Monoubiquitination Is Critical for Ovarian Tumor Domain-containing Ubiquitin Aldehyde Binding Protein 1 (Otub1) to Suppress UbcH5 Enzyme and Stabilize p53 Protein

Ovarian tumor domain-containing ubiquitin (Ub) aldehyde binding protein 1 (Otub1) regulates p53 stability and activity via non-canonical inhibition of the MDM2 cognate Ub-conjugating enzyme (E2) UbcH5. However, it is not clear how this activity of Otub1 is regulated in cells. Here we report that Otub1 is monoubiquitinated by UbcH5 in cells and in vitro, primarily at the lysine 59 and 109 residues. This monoubiquitination, in turn, contributes to the activity of Otub1 to suppress UbcH5. The lysine-free Otub1 mutant (Otub1^K0) fails to be monoubiquitinated and is unable to suppress the Ub-conjugating activity of UbcH5 in vitro and the MDM2-mediated p53 ubiquitination in cells. Consistently, this mutant is unable to stabilize p53, induce apoptosis, and suppress cell proliferation. Overexpression of Otub1^K0 inhibits DNA-damage induced apoptosis. Adding either Lys-59 or Lys-109 back to the Otub1^K0 mutant restores the monoubiquitination of Otub1 and its function to stabilize and activate p53. We further show that UbcH5 preferentially binds to the monoubiquitinated Otub1 via Ub interaction with its backside donor Ub-interacting surface, suggesting that this binding interferes with the self-assembly of Ub-charged UbcH5 (UbcH5∼Ub) conjugates, which is critical for Ub transfer. Thus, our data reveal novel insights into the Otub1 inhibition of E2 wherein monoubiquitination promotes the interaction of Otub1 with UbcH5 and the function to suppress it.     

High-throughput crystallization of membrane proteins using the lipidic bicelle method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent's inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization. Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. Bicelles have been successfully used to crystallize several membrane proteins (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer's arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.     

An interdomain binding site on HIV-1 Nef interacts with PACS-1 and PACS-2 on endosomes to down-regulate MHC-I

The human immunodeficiency virus type 1 (HIV-1) accessory protein Nef directs virus escape from immune surveillance by subverting host cell intracellular signaling and membrane traffic to down-regulate cell-surface major histocompatibility complex class I (MHC-I). The interaction of Nef with the sorting proteins PACS-1 and PACS-2 mediates key signaling and trafficking steps required for Nef-mediated MHC-I down-regulation. Little is known, however, about the molecular basis underlying the Nef-PACS interaction. Here we identify the sites on Nef and the PACS proteins required for their interaction and describe the consequences of disrupting this interaction for Nef action. A previously unidentified cargo subsite on PACS-1 and PACS-2 interacted with a bipartite site on Nef formed by the EEEE(65) acidic cluster on the N-terminal domain and W(113) in the core domain. Mutation of these sites prevented the interaction between Nef and the PACS proteins on Rab5 (PACS-2 and PACS-1)- or Rab7 (PACS-1)-positive endosomes as determined by bimolecular fluorescence complementation and caused a Nef mutant defective in PACS binding to localize to distorted endosomal compartments. Consequently, disruption of the Nef-PACS interaction repressed Nef-induced MHC-I down-regulation in peripheral blood mononuclear cells. Our results provide insight into the molecular basis of Nef action and suggest new strategies to combat HIV-1.     

Targeting of aberrant mRNAs to cytoplasmic processing bodies

In eukaryotes, a specialized pathway of mRNA degradation termed nonsense-mediated decay (NMD) functions in mRNA quality control by recognizing and degrading mRNAs with aberrant termination codons. We demonstrate that NMD in yeast targets premature termination codon (PTC)-containing mRNA to P-bodies. Upf1p is sufficient for targeting mRNAs to P-bodies, whereas Upf2p and Upf3p act, at least in part, downstream of P-body targeting to trigger decapping. The ATPase activity of Upf1p is required for NMD after the targeting of mRNAs to P-bodies. Moreover, Upf1p can target normal mRNAs to P-bodies but not promote their degradation. These observations lead us to propose a new model for NMD wherein two successive steps are used to distinguish normal and aberrant mRNAs.     

