S. pombe Uba1-Ubc15 Structure Reveals a Novel Regulatory Mechanism of Ubiquitin E2 Activity.

Ubiquitin (Ub) E1 initiates the Ub conjugation cascade by activating and transferring Ub to tens of different E2s. How Ub E1 cooperates with E2s that differ substantially in their predicted E1-interacting residues is unknown. Here, we report the structure of S. pombe Uba1 in complex with Ubc15, a Ub E2 with intrinsically low E1-E2 Ub thioester transfer activity. The structure reveals a distinct Ubc15 binding mode that substantially alters the network of interactions at the E1-E2 interface compared to the only other available Ub E1-E2 structure. Structure-function analysis reveals that the intrinsically low activity of Ubc15 largely results from the presence of an acidic residue at its N-terminal region. Notably, Ub E2 N termini are serine/threonine rich in many other Ub E2s, leading us to hypothesize that phosphorylation of these sites may serve as a novel negative regulatory mechanism of Ub E2 activity, which we demonstrate biochemically and in cell-based assays.

Crystal structure of a human ubiquitin E1-ubiquitin complex reveals conserved functional elements essential for activity.

Ubiquitin (Ub) signaling plays a key regulatory role in nearly every aspect of eukaryotic biology and is initiated by E1 enzymes that activate and transfer Ub to E2 Ub-conjugating enzymes. Despite Ub E1's fundamental importance to the cell and its attractiveness as a target for therapeutic intervention in cancer and other diseases, its only available structural information is derived from yeast orthologs of human ubiquitin-like modifier-activating enzyme 1 (hUBA1). To illuminate structural differences between yeast and hUBA1 structures that might be exploited for the development of small-molecule therapeutics, we determined the first crystal structure of a hUBA1-Ub complex. Using structural analysis, molecular modeling, and biochemical analysis, we demonstrate that hUBA1 shares a conserved overall structure and mechanism with previously characterized yeast orthologs, but displays subtle structural differences, particularly within the active site. Computational analysis revealed four potential ligand-binding hot spots on the surface of hUBA1 that might serve as targets to inhibit hUBA1 at the level of Ub activation or E2 recruitment or that might potentially be used in approaches such as protein-targeting chimeric molecules. Taken together, our work enhances our understanding of the hUBA1 mechanism, provides an improved framework for the development of small-molecule inhibitors of UBA1, and serves as a stepping stone for structural studies that involve the enzymes of the human Ub system at the level of both E1 and E2.

Interleukin-12 enhances the function and anti-tumor activity in murine and human CD8(+) T cells.

Mouse CD8(+) T cells conditioned with interleukin (IL)-12 ex vivo mediate the potent regression of established melanoma when transferred into lymphodepleted mice. However, the quantitative and qualitative changes induced by IL-12 in the responding mouse CD8(+) T cells have not been well defined. Moreover, the mechanisms by which IL-12-conditioning impacts human CD8(+) T cells, and how such cells might be expanded prior to infusion into patients is not known. We found that ex vivo IL-12-conditioning of mouse CD8(+) T cells led to a tenfold-100-fold increase in persistence and anti-tumor efficacy upon adoptive transfer into lymphodepleted mice. The enhancing effect of IL-12 was associated with maintenance of functional avidity. Importantly, in the context of ongoing ACT clinical trials, human CD8(+) T cells genetically modified with a tyrosinase-specific T cell receptor (TCR) exhibited significantly enhanced functional activity when conditioned with IL-12 as indicated by heightened granzyme B expression and elevated peptide-specific CD107a degranulation. This effect was sustainable despite the 20 days of in vitro cellular expansion required to expand cells over 1,000-fold allowing adequate cell numbers for administration to cancer patients. Overall, these findings support the efficacy and feasibility of ex vivo IL-12-conditioning of TCR-modified human CD8(+) T cells for adoptive transfer and cancer therapy.

Fluorescent silica particles for monitoring oxygen levels in three-dimensional heterogeneous cellular structures.

