PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-25 (130)
 

Clipboard (0)
None

Select a Filter Below

Journals
more »
Year of Publication
more »
1.  Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1 
The EMBO Journal  2015;34(22):2840-2861.
Abstract
Mutations in the PTEN‐induced kinase 1 (PINK1) are causative of autosomal recessive Parkinson's disease (PD). We have previously reported that PINK1 is activated by mitochondrial depolarisation and phosphorylates serine 65 (Ser65) of the ubiquitin ligase Parkin and ubiquitin to stimulate Parkin E3 ligase activity. Here, we have employed quantitative phosphoproteomics to search for novel PINK1‐dependent phosphorylation targets in HEK (human embryonic kidney) 293 cells stimulated by mitochondrial depolarisation. This led to the identification of 14,213 phosphosites from 4,499 gene products. Whilst most phosphosites were unaffected, we strikingly observed three members of a sub‐family of Rab GTPases namely Rab8A, 8B and 13 that are all phosphorylated at the highly conserved residue of serine 111 (Ser111) in response to PINK1 activation. Using phospho‐specific antibodies raised against Ser111 of each of the Rabs, we demonstrate that Rab Ser111 phosphorylation occurs specifically in response to PINK1 activation and is abolished in HeLa PINK1 knockout cells and mutant PINK1 PD patient‐derived fibroblasts stimulated by mitochondrial depolarisation. We provide evidence that Rab8A GTPase Ser111 phosphorylation is not directly regulated by PINK1 in vitro and demonstrate in cells the time course of Ser111 phosphorylation of Rab8A, 8B and 13 is markedly delayed compared to phosphorylation of Parkin at Ser65. We further show mechanistically that phosphorylation at Ser111 significantly impairs Rab8A activation by its cognate guanine nucleotide exchange factor (GEF), Rabin8 (by using the Ser111Glu phosphorylation mimic). These findings provide the first evidence that PINK1 is able to regulate the phosphorylation of Rab GTPases and indicate that monitoring phosphorylation of Rab8A/8B/13 at Ser111 may represent novel biomarkers of PINK1 activity in vivo. Our findings also suggest that disruption of Rab GTPase‐mediated signalling may represent a major mechanism in the neurodegenerative cascade of Parkinson's disease.
doi:10.15252/embj.201591593
PMCID: PMC4654935  PMID: 26471730
Parkinson's disease; phosphoproteomics; PINK1; Rab GTPases; Membrane & Intracellular Transport; Methods & Resources; Post-translational Modifications, Proteolysis & Proteomics
2.  mTOR activates the VPS34–UVRAG complex to regulate autolysosomal tubulation and cell survival 
The EMBO Journal  2015;34(17):2272-2290.
Lysosomes are essential organelles that function to degrade and recycle unwanted, damaged and toxic biological components. Lysosomes also act as signalling platforms in activating the nutrient-sensing kinase mTOR. mTOR regulates cellular growth, but it also helps to maintain lysosome identity by initiating lysosomal tubulation through a process termed autophagosome-lysosome reformation (ALR). Here we identify a lysosomal pool of phosphatidylinositol 3-phosphate that, when depleted by specific inhibition of the class III phosphoinositide 3-kinase VPS34, results in prolonged lysosomal tubulation. This tubulation requires mTOR activity, and we identified two direct mTOR phosphorylation sites on UVRAG (S550 and S571) that activate VPS34. Loss of these phosphorylation sites reduced VPS34 lipid kinase activity and resulted in an increase in number and length of lysosomal tubules. In cells in which phosphorylation at these UVRAG sites is disrupted, the result of impaired lysosomal tubulation alongside ALR activation is massive cell death. Our data imply that ALR is critical for cell survival under nutrient stress and that VPS34 is an essential regulatory element in this process.
doi:10.15252/embj.201590992
PMCID: PMC4585463  PMID: 26139536
lysosome; mTOR; tubule; UVRAG; VPS34
3.  Primary care physicians’ perceived barriers and facilitators to conservative care for older adults with chronic kidney disease: design of a mixed methods study 
Background
Guideline committees have identified the need for research to inform the provision of conservative care for older adults with stage 5 chronic kidney disease (CKD) who have a high burden of comorbidity or functional impairment. We will use both qualitative and quantitative methodologies to provide a comprehensive understanding of barriers and facilitators to care for these patients in primary care.
Objectives
Our objectives are to (1) interview primary care physicians to determine their perspectives of conservative care for older adults with stage 5 CKD and (2) survey primary care physicians to determine the prevalence of key barriers and facilitators to provision of conservative care for older adults with stage 5 CKD.
Design
A sequential exploratory mixed methods design was adopted for this study. The first phase of the study will involve fundamental qualitative description and the second phase will be a cross-sectional population-based survey.
Setting
The research is conducted in Alberta, Canada.
Participants
The participants are primary care physicians with experience in providing care for older adults with stage 5 CKD not planning on initiating dialysis.
Methods
The first objective will be achieved by undertaking interviews with primary care physicians from southern Alberta. Participants will be selected purposively to include physicians with a range of characteristics (e.g., age, gender, and location of clinical practice). Interviews will be recorded, transcribed verbatim, and analyzed using conventional content analysis to generate themes. The second objective will be achieved by undertaking a population-based survey of primary care physicians in Alberta. The questionnaire will be developed based on the findings from the qualitative interviews and pilot tested for face and content validity. Physicians will be provided multiple options to complete the questionnaire including mail, fax, and online methods. Descriptive statistics and associations between demographic factors and barriers and facilitators to care will be analyzed using regression models.
