Structural Characterization Of Human Uch37

Uch37 is a de-ubiquitylating enzyme that is functionally linked with the 26S proteasome via Rpn13, and is essential for metazoan development. Here, we report the X-ray crystal structure of full-length human Uch37 at 2.95 Å resolution. Uch37\u27s catalytic domain is similar to those of all UCH enzymes characterized to date. The C-terminal extension is elongated, predominantly helical and contains coiled coil interactions. Additionally, we provide an initial characterization of Uch37\u27s oligomeric state and identify a systematic error in previous analyses of Uch37 activity. Taken together, these data provide a strong foundation for further analysis of Uch37\u27s several functions

Proteins

Uch37 is a de-ubiquitylating enzyme that is functionally linked with the 26S proteasome via Rpn13, and is essential for metazoan development. Here, we report the X-ray crystal structure of full-length human Uch37 at 2.95 \uc5 resolution. Uch37's catalytic domain is similar to those of all UCH enzymes characterized to date. The C-terminal extension is elongated, predominantly helical and contains coiled coil interactions. Additionally, we provide an initial characterization of Uch37's oligomeric state and identify a systematic error in previous analyses of Uch37 activity. Taken together, these data provide a strong foundation for further analysis of Uch37's several functions.T15 LM007359-06/LM/NLM NIH HHS/United StatesR01 LM008796-04/LM/NLM NIH HHS/United StatesS10 RR013790/RR/NCRR NIH HHS/United StatesGM074901/GM/NIGMS NIH HHS/United StatesU54 GM074901-03/GM/NIGMS NIH HHS/United StatesT15 LM007359-06S2/LM/NLM NIH HHS/United StatesT15 LM007359-09/LM/NLM NIH HHS/United StatesS10 RR13790/RR/NCRR NIH HHS/United StatesT15 LM007359-09S1/LM/NLM NIH HHS/United StatesR01 LM008796-03/LM/NLM NIH HHS/United StatesT15-LM007359/LM/NLM NIH HHS/United StatesT15 LM007359/LM/NLM NIH HHS/United StatesU54 GM074901-02S1/GM/NIGMS NIH HHS/United StatesT15 LM007359-08/LM/NLM NIH HHS/United StatesT15 LM007359-06S1/LM/NLM NIH HHS/United StatesT15 LM007359-07/LM/NLM NIH HHS/United StatesU54 GM074901-05/GM/NIGMS NIH HHS/United StatesDE-AC02-06CH11357/CH/OID CDC HHS/United StatesR01 LM008796-02/LM/NLM NIH HHS/United StatesU54 GM074901-01/GM/NIGMS NIH HHS/United StatesU54 GM074901-01S1/GM/NIGMS NIH HHS/United StatesT15 LM007359-10/LM/NLM NIH HHS/United StatesU54 GM074901-02/GM/NIGMS NIH HHS/United StatesR01-LM008796/LM/NLM NIH HHS/United StatesU54 GM074901-03S1/GM/NIGMS NIH HHS/United StatesR01 LM008796-01/LM/NLM NIH HHS/United StatesR01 LM008796/LM/NLM NIH HHS/United StatesU54 GM074901/GM/NIGMS NIH HHS/United StatesU54 GM074901-04/GM/NIGMS NIH HHS/United States2013-02-02T00:00:00Z21953935PMC3251636vault:729

Creating Protein Models from Electron-Density Maps using Particle-Filtering Methods

Motivation: One bottleneck in high-throughput protein crystallography is interpreting an electron-density map; that is, fitting a molecular model to the 3D picture crystallography produces. Previously, we developed ACMI, an algorithm that uses a probabilistic model to infer an accurate protein backbone layout. Here we use a sampling method known as particle filtering to produce a set of all-atom protein models. We use the output of ACMI to guide the particle filter’s sampling, producing an accurate, physically feasible set of structures. Results: We test our algorithm on ten poor-quality experimental density maps. We show that particle filtering produces accurate allatom models, resulting in fewer chains, lower sidechain RMS error, and reduced R factor, compared to simply placing the best-matching sidechains on ACMI’s trace. We show that our approach produces a more accurate model than three leading methods – TEXTAL, RESOLVE, and ARP/WARP – in terms of main chain completeness, sidechain identification, and crystallographic R factor. Availability: Source code and experimental density maps available a

What Controls the Controller: Structure and Function Characterizations of Transcription Factor PU.1 Uncover Its Regulatory Mechanism

