yeast. With the use of a simple dynamical model, it was demonstrated that the cell-cycle network is extremely stable and robust for its function. The biological stationary state, the G1 state, is a global attractor of the dynamics. The biological pathway, the cell-cycle sequence of protein states, is a globally attracting trajectory of the dynamics. These properties are largely preserved with respect to small perturbations to the network. These results suggest that cellular regulatory networks are robustly designed for their functions.