A cell's behavior is a consequence of the complex interactions between its numerous constituents, such as DNA, RNA, proteins and small molecules. Cells use signaling pathways and regulatory mechanisms to coordinate multiple processes, allowing them to respond to and adapt to an ever-changing environment. The large number of components, the degree of interconnectivity and the complex control of cellular networks are becoming evident in the integrated genomic and proteomic analyses that are emerging. It is increasingly recognized that the understanding of properties that arise from whole-cell function require integrated, theoretical descriptions of the relationships between different cellular components. Recent theoretical advances allow us to describe cellular network structure with graph concepts and have revealed organizational features shared with numerous non-biological networks. We now have the opportunity to describe quantitatively a network of hundreds or thousands of interacting components. Moreover, the observed topologies of cellular networks give us clues about their evolution and how their organization influences their function and dynamic responses.

/pdf/scale-free-networks-in-cell-biology-2kkl3wlt5r.pdf

Scale-free networks in cell biology

Gene regulatory networks have an important role in every process of life, including cell differentiation, metabolism, the cell cycle and signal transduction. By understanding the dynamics of these networks we can shed light on the mechanisms of diseases that occur when these cellular processes are dysregulated. Accurate prediction of the behaviour of regulatory networks will also speed up biotechnological projects, as such predictions are quicker and cheaper than lab experiments. Computational methods, both for supporting the development of network models and for the analysis of their functionality, have already proved to be a valuable research tool.

http://www.cs.rice.edu/~nakhleh/COMP572/Papers/ModelGeneRegNets.pdf

Modelling and analysis of gene regulatory networks

Small-molecule drug discovery has traditionally focused on occupancy of a binding site that directly affects protein function, and this approach typically precludes targeting proteins that lack such amenable sites. Furthermore, high systemic drug exposures may be needed to maintain sufficient target inhibition in vivo, increasing the risk of undesirable off-target effects. Induced protein degradation is an alternative approach that is event-driven: upon drug binding, the target protein is tagged for elimination. Emerging technologies based on proteolysis-targeting chimaeras (PROTACs) that exploit cellular quality control machinery to selectively degrade target proteins are attracting considerable attention in the pharmaceutical industry owing to the advantages they could offer over traditional small-molecule strategies. These advantages include the potential to reduce systemic drug exposure, the ability to counteract increased target protein expression that often accompanies inhibition of protein function and the potential ability to target proteins that are not currently therapeutically tractable, such as transcription factors, scaffolding and regulatory proteins.

/pdf/induced-protein-degradation-an-emerging-drug-discovery-4jbmzgyyxh.pdf

Induced protein degradation: an emerging drug discovery paradigm

Ubiquitin E3 ligases control every aspect of eukaryotic biology by promoting protein ubiquitination and degradation. At the end of a three-enzyme cascade, ubiquitin ligases mediate the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to specific substrate proteins. Early investigations of E3s of the RING (really interesting new gene) and HECT (homologous to the E6AP carboxyl terminus) types shed light on their enzymatic activities, general architectures, and substrate degron-binding modes. Recent studies have provided deeper mechanistic insights into their catalysis, activation, and regulation. In this review, we summarize the current progress in structure-function studies of ubiquitin ligases as well as exciting new discoveries of novel classes of E3s and diverse substrate recognition mechanisms. Our increased understanding of ubiquitin ligase function and regulation has provided the rationale for developing E3-targeting therapeutics for the treatment of human diseases.