Bacterial biofilms are a major obstacle challenging the development of more effective therapies to treat implant infections. Oxygen availability to bacterial cells has been implicated in biofilm formation and planktonic cell detachment; however, there are insufficient tools available to measure oxygen concentrations within complex three-dimensional structures with ∼ 1 µm resolution. Such measurements may complement measures of biofilm structure and cell activity to provide a more comprehensive understanding of biofilm biology. Thus, we developed oxygen-sensing microparticles specifically designed to characterize oxygen transport through the volume of bacterial biofilms. The Stöber method was used to synthesize monodisperse silica microparticles of approximately the same size as a bacterium (∼ 1 µm). Two fluorophores, oxygen-sensitive Ru(Ph(2) phen(3))Cl(2), and the reference fluorophore Nile blue chloride were immobilized on the surface of the particles. We demonstrate application of the microparticles toward measuring the oxygen concentration profiles within a live Staphylococcus aureus biofilm.

Crystal structures reveal catalytic and regulatory mechanisms of the dual-specificity ubiquitin/FAT10 E1 enzyme Uba6.

The E1 enzyme Uba6 initiates signal transduction by activating ubiquitin and the ubiquitin-like protein FAT10 in a two-step process involving sequential catalysis of adenylation and thioester bond formation. To gain mechanistic insights into these processes, we determined the crystal structure of a human Uba6/ubiquitin complex. Two distinct architectures of the complex are observed: one in which Uba6 adopts an open conformation with the active site configured for catalysis of adenylation, and a second drastically different closed conformation in which the adenylation active site is disassembled and reconfigured for catalysis of thioester bond formation. Surprisingly, an inositol hexakisphosphate (InsP6) molecule binds to a previously unidentified allosteric site on Uba6. Our structural, biochemical, and biophysical data indicate that InsP6 allosterically inhibits Uba6 activity by altering interconversion of the open and closed conformations of Uba6 while also enhancing its stability. In addition to revealing the molecular mechanisms of catalysis by Uba6 and allosteric regulation of its activities, our structures provide a framework for developing Uba6-specific inhibitors and raise the possibility of allosteric regulation of other E1s by naturally occurring cellular metabolites.

Effects of steroids and angiotensin converting enzyme inhibition on circumferential strain in boys with Duchenne muscular dystrophy: a cross-sectional and longitudinal study utilizing cardiovascular magnetic resonance.

BACKGROUND: Steroid use has prolonged ambulation in Duchenne muscular dystrophy (DMD) and combined with advances in respiratory care overall management has improved such that cardiac manifestations have become the major cause of death. Unfortunately, there is no consensus for DMD-associated cardiac disease management. Our purpose was to assess effects of steroid use alone or in combination with angiotensin converting enzyme inhibitors (ACEI) or angiotension receptor blocker (ARB) on cardiovascular magnetic resonance (CMR) derived circumferential strain (εcc). METHODS: We used CMR to assess effects of corticosteroids alone (Group A) or in combination with ACEI or ARB (Group B) on heart rate (HR), left ventricular ejection fraction (LVEF), mass (LVM), end diastolic volume (LVEDV) and circumferential strain (εcc) in a cohort of 171 DMD patients >5 years of age. Treatment decisions were made independently by physicians at both our institution and referral centers and not based on CMR results. RESULTS: Patients in Group A (114 studies) were younger than those in Group B (92 studies)(10 ± 2.4 vs. 12.4 ± 3.2 years, p < 0.0001), but HR, LVEF, LVEDV and LVM were not different. Although εcc magnitude was lower in Group B than Group A (-13.8 ± 1.9 vs. -12.8 ± 2.0, p = 0.0004), age correction using covariance analysis eliminated this effect. In a subset of patients who underwent serial CMR exams with an inter-study time of ~15 months, εcc worsened regardless of treatment group. CONCLUSIONS: These results support the need for prospective clinical trials to identify more effective treatment regimens for DMD associated cardiac disease.

Structural insights into E1 recognition and the ubiquitin-conjugating activity of the E2 enzyme Cdc34.

Ubiquitin (Ub) signaling requires the sequential interactions and activities of three enzymes, E1, E2, and E3. Cdc34 is an E2 that plays a key role in regulating cell cycle progression and requires unique structural elements to function. The molecular basis by which Cdc34 engages its E1 and the structural mechanisms by which its unique C-terminal extension functions in Cdc34 activity are unknown. Here, we present crystal structures of Cdc34 alone and in complex with E1, and a Cdc34~Ub thioester mimetic that represents the product of Uba1-Cdc34 Ub transthiolation. These structures reveal conformational changes in Uba1 and Cdc34 and a unique binding mode that are required for transthiolation. The Cdc34~Ub structure reveals contacts between the Cdc34 C-terminal extension and Ub that stabilize Cdc34~Ub in a closed conformation and are critical for Ub discharge. Altogether, our structural, biochemical, and cell-based studies provide insights into the molecular mechanisms by which Cdc34 function in cells.