Limitations
A potential limitation of this mixed methods study is its cross-sectional nature.
Conclusions
This work will inform development of clinical resources and tools for care of older adults with stage 5 CKD, to address barriers and enable facilitators to community-based conservative care.
Electronic supplementary material
The online version of this article (doi:10.1186/s40697-016-0110-0) contains supplementary material, which is available to authorized users.
doi:10.1186/s40697-016-0110-0
PMCID: PMC4819283  PMID: 27047667
Chronic kidney disease; Conservative care; Mixed methods; Non-dialysis care; Older adults; Primary care physicians
4.  The State of the Human Proteome in 2014/2015 as viewed through PeptideAtlas: enhancing accuracy and coverage through the AtlasProphet 
Journal of proteome research  2015;14(9):3461-3473.
The Human PeptideAtlas is a compendium of the highest quality peptide identifications from over 1000 shotgun mass spectrometry proteomics experiments collected from many different labs, all reanalyzed through a uniform processing pipeline. The latest 2015-03 build contains substantially more input data than past releases, is mapped to a recent version of our merged reference proteome, and uses improved informatics processing and the development of the AtlasProphet to provide the highest quality results. Within the set of ~20,000 neXtProt primary entries, 14,070 (70%) are confidently detected in the latest build, 5% are ambiguous, 9% are redundant, leaving the total percentage of proteins for which there are no mapping detections at just 16% (3166), all derived from over 133 million peptide-spectrum matches identifying more than 1 million distinct peptides using AtlasProphet to characterize and classify the protein matches. Improved handling for detection and presentation of single amino-acid variants (SAAVs) reveals the detection of 5,326 uniquely mapping SAAVs across 2,794 proteins. With such a large amount of data, the control of false positives is a challenge. We present the methodology and results for maintaining rigorous quality, along with a discussion of the implications of the remaining sources of errors in the build. We check our uncertainty estimates against a set of olfactory receptor proteins not expected to be present in the set. We show how the use of synthetic reference spectra can provide confirmatory evidence for claims of detection of proteins with weak evidence.
doi:10.1021/acs.jproteome.5b00500
PMCID: PMC4755269  PMID: 26139527
shotgun proteomics; tandem mass spectrometry; repositories; PeptideAtlas; Human Proteome Project; observed proteome
5.  Computer-assisted total knee arthroplasty using mini midvastus or medial parapatellar approach technique 
Background
Despite the growing evidence in the literature there is still a lack of consensus regarding the use of minimally invasive surgical technique (MIS) in total knee arthroplasty (TKA).
Methods
A prospective, randomized, international multicentre trial including 69 patients was performed to compare computer-assisted TKA (CAS-TKA) using either mini-midvastus (MIS group) or standard medial parapatellar approach (conventional group).
Patients from 3 centers (Maastricht, Zwickau, Adelaide) with end-stage osteoarthritis of the knee were randomized to either an MIS group with dedicated instrumentation or a conventional group to receive cruciate retaining CAS-TKA without patella resurfacing. The primary outcome was to compare post operative pain and range of motion (ROM). The secondary outcome was to measure the duration of surgery, blood loss, chair rise test, quadriceps strength, anterior knee pain, Knee Society Score (KSS),WOMAC scores, mechanical leg axis and component alignment.
Results
Patients in the MIS group (3.97 ± 2.16) had significant more pain at 2 weeks than patients in the conventional group (2.77 ± 1.43) p = 0.003. There was no significant difference in any of the other primary outcome parameters. Surgery time was significantly longer (p < 0.001) and there were significantly higher blood loss (p = 0.002) in the MIS group as compared to the conventional group. The difference of the mean mechanical leg alignment between the groups was not statistically significant (–0.43° (95 % CI –1.50 – 0.64); p = 0.43).
There was no significant difference of component alignment between the two surgical groups with respect to flexion/extension (p = 0.269), varus/valgus (p = 0.653) or rotational alignment (p = 0.485) of the femur component and varus valgus alignment (p = 0.778) or posterior slope (p = 0.164) of the tibial component.
Conclusion
There was no advantage of the MIS approach compared to a conventional approach CAS-TKA in any of the primary outcome measurements assessed, however the MIS approach was associated with longer surgical time and greater blood loss. MIS-TKA in combination with computer navigation is safe in terms of implant positioning.
Trial registration number
ClinicalTrials.gov NCT02625311 8 December 2015
doi:10.1186/s12891-016-0872-7
PMCID: PMC4711101  PMID: 26762175
Total knee arthroplasty; Navigation; Minimally invasive surgery; Blood loss; Accuracy
6.  Spliced leader RNA trans-splicing discovered in copepods 
Scientific Reports  2015;5:17411.