Ubiquitin Ligases: Structure, Function, and Regulation

Computation underlies the organization of cells into higher-order structures, for example during development or the spatial association of bacteria in a biofilm. Each cell performs a simple computational operation, but when combined with cell-cell communication, intricate patterns emerge. Here we study this process by combining a simple genetic circuit with quorum sensing to produce more complex computations in space. We construct a simple NOR logic gate in Escherichia coli by arranging two tandem promoters that function as inputs to drive the transcription of a repressor. The repressor inactivates a promoter that serves as the output. Individual colonies of E. coli carry the same NOR gate, but the inputs and outputs are wired to different orthogonal quorum-sensing 'sender' and 'receiver' devices. The quorum molecules form the wires between gates. By arranging the colonies in different spatial configurations, all possible two-input gates are produced, including the difficult XOR and EQUALS functions. The response is strong and robust, with 5- to >300-fold changes between the 'on' and 'off' states. This work helps elucidate the design rules by which simple logic can be harnessed to produce diverse and complex calculations by rewiring communication between cells.

/pdf/robust-multicellular-computing-using-genetically-encoded-nor-308ugsbonc.pdf

Robust multicellular computing using genetically encoded NOR gates and chemical ‘wires’

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

/pdf/the-yeast-cell-cycle-network-is-robustly-designed-1cisbowd6o.pdf

The yeast cell-cycle network is robustly designed.

The proteasome is an ATP-dependent, 2.5-megadalton machine responsible for selective protein degradation in eukaryotic cells. Here we present cryo-EM structures of the substrate-engaged human proteasome in seven conformational states at 2.8-3.6 A resolution, captured during breakdown of a polyubiquitylated protein. These structures visualize a continuum of dynamic substrate-proteasome interactions from ubiquitin recognition to substrate translocation, during which ATP hydrolysis sequentially navigates through all six ATPases. Three principal modes of coordinated hydrolysis are observed, featuring hydrolytic events in two oppositely positioned ATPases, in two adjacent ATPases, and in one ATPase at a time. These hydrolytic modes regulate deubiquitylation, translocation initiation and processive unfolding of substrates, respectively. ATP hydrolysis powers a hinge-like motion in each ATPase that regulates its substrate interaction. Synchronization of ATP binding, ADP release and ATP hydrolysis in three adjacent ATPases drives rigid-body rotations of substrate-bound ATPases that are propagated unidirectionally in the ATPase ring and unfold the substrate.

/pdf/cryo-em-structures-and-dynamics-of-substrate-engaged-human-205w1zw82r.pdf

Cryo-EM structures and dynamics of substrate-engaged human 26S proteasome.

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.

https://science.sciencemag.org/content/sci/348/6231/1250834.full.pdf

Substrate degradation by the proteasome: A single-molecule kinetic analysis

USP14 is a major regulator of the proteasome and one of three proteasome-associated deubiquitinating enzymes. 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.

/pdf/usp14-deubiquitinates-proteasome-bound-substrates-that-are-79aqsrjc0n.pdf

USP14 deubiquitinates proteasome-bound substrates that are ubiquitinated at multiple sites

The proteasome is the major engine of protein degradation in all eukaryotic cells. At the heart of this machine is a heterohexameric ring of AAA (ATPases associated with diverse cellular activities) proteins that unfolds ubiquitylated target proteins that are concurrently translocated into a proteolytic chamber and degraded into peptides. Using cryoelectron microscopy, we determined a near–atomic-resolution structure of the 2.5-MDa human proteasome in its ground state, as well as subnanometer-resolution structures of the holoenzyme in three alternative conformational states. The substrate-unfolding AAA-ATPase channel is narrowed by 10 inward-facing pore loops arranged into two helices that run in parallel with each other, one hydrophobic in character and the other highly charged. The gate of the core particle was unexpectedly found closed in the ground state and open in only one of the alternative states. Coordinated, stepwise conformational changes of the regulatory particle couple ATP hydrolysis to substrate translocation and regulate gating of the core particle, leading to processive degradation.

Ying Lu

Papers

The yeast cell-cycle network is robustly designed.

Cryo-EM structures and dynamics of substrate-engaged human 26S proteasome.

Substrate degradation by the proteasome: A single-molecule kinetic analysis

USP14 deubiquitinates proteasome-bound substrates that are ubiquitinated at multiple sites

Structural basis for dynamic regulation of the human 26S proteasome.