The ETS family transcription factor PU.1/Spi-1 is a master regulator of the self-renewal of hematopoietic stem cells and their differentiation along both major lymphoid and myeloid branches. PU.1 activity is determined in a dosage-dependent manner as a function of both its expression and real-time regulation at the DNA level. While control of PU.1 expression is well established, the molecular mechanisms of its real-time regulation remain elusive. Our work is focused on discovering a complete regulatory mechanism that governs the molecular interactions of PU.1. Structurally, PU.1 exhibits a classic transcription factor architecture in which intrinsically disordered regions (IDR), consisting of 66% of its primary structure, are tethered to a well-structured DNA binding domain. The transcriptionally active form of PU.1 is a monomer that binds target DNA sites as a 1:1 complex. Our investigations show that IDRs of PU.1 reciprocally control two separate inactive dimeric forms, with and without DNA. At high concentrations, PU.1 forms a non-canonical 2:1 complex at a single DNA specific site. In the absence of DNA, PU.1 also forms a dimer, but it is incompatible with DNA binding. The DNA-free PU.1 dimer is further promoted by phosphomimetic mutants of IDR residues that are phosphorylated in B-lymphocytic activation. These results lead us to postulate a model of real-time PU.1 regulation, unknown in the ETS family, where independent dimeric forms antagonize each other to control the dosage of active PU.1 monomer at its target DNA sites. To demonstrate the biological relevance of our model, cellular assays probing PU.1-specific reporters and native target genes show that PU.1 transactivation exhibits a distinct dose response consistent with negative feedback. In summary, we have established the first model for the general real-time regulation of PU.1 at the DNA/protein level, without the need for recruiting specific binding partners. These novel interactions present potential therapeutic targets for correcting de-regulated PU.1 dosage in hematologic disorders, including leukemia, lymphoma, and myeloma

Developing a New Drug Delivery System for the Brain

Parkinson’s Disease (PD) is a neurodegenerative disease with limited, symptomatic treatment options. The gradual progression of PD is characterised by the death of dopaminergic neurons in the substantia nigra; the slow progression of PD ultimately resulting in loss of movement and co-ordination is responsible for its high socioeconomic burden. A significant barrier to PD research is the lack of animal or cell-based models for the disease which limits the development of effective treatment. The SH-SY5Y cell line is frequently used for PD research due to its catecholaminergic phenotype and ease of maintenance. SH-SY5Y cells can be differentiated to a neuronal phenotype using differentiating agents including retinoic acid (RA) and Brain Derived Neurotrophic Factor (BDNF). Cells were differentiated to a neuronal phenotype through the addition of RA and gradual Foetal Calf Serum (FCS) starvation from the cell media. The process of cell differentiation was assessed and imaged using inverted microscopy with an attached camera. The SH-SY5Y cells were successfully differentiated to a neuronal phenotype using FCS depravation and addition of RA, protocol demonstrated to be consistently reproducible producing mature, differentiated SH-SY5Y cells within 14 days. For SH-SY5Y cells to be reflective of the substantia nigra and act as an effective PD model the cells must be differentiated to a neuronal phenotype; however, many research papers do not provide information regarding the dopaminergic phenotype of the cells or explain why the cell line was selected as a model. In this research project we set out to investigate the reproducibility of SH-SY5Y cell differentiation protocols using RA, to assess mitochondrial activity of differentiated cells using cell viability assays and to confirm that differentiated SH-SY5Y cells can successfully synthesise dopamine. 3-(4,5-dimethyldiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and PrestoBlue cellular viability assays were conducted to assess cellular metabolism and enzyme activity for the cell population. The ability of differentiated cells to produce dopamine was assessed by dopamine ELISA. The results of the MTT and PrestoBlue cell viability assays confirmed that undifferentiated and differentiated SH-SY5Y cells are metabolically active, however fully differentiated cells proliferate at a reduced rate, approximately one fifth of that of their undifferentiated counterparts, indicating that differentiation is stressful to cells and can result in cell death. ELISA results demonstrate that both undifferentiated and differentiated SH-SY5Y cells synthesise dopamine with the production being significantly upregulated, by an average of six-fold, after differentiation. The findings of this investigation demonstrate that SH-SY5Y cells can be used as an effective model for PD, providing a suitable differentiation protocol has been used to drive the cells towards a neuronal, dopaminergic phenotype. However, due to a gap in the literature the level of dopamine production by differentiated SH-SY5Y cells cannot be compared against in vivo neurons found in the substantia nigra. Results indicate that SH�SY5Y cells, differentiated by FCS deprivation and addition of RA, can be used as a PD model however the dopaminergic phenotype of cells should be assessed on a batch-by-batch basis to ensure that cells continue to act as an effective mod