Copepods are one of the most abundant metazoans in the marine ecosystem, constituting a critical link in aquatic food webs and contributing significantly to the global carbon budget, yet molecular mechanisms of their gene expression are not well understood. Here we report the detection of spliced leader (SL) trans-splicing in calanoid copepods. We have examined nine species of wild-caught copepods from Jiaozhou Bay, China that represent the major families of the calanoids. All these species contained a common 46-nt SL (CopepodSL). We further determined the size of CopepodSL precursor RNA (slRNA; 108-158 nt) through genomic analysis and 3′-RACE technique, which was confirmed by RNA blot analysis. Structure modeling showed that the copepod slRNA folded into typical slRNA secondary structures. Using a CopepodSL-based primer set, we selectively enriched and sequenced copepod full-length cDNAs, which led to the characterization of copepod transcripts and the cataloging of the complete set of 79 eukaryotic cytoplasmic ribosomal proteins (cRPs) for a single copepod species. We uncovered the SL trans-splicing in copepod natural populations, and demonstrated that CopepodSL was a sensitive and specific tool for copepod transcriptomic studies at both the individual and population levels and that it would be useful for metatranscriptomic analysis of copepods.
doi:10.1038/srep17411
PMCID: PMC4664967  PMID: 26621068
7.  mTOR activates the VPS34–UVRAG complex to regulate autolysosomal tubulation and cell survival 
The EMBO Journal  2015;34(17):2272-2290.
Lysosomes are essential organelles that function to degrade and recycle unwanted, damaged and toxic biological components. Lysosomes also act as signalling platforms in activating the nutrient-sensing kinase mTOR. mTOR regulates cellular growth, but it also helps to maintain lysosome identity by initiating lysosomal tubulation through a process termed autophagosome-lysosome reformation (ALR). Here we identify a lysosomal pool of phosphatidylinositol 3-phosphate that, when depleted by specific inhibition of the class III phosphoinositide 3-kinase VPS34, results in prolonged lysosomal tubulation. This tubulation requires mTOR activity, and we identified two direct mTOR phosphorylation sites on UVRAG (S550 and S571) that activate VPS34. Loss of these phosphorylation sites reduced VPS34 lipid kinase activity and resulted in an increase in number and length of lysosomal tubules. In cells in which phosphorylation at these UVRAG sites is disrupted, the result of impaired lysosomal tubulation alongside ALR activation is massive cell death. Our data imply that ALR is critical for cell survival under nutrient stress and that VPS34 is an essential regulatory element in this process.
doi:10.15252/embj.201590992
PMCID: PMC4585463  PMID: 26139536
lysosome; mTOR; tubule; UVRAG; VPS34
8.  Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1 
The EMBO Journal  2015;34(22):2840-2861.
Mutations in the PTEN-induced kinase 1 (PINK1) are causative of autosomal recessive Parkinson's disease (PD). We have previously reported that PINK1 is activated by mitochondrial depolarisation and phosphorylates serine 65 (Ser65) of the ubiquitin ligase Parkin and ubiquitin to stimulate Parkin E3 ligase activity. Here, we have employed quantitative phosphoproteomics to search for novel PINK1-dependent phosphorylation targets in HEK (human embryonic kidney) 293 cells stimulated by mitochondrial depolarisation. This led to the identification of 14,213 phosphosites from 4,499 gene products. Whilst most phosphosites were unaffected, we strikingly observed three members of a sub-family of Rab GTPases namely Rab8A, 8B and 13 that are all phosphorylated at the highly conserved residue of serine 111 (Ser111) in response to PINK1 activation. Using phospho-specific antibodies raised against Ser111 of each of the Rabs, we demonstrate that Rab Ser111 phosphorylation occurs specifically in response to PINK1 activation and is abolished in HeLa PINK1 knockout cells and mutant PINK1 PD patient-derived fibroblasts stimulated by mitochondrial depolarisation. We provide evidence that Rab8A GTPase Ser111 phosphorylation is not directly regulated by PINK1 in vitro and demonstrate in cells the time course of Ser111 phosphorylation of Rab8A, 8B and 13 is markedly delayed compared to phosphorylation of Parkin at Ser65. We further show mechanistically that phosphorylation at Ser111 significantly impairs Rab8A activation by its cognate guanine nucleotide exchange factor (GEF), Rabin8 (by using the Ser111Glu phosphorylation mimic). These findings provide the first evidence that PINK1 is able to regulate the phosphorylation of Rab GTPases and indicate that monitoring phosphorylation of Rab8A/8B/13 at Ser111 may represent novel biomarkers of PINK1 activity in vivo. Our findings also suggest that disruption of Rab GTPase-mediated signalling may represent a major mechanism in the neurodegenerative cascade of Parkinson's disease.
doi:10.15252/embj.201591593
PMCID: PMC4654935  PMID: 26471730
Parkinson's disease; phosphoproteomics; PINK1; Rab GTPases
9.  Primary healthcare needs and barriers to care among Calgary’s homeless populations 
BMC Family Practice  2015;16:139.
Background
Despite Canada’s universal healthcare system, significant barriers impede individuals experiencing homelessness from accessing health services. Furthermore, there is a paucity in the qualitative literature describing how Canadians experiencing homelessness access health care services. Our objective was to qualitatively explore perceived healthcare needs and barriers among individuals experiencing homelessness in one large Canadian city – Calgary, Alberta.
Methods
We conducted a qualitative descriptive study that included open-ended interviews and focus groups with a variety of stakeholders who are involved in healthcare among Calgary’s homeless populations. These included individuals experiencing homelessness (n = 11) as well as employees from several healthcare service providers for those experiencing homelessness (n = 11). Transcripts from these interviews were thematically analyzed by two analysts.