Development of a Fatal Noncompressible Truncal Hemorrhage Model with Combined Hepatic and Portal Venous Injury in Normothermic Normovolemic Swine

Noncompressible truncal hemorrhage and brain injury currently account for most early mortality of warfighters on the battlefield. There is no effective treatment for noncompressible truncal hemorrhage, other than rapid evacuation to a surgical facility. The availability of an effective field treatment for noncompressible truncal hemorrhage could increase the number of warfighters salvaged from this frequently-lethal scenario. Our intent was to develop a porcine model of noncompressible truncal hemorrhage with a ∼50% one-hour mortality so that we could develop new treatments for this difficult problem. Normovolemic normothermic domestic swine (barrows, 3 months old, 34–36 kg) underwent one of three injury types through a midline incision: 1) central stellate injury (N = 6); 2) excision of a portal vein branch distal to the main PV trunk (N = 6); or 3) hemi-transection of the left lateral lobe of the liver at its base (N = 10). The one-hour mortality of these injuries was 0, 82, and 40%, respectively; the final mean arterial pressure was 65, 24, and 30 mm Hg, respectively; and the final hemoglobin was 8.3, 2.3, and 3.6 g/dL, respectively. Hemi-transection of the left lateral lobe of the liver appeared to target our desired mortality rate better than the other injury mechanisms.     

Processing bodies require RNA for assembly and contain nontranslating mRNAs

Recent experiments have defined cytoplasmic foci, referred to as processing bodies (P-bodies), wherein mRNA decay factors are concentrated and where mRNA decay can occur. However, the physical nature of P-bodies, their relationship to translation, and possible roles of P-bodies in cellular responses remain unclear. We describe four properties of yeast P-bodies that indicate that P-bodies are dynamic structures that contain nontranslating mRNAs and function during cellular responses to stress. First, in vivo and in vitro analysis indicates that P-bodies are dependent on RNA for their formation. Second, the number and size of P-bodies vary in response to glucose deprivation, osmotic stress, exposure to ultraviolet light, and the stage of cell growth. Third, P-bodies vary with the status of the cellular translation machinery. Inhibition of translation initiation by mutations, or cellular stress, results in increased P-bodies. In contrast, inhibition of translation elongation, thereby trapping the mRNA in polysomes, leads to dissociation of P-bodies. Fourth, multiple translation factors and ribosomal proteins are lacking from P-bodies. These results suggest additional biological roles of P-bodies in addition to being sites of mRNA degradation.     

Folding pathway mediated by an intramolecular chaperone: intrinsically unstructured propeptide modulates stochastic activation of subtilisin

Several secreted proteases are synthesized with N-terminal propeptides that function as intramolecular chaperones (IMCs) and direct the folding of proteases to their native functional states. Using subtilisin E as our model system, we had earlier established that (i) release and degradation of the IMC from its complex with the protease upon completion of folding is the rate-determining step to protease maturation and, (ii) IMC of SbtE is an extremely charged, intrinsically unstructured polypeptide that adopts an alpha-beta structure only in the presence of the protease. Here, we explore the mechanism of IMC release and the intricate relationship between IMC structure and protease activation. We establish that the release of the first IMC from its protease domain is a non-deterministic event that subsequently triggers an activation cascade through trans-proteolysis. By in silico simulation of the protease maturation pathway through application of stochastic algorithms, we further analyze the sub-stages of the release step. Our work shows that modulating the structure of the IMC domain through external solvent conditions can vary both the time and randomness of protease activation. This behavior of the protease can be correlated to varying the release-rebinding equilibrium of IMC, through simulation. Thus, a delicate balance underlies IMC structure, release, and protease activation. Proteases are ubiquitous enzymes crucial for fundamental cellular processes and require deterministic activation mechanisms. Our work on SbtE establishes that through selection of an intrinsically unstructured IMC domain, nature appears to have selected for a viable deterministic handle that controls a fundamentally random event. While this outlines an important mechanism for regulation of protease activation, it also provides a unique approach to maintain industrially viable subtilisins in extremely stable states that can be activated at will.     

Identification of the Intracellular Gate for a Member of the Equilibrative Nucleoside Transporter (ENT) Family

Equilibrative nucleoside transporters of the SLC29 family play important roles in many physiological and pharmacological processes, including import of drugs for treatment of cancer, AIDS, cardiovascular, and parasitic diseases. However, no crystal structure is available for any member of this family. In previous studies we generated a computational model of the Leishmania donovani nucleoside transporter 1.1 (LdNT1.1) that captured this permease in the outward-closed conformation, and we identified the extracellular gate. In the present study we have modeled the inward-closed conformation of LdNT1.1 using the crystal structure of the Escherichia coli fucose transporter FucP and have identified four transmembrane helices whose ends close to form a predicted intracellular gate. We have tested this prediction by site-directed mutagenesis of relevant helix residues and by cross-linking of introduced cysteine pairs. The results are consistent with the predictions of the computational model and suggest that a similarly constituted gate operates in other members of the equilibrative nucleoside transporter family.