Molecular mechanism of a covalent allosteric inhibitor of SUMO E1 activating enzyme.

E1 enzymes activate ubiquitin (Ub) and ubiquitin-like modifiers (Ubls) in the first step of Ub/Ubl conjugation cascades and represent potential targets for therapeutic intervention in cancer and other life-threatening diseases. Here, we report the crystal structure of the E1 enzyme for the Ubl SUMO in complex with a recently discovered and highly specific covalent allosteric inhibitor (COH000). The structure reveals that COH000 targets a cryptic pocket distinct from the active site that is completely buried in all previous SUMO E1 structures and that COH000 binding to SUMO E1 is accompanied by a network of structural changes that altogether lock the enzyme in a previously unobserved inactive conformation. These structural changes include disassembly of the active site and a 180° rotation of the catalytic cysteine-containing SCCH domain, relative to conformational snapshots of SUMO E1 poised to catalyze adenylation. Altogether, our study provides a molecular basis for the inhibitory mechanism of COH000 and its SUMO E1 specificity, and also establishes a framework for potential development of molecules targeting E1 enzymes for other Ubls at a cryptic allosteric site.

Meeting radiation therapy patients informational needs through educational videos augmented by 3D visualisation software.

INTRODUCTION: Misconceptions and uncertainties about radiotherapy compound the anxiety patients experience at the commencement of treatment. This project investigated the utility of locally produced treatment process videos in meeting patients' informational needs. METHODS: In-house video production was conducted on a voluntary basis by staff and patients at a regional Australian radiotherapy centre. Videos included real footage and animated sections created with PEARLTM 3D visualisation software (Vertual Ltd, UK) to meet specific key content objectives. Quantitative cross sectional analysis was conducted. Patients attending for simulation watched a relevant video. After their first fraction of radiotherapy they were asked to complete an ethics-reviewed questionnaire about how well the video addressed their information needs. RESULTS: The survey completion rate was 29% (n = 61/212). Surveys were collected over 9 months from August 2014 to April 2015. Statistical analysis found 98% of patients reported that the video was useful in meeting one or more of the learning objectives. Forty-nine percent of patients also reported a reduction in fear and anxiety as a result of watching the video. Patients reported subsequent review of videos at home (39%), primarily to explain treatment processes to loved ones (46%). CONCLUSION: The combination of real footage and 3D visualisation software assisted in meeting learning objectives regarding the treatment process. Standardised videos provided consistency of information provision to patients and facilitated multiple viewings of the video if desired.

Clients and Oncogenic Roles of Molecular Chaperone gp96/grp94.

As an endoplasmic reticulum heat shock protein (HSP) 90 paralogue, glycoprotein (gp) 96 possesses immunological properties by chaperoning antigenic peptides for activation of T cells. Genetic studies in the last decade have unveiled that gp96 is also an essential master chaperone for multiple receptors and secreting proteins including Toll-like receptors (TLRs), integrins, the Wnt coreceptor, Low Density Lipoprotein Receptor-Related Protein 6 (LRP6), the latent TGFß docking receptor, Glycoprotein A Repetitions Predominant (GARP), Glycoprotein (GP) Ib and insulin-like growth factors (IGF). Clinically, elevated expression of gp96 in a variety of cancers correlates with the advanced stage and poor survival of cancer patients. Recent preclinical studies have also uncovered that gp96 expression is closely linked to cancer progression in multiple myeloma, hepatocellular carcinoma, breast cancer and inflammation-associated colon cancer. Thus, gp96 is an attractive therapeutic target for cancer treatment. The chaperone function of gp96 depends on its ATPase domain, which is structurally distinct from other HSP90 members, and thus favors the design of highly selective gp96-targeted inhibitors against cancer. We herein discuss the strategically important oncogenic clients of gp96 and their underlying biology. The roles of cell-intrinsic gp96 in T cell biology are also discussed, in part because it offers another opportunity of cancer therapy by manipulating levels of gp96 in T cells to enhance host immune defense.