Results
Stakeholder interviews yielded several pervasive themes surrounding the health care needs of the homeless and barriers to accessing care. Some of the primary health care needs which were identified included mental health, addictions, and allied health as well as care that addresses the social determinants of health. Notably, it was difficult for many stakeholders to pinpoint specific health care priorities, as they identified that the health care needs among Calgary’s homeless populations are diverse and complex, often even describing the needs as overwhelming. Types of barriers to primary care that were identified by stakeholders included: emotional, educational, geographical, financial and structural barriers, as well as discrimination.
Conclusions
Our findings highlight the diverse primary health care needs of Calgary’s homeless populations. Despite the fact that Canada has a universal publicly funded health care system, individuals experiencing homelessness face significant barriers in accessing primary care.
doi:10.1186/s12875-015-0361-3
PMCID: PMC4603688  PMID: 26463577
Homeless; Poverty; Qualitative research; Needs assessment
10.  An open-source computational and data resource to analyze digital maps of immunopeptidomes 
eLife  null;4:e07661.
We present a novel mass spectrometry-based high-throughput workflow and an open-source computational and data resource to reproducibly identify and quantify HLA-associated peptides. Collectively, the resources support the generation of HLA allele-specific peptide assay libraries consisting of consensus fragment ion spectra, and the analysis of quantitative digital maps of HLA peptidomes generated from a range of biological sources by SWATH mass spectrometry (MS). This study represents the first community-based effort to develop a robust platform for the reproducible and quantitative measurement of the entire repertoire of peptides presented by HLA molecules, an essential step towards the design of efficient immunotherapies.
DOI: http://dx.doi.org/10.7554/eLife.07661.001
eLife digest
The cells of the immune system protect us by recognizing telltale molecules produced by damaged and diseased cells, or by infection-causing microorganisms (which are also called pathogens). To help with this process, the cells in our bodies display small fragments of proteins (called peptides) on their surface that are then checked by the immune cells. Collectively, these peptides are referred to as the ‘immunopeptidome’, and deciphering the complexity of the human immunopeptidome is important for both basic research and medical science. Such an achievement would help to guide the development of next-generation vaccines and therapies against autoimmune disorders, infectious diseases and cancers.
In the past, immune peptides were mostly identified using a technique that is commonly called ‘shotgun’ mass spectrometry. However, this approach doesn't always provide reproducible results. In 2012, researchers reported the development of a new approach—which they called ‘SWATH’ mass spectrometry—that could yield more reproducible data.
Now, Caron et al.—including many of the researchers involved in the 2012 study—have developed a large collection of standardized tests that use SWATH mass spectrometry to analyze the human immunopeptidome. The workflow and the computational and data resources developed as part of this international effort are the first steps toward highly reproducible and measurable analyses of the immunopeptidome across many samples. Moreover, the large repository of assays generated by the project has been made public and will serve a large community of researchers, which should enable better collaborations.
In the future, SWATH mass spectrometry could be used as a robust technology for the reproducible detection and measurement of pathogen-specific or cancer-specific immune peptides. This could greatly help in the design of personalized immune-based therapies.
DOI: http://dx.doi.org/10.7554/eLife.07661.002
doi:10.7554/eLife.07661
PMCID: PMC4507788  PMID: 26154972
human leukocytes antigen; immunopeptidome; targeted mass spectrometry; SWATH-MS; DIA; human
11.  Using PeptideAtlas, SRMAtlas and PASSEL – Comprehensive Resources for discovery and targeted proteomics 
PeptideAtlas, SRMAtlas and PASSEL are web-accessible resources to support discovery and targeted proteomics research. PeptideAtlas is a multi-species compendium of shotgun proteomic data provided by the scientific community, SRMAtlas is a resource of high-quality, complete proteome SRM assays generated in a consistent manner for the targeted identification and quantification of proteins, and PASSEL is a repository that compiles and represents selected reaction monitoring data, all in an easy to use interface. The databases are generated from native mass spectrometry data files that are analyzed in a standardized manner including statistical validation of the results. Each resource offers search functionalities and can be queried by user defined constraints; the query results are provided in tables or are graphically displayed. PeptideAtlas, SRMAtlas and PASSEL are publicly available freely via the website http://www.peptideatlas.org. In this protocol, we describe the use of these resources, we highlight how to submit, search, collate and download data.
doi:10.1002/0471250953.bi1325s46
PMCID: PMC4331073  PMID: 24939129
discovery proteomics; targeted proteomics; selected reaction monitoring (SRM); data repository; data resource; complete proteome library
12.  Development of a replication-competent lentivirus assay for dendritic cell-targeting lentiviral vectors 
It is a current regulatory requirement to demonstrate absence of detectable replication-competent lentivirus (RCL) in lentiviral vector products prior to use in clinical trials. Immune Design previously described an HIV-1-based integration-deficient lentiviral vector for use in cancer immunotherapy (VP02). VP02 is enveloped with E1001, a modified Sindbis virus glycoprotein which targets dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) expressed on dendritic cells in vivo. Vector enveloped with E1001 does not transduce T-cell lines used in standard HIV-1-based RCL assays, making current RCL testing formats unsuitable for testing VP02. We therefore developed a novel assay to test for RCL in clinical lots of VP02. This assay, which utilizes a murine leukemia positive control virus and a 293F cell line expressing the E1001 receptor DC-SIGN, meets a series of evaluation criteria defined in collaboration with US regulatory authorities and demonstrates the ability of the assay format to amplify and detect a hypothetical RCL derived from VP02 vector components. This assay was qualified and used to test six independent GMP production lots of VP02, in which no RCL was detected. We propose that the evaluation criteria used to rationally design this novel method should be considered when developing an RCL assay for any lentiviral vector.
doi:10.1038/mtm.2015.17
PMCID: PMC4445008  PMID: 26029728
13.  Small molecule inhibition of group I p21-activated kinases in breast cancer induces apoptosis and potentiates the activity of microtubule stabilizing agents 
Introduction
Breast cancer, the most common cause of cancer-related deaths worldwide among women, is a molecularly and clinically heterogeneous disease. Extensive genetic and epigenetic profiling of breast tumors has recently revealed novel putative driver genes, including p21-activated kinase (PAK)1. PAK1 is a serine/threonine kinase downstream of small GTP-binding proteins, Rac1 and Cdc42, and is an integral component of growth factor signaling networks and cellular functions fundamental to tumorigenesis.
Methods
PAK1 dysregulation (copy number gain, mRNA and protein expression) was evaluated in two cohorts of breast cancer tissues (n = 980 and 1,108). A novel small molecule inhibitor, FRAX1036, and RNA interference were used to examine PAK1 loss of function and combination with docetaxel in vitro. Mechanism of action for the therapeutic combination, both cellular and molecular, was assessed via time-lapse microscopy and immunoblotting.
Results
We demonstrate that focal genomic amplification and overexpression of PAK1 are associated with poor clinical outcome in the luminal subtype of breast cancer (P = 1.29 × 10−4 and P = 0.015, respectively). Given the role for PAK1 in regulating cytoskeletal organization, we hypothesized that combination of PAK1 inhibition with taxane treatment could be combined to further interfere with microtubule dynamics and cell survival. Consistent with this, administration of docetaxel with either a novel small molecule inhibitor of group I PAKs, FRAX1036, or PAK1 small interfering RNA oligonucleotides dramatically altered signaling to cytoskeletal-associated proteins, such as stathmin, and induced microtubule disorganization and cellular apoptosis. Live-cell imaging revealed that the duration of mitotic arrest mediated by docetaxel was significantly reduced in the presence of FRAX1036, and this was associated with increased kinetics of apoptosis.
Conclusions
Taken together, these findings further support PAK1 as a potential target in breast cancer and suggest combination with taxanes as a viable strategy to increase anti-tumor efficacy.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-015-0564-5) contains supplementary material, which is available to authorized users.
doi:10.1186/s13058-015-0564-5
PMCID: PMC4445529  PMID: 25902869
14.  K29-Selective Ubiquitin Binding Domain Reveals Structural Basis of Specificity and Heterotypic Nature of K29 Polyubiquitin 
Molecular Cell  2015;58(1):83-94.
Summary
Polyubiquitin chains regulate diverse cellular processes through the ability of ubiquitin to form chains of eight different linkage types. Although detected in yeast and mammals, little is known about K29-linked polyubiquitin. Here we report the generation of K29 chains in vitro using a ubiquitin chain-editing complex consisting of the HECT E3 ligase UBE3C and the deubiquitinase vOTU. We determined the crystal structure of K29-linked diubiquitin, which adopts an extended conformation with the hydrophobic patches on both ubiquitin moieties exposed and available for binding. Indeed, the crystal structure of the NZF1 domain of TRABID in complex with K29 chains reveals a binding mode that involves the hydrophobic patch on only one of the ubiquitin moieties and exploits the flexibility of K29 chains to achieve linkage selective binding. Further, we establish methods to study K29-linked polyubiquitin and find that K29 linkages exist in cells within mixed or branched chains containing other linkages.
Graphical Abstract
Highlights
•Large-scale enzymatic assembly and purification of K29-linked polyubiquitin chains•K29 diubiquitin adopts extended conformation in crystal structure•Crystal structure of K29 diubiquitin in complex with selective binding domain•Presence of K29 chains within mixed/branched chains containing other linkages
Kristariyanto et al. find that K29-linked ubiquitin chains are present within ubiquitin chains containing other linkage types. They describe a method to assemble K29 chains, and they characterize a protein domain that selectively binds to these chains.
doi:10.1016/j.molcel.2015.01.041
PMCID: PMC4386640  PMID: 25752573
15.  Assembly and structure of Lys33-linked polyubiquitin reveals distinct conformations 
Biochemical Journal  2015;467(Pt 2):345-352.
Ubiquitylation regulates a multitude of biological processes and this versatility stems from the ability of ubiquitin (Ub) to form topologically different polymers of eight different linkage types. Whereas some linkages have been studied in detail, other linkage types including Lys33-linked polyUb are poorly understood. In the present study, we identify an enzymatic system for the large-scale assembly of Lys33 chains by combining the HECT (homologous to the E6–AP C-terminus) E3 ligase AREL1 (apoptosis-resistant E3 Ub protein ligase 1) with linkage selective deubiquitinases (DUBs). Moreover, this first characterization of the chain selectivity of AREL1 indicates its preference for assembling Lys33- and Lys11-linked Ub chains. Intriguingly, the crystal structure of Lys33-linked diUb reveals that it adopts a compact conformation very similar to that observed for Lys11-linked diUb. In contrast, crystallographic analysis of Lys33-linked triUb reveals a more extended conformation. These two distinct conformational states of Lys33-linked polyUb may be selectively recognized by Ub-binding domains (UBD) and enzymes of the Ub system. Importantly, our work provides a method to assemble Lys33-linked polyUb that will allow further characterization of this atypical chain type.
Of the eight different polyubiquitin linkage types, very little is known about Lys33-linked polyubiquitin. Here the authors reveal that the HECT E3 ligase AREL1 assembles Lys33-linked polyubiquitin, and establish a method for large-scale assembly that enabled structural and biochemical studies.
doi:10.1042/BJ20141502
PMCID: PMC4390085  PMID: 25723849
deubiquitinase; homologous to the E6–AP C-terminus (HECT) E3 ligase; polyubiquitin; ubiquitin linkage; AREL1, apoptosis-resistant E3 ubiquitin protein ligase 1; ASU, asymmetric unit; Crn7, coronin-7; DUB, deubiquitinase; HECT, homologous to the E6–AP C-terminus; pRM, parallel reaction monitoring; PTM, post-translational modification; RBR, RING-between-RING; RING, really interesting new gene; TCR, T-cell antigen receptor; Ub, ubiquitin; UBD, ubiquitin-binding domain
16.  Wear of highly crosslinked polyethylene acetabular components 
Acta Orthopaedica  2015;86(2):159-168.
Background and purpose
Wear rates of highly crosslinked polyethylene (XLPE) acetabular components have varied considerably between different published studies. This variation is in part due to the different techniques used to measure wear and to the errors inherent in measuring the relatively low amounts of wear in XLPE bearings. We undertook a scoping review of studies that have examined the in vivo wear of XLPE acetabular components using the most sensitive method available, radiostereometric analysis (RSA).
Methods
A systematic search of the PubMed, Scopus, and Cochrane databases was performed to identify published studies in which RSA was used to measure wear of XLPE components in primary total hip arthroplasty (THA).
Results
18 publications examined 12 primary THA cohorts, comprising only 260 THAs at 2–10 years of follow-up. The mean or median proximal wear rate reported ranged from 0.00 to 0.06 mm/year. However, differences in the manner in which wear was determined made it difficult to compare some studies. Furthermore, differences in RSA methodology between studies, such as the use of supine or standing radiographs and the use of beaded or unbeaded reference segments, may limit future meta-analyses examining the effect of patient and implant variables on wear rates.
Interpretation
This scoping review confirmed the low wear rates of XLPE in THA, as measured by RSA. We make recommendations to enhance the standardization of reporting of RSA wear results, which will facilitate early identification of poorly performing implants and enable a better understanding of the effects of surgical and patient factors on wear.
doi:10.3109/17453674.2014.972890
PMCID: PMC4404765  PMID: 25301435
17.  Minimum Information Specification For In Situ Hybridization and Immunohistochemistry Experiments (MISFISHIE) 
Nature biotechnology  2008;26(3):305-312.
One purpose of the biomedical literature is to report results in sufficient detail so that the methods of data collection and analysis can be independently replicated and verified. Here we present for consideration a minimum information specification for gene expression localization experiments, called the “Minimum Information Specification For In Situ Hybridization and Immunohistochemistry Experiments (MISFISHIE)”. It is modelled after the MIAME (Minimum Information About a Microarray Experiment) specification for microarray experiments. Data specifications like MIAME and MISFISHIE specify the information content without dictating a format for encoding that information. The MISFISHIE specification describes six types of information that should be provided for each experiment: Experimental Design, Biomaterials and Treatments, Reporters, Staining, Imaging Data, and Image Characterizations. This specification has benefited the consortium within which it was initially developed and is expected to benefit the wider research community. We welcome feedback from the scientific community to help improve our proposal.
doi:10.1038/nbt1391
PMCID: PMC4367930  PMID: 18327244
18.  Procedural skills practice and training needs of doctors, nurses, midwives and paramedics in rural Victoria 
Introduction
Procedural skills are a significant component of clinical practice. Doctors, nurses, midwives and paramedics are trained to use a variety of procedural skills. Rural clinicians in particular are often required to maintain competence in some procedural skills that are used infrequently, and which may require regular and repeated rehearsal. This paper reports on a research project conducted in Gippsland, Victoria, to ascertain the frequency of use, and relevance to clinical practice, of a range of skills in the fields of medicine, nursing, midwifery, and paramedic practice. The project also gathered data on the attitudes of clinicians regarding how frequently and by what means they thought they needed to practice these skills with a particular focus on the use of simulation as an educational method.
Methods
The research was conducted following identification of a specific set of procedural skills for each professional group. Skills were identified by an expert steering committee. We developed online questionnaires that consisted of two parts: 1) demographic and professional characteristics, and 2) experience of procedural skills and perceived training needs. We sought to invite all practicing clinicians (doctors, nurses, midwives, paramedics) working in Gippsland. Online surveys were distributed between November 2011 and April 2012 with three follow-up attempts. The Monash University Human Research Ethics Committee approved the study.
Results
Valid responses were received from 58 doctors, 94 nurses, 46 midwives, and 30 paramedics, whom we estimate to represent not more than 20% of current clinicians within these professions. This response rate reflected some of the difficulties experienced in the conduct of the research. Results were tabulated for each professional group across the range of skills. There was significant correlation between the frequency of certain skills and confidence with maintenance of these skills. This did not necessarily correlate with perceptions of respondents as to how often they need to practice each skill to maintain mastery. The more complex the skill, the more likely the respondents were to report a need for frequent rehearsal of the skill. There was variation between the professional groups as to how to retain mastery; for some skills, professional groups reported skill maintenance through clinical observation and clinical practice; for other skills, simulation was seen to be more appropriate.
Conclusion
This project provided insight into the clinical application of procedural skills for clinicians comprising a relatively large professional population within a defined geographical region in rural Victoria, as well as attitudes to skills maintenance and competency. Although not the focus of the study, an unexpected outcome was the design of questionnaires on procedural skills. We believe that the questionnaires may have value in other rural settings. We acknowledge the limitations of the study in the text. The project provides some information on which to base planning for procedural skills education, including simulation-based training, and directions for further research.
doi:10.2147/AMEP.S77779
PMCID: PMC4372009  PMID: 25834473
procedural skills; simulation; rural health education; skills maintenance
19.  Phosphorylation of Synaptic Vesicle Protein 2A at Thr84 by Casein Kinase 1 Family Kinases Controls the Specific Retrieval of Synaptotagmin-1 
The Journal of Neuroscience  2015;35(6):2492-2507.
Synaptic vesicle protein 2A (SV2A) is a ubiquitous component of synaptic vesicles (SVs). It has roles in both SV trafficking and neurotransmitter release. We demonstrate that Casein kinase 1 family members, including isoforms of Tau–tubulin protein kinases (TTBK1 and TTBK2), phosphorylate human SV2A at two constellations of residues, namely Cluster-1 (Ser42, Ser45, and Ser47) and Cluster-2 (Ser80, Ser81, and Thr84). These residues are also phosphorylated in vivo, and the phosphorylation of Thr84 within Cluster-2 is essential for triggering binding to the C2B domain of human synaptotagmin-1. We show by crystallographic and other analyses that the phosphorylated Thr84 residue binds to a pocket formed by three conserved Lys residues (Lys314, Lys326, and Lys328) on the surface of the synaptotagmin-1 C2B domain. Finally, we observed dysfunctional synaptotagmin-1 retrieval during SV endocytosis by ablating its phospho-dependent interaction with SV2A, knockdown of SV2A, or rescue with a phosphorylation-null Thr84 SV2A mutant in primary cultures of mouse neurons. This study reveals fundamental details of how phosphorylation of Thr84 on SV2A controls its interaction with synaptotagmin-1 and implicates SV2A as a phospho-dependent chaperone required for the specific retrieval of synaptotagmin-1 during SV endocytosis.
doi:10.1523/JNEUROSCI.4248-14.2015
PMCID: PMC4323530  PMID: 25673844
CK1; SV2A; synaptotagmin
20.  Group I Paks as therapeutic targets in NF2-deficient meningioma 
Oncotarget  2015;6(4):1981-1994.
Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder characterized by the development of multiple tumors in the central nervous system, most notably schwannomas and meningiomas. Mutational inactivation of NF2 is found in 40–60% of sporadic meningiomas, but the molecular mechanisms underlying malignant changes of meningioma cells remain unclear. Because group I p21-activated kinases (Paks) bind to and are inhibited by the NF2-encoded protein Merlin, we assessed the signaling and anti-tumor effects of three group-I specific Pak inhibitors - Frax597, 716 and 1036 - in NF2−/− meningiomas in vitro and in an orthotopic mouse model. We found that these Pak inhibitors suppressed the proliferation and motility of both benign (Ben-Men1) and malignant (KT21-MG1) meningiomas cells. In addition, we found a strong reduction in phosphorylation of Mek and S6, and decreased cyclin D1 expression in both cell lines after treatment with Pak inhibitors. Using intracranial xenografts of luciferase-expressing KT21-MG1 cells, we found that treated mice showed significant tumor suppression for all three Pak inhibitors. Similar effects were observed in Ben-Men1 cells. Tumors dissected from treated animals exhibited an increase in apoptosis without notable change in proliferation. Collectively, these results suggest that Pak inhibitors might be useful agents in treating NF2-deficient meningiomas.
PMCID: PMC4385830  PMID: 25596744
protein kinases; p21-activated kinase; neurofibromatosis; meningioma; signal transduction; small molecule inhibitors
21.  Trypanosoma brucei Translation Initiation Factor Homolog EIF4E6 Forms a Tripartite Cytosolic Complex with EIF4G5 and a Capping Enzyme Homolog 
Eukaryotic Cell  2014;13(7):896-908.
Trypanosomes lack the transcriptional control characteristic of the majority of eukaryotes that is mediated by gene-specific promoters in a one-gene–one-promoter arrangement. Rather, their genomes are transcribed in large polycistrons with no obvious functional linkage. Posttranscriptional regulation of gene expression must thus play a larger role in these organisms. The eIF4E homolog TbEIF4E6 binds mRNA cap analogs in vitro and is part of a complex in vivo that may fulfill such a role. Knockdown of TbEIF4E6 tagged with protein A-tobacco etch virus protease cleavage site-protein C to approximately 15% of the normal expression level resulted in viable cells that displayed a set of phenotypes linked to detachment of the flagellum from the length of the cell body, if not outright flagellum loss. While these cells appeared and behaved as normal under stationary liquid culture conditions, standard centrifugation resulted in a marked increase in flagellar detachment. Furthermore, the ability of TbEIF4E6-depleted cells to engage in social motility was reduced. The TbEIF4E6 protein forms a cytosolic complex containing a triad of proteins, including the eIF4G homolog TbEIF4G5 and a hypothetical protein of 70.3 kDa, referred to as TbG5-IP. The TbG5-IP analysis revealed two domains with predicted secondary structures conserved in mRNA capping enzymes: nucleoside triphosphate hydrolase and guanylyltransferase. These complex members have the potential for RNA interaction, either via the 5′ cap structure for TbEIF4E6 and TbG5-IP or through RNA-binding domains in TbEIF4G5. The associated proteins provide a signpost for future studies to determine how this complex affects capped RNA molecules.
doi:10.1128/EC.00071-14
PMCID: PMC4135740  PMID: 24839125
22.  Low Wear of a Second-generation Highly Crosslinked Polyethylene Liner: A 5-year Radiostereometric Analysis Study 
Background
A sequentially irradiated and annealed, second-generation highly crosslinked polyethylene (XLPE) liner was introduced clinically in 2005 to reduce in vivo oxidation. This liner design has also been shown to reduce wear in vitro when compared with conventional and first-generation crosslinked liners. To date, there is only one study reporting an in vivo wear rate of this liner at 5 years’ followup. However, that study used measurements made from plain radiographs, which have limited sensitivity, particularly when monitoring very low amounts of wear.
Questions/purposes
What is the amount and direction of wear at 5 years using radiostereometric analysis (RSA) in patients who had THAs that included second-generation XLPE?
Methods
We prospectively reviewed 21 patients who underwent primary cementless THA with the same design of XLPE acetabular liner and 32-mm articulation. Tantalum markers were inserted during surgery and all patients had RSA radiographs at 1 week, 6 months, and 1, 2, and 5 years postoperatively. Femoral head penetration within the acetabular component was measured with UmRSA® software. One patient died and two had incomplete radiographs leaving 18 radiographic series for analysis.
Results
The mean amounts of proximal, two-dimensional, and three-dimensional head penetration between 1 week and 5 years were 0.018, 0.071, and 0.149 mm, respectively. The mean proximal, two-dimensional, and three-dimensional wear rates calculated between 1 year and 5 years were all less than 0.001 mm/year with no patient recording a wear rate of more than 0.040 mm/year.
Conclusions
The head penetration of a second-generation XLPE liner remained low at 5 years and the wear rate calculated after the first year was low in all directions. This low level of wear remains encouraging for the future clinical performance of this material.
Level of Evidence
Level IV, therapeutic study. See Instructions for Authors for a complete description of levels of evidence.
doi:10.1007/s11999-013-3188-z
PMCID: PMC3792282  PMID: 23893361
23.  Age modification of diabetes-related hospitalization among First Nations adults in Alberta, Canada 
Background
We sought to determine the modifying effects of age and multimorbidity on the association between First Nations status and hospitalizations for diabetes-specific ambulatory care sensitive conditions (ACSC).
Findings
We identified 183,654 adults with diabetes from Alberta Canada, and followed them for one year for the outcome of hospitalization or emergency department (ED) visit for a diabetes-specific ACSC. We used logistic regression to determine the association between First Nations status and the outcome, assessing for effect modification by age and multimorbidity with interaction terms. In a model adjusting for age, age2, baseline A1c, duration of diabetes, and multimorbidity, First Nations people were at greater risk than non-First Nations to experience a diabetes-specific hospitalization or ED visit (unadjusted odds ratio [OR] 3.74; 95% confidence interval [CI]: 3.45-4.07). After adjustment for relevant covariates, this association varied by age (interaction: p = 0.018): adjusted OR 3.94 (95% CI: 3.11-4.99) and 5.74 (95% CI: 3.36-9.80) for First Nations compared to non-First Nations at ages 30 and 80 years, respectively.
Conclusions
Compared with non-First Nations, older First Nations patients with diabetes are at greater risk for diabetes-specific hospitalizations. Older First Nations patients with diabetes should be given priority access to primary care services as they are at greatest risk for requiring hospitalization for stabilization of their condition.
doi:10.1186/1758-5996-6-108
PMCID: PMC4192759  PMID: 25309626
American indian; First Nations; Hospitalization; Diabetes mellitus; Risk adjustment
24.  A repository of assays to quantify 10,000 human proteins by SWATH-MS 
Scientific Data  2014;1:140031.
Mass spectrometry is the method of choice for deep and reliable exploration of the (human) proteome. Targeted mass spectrometry reliably detects and quantifies pre-determined sets of proteins in a complex biological matrix and is used in studies that rely on the quantitatively accurate and reproducible measurement of proteins across multiple samples. It requires the one-time, a priori generation of a specific measurement assay for each targeted protein. SWATH-MS is a mass spectrometric method that combines data-independent acquisition (DIA) and targeted data analysis and vastly extends the throughput of proteins that can be targeted in a sample compared to selected reaction monitoring (SRM). Here we present a compendium of highly specific assays covering more than 10,000 human proteins and enabling their targeted analysis in SWATH-MS datasets acquired from research or clinical specimens. This resource supports the confident detection and quantification of 50.9% of all human proteins annotated by UniProtKB/Swiss-Prot and is therefore expected to find wide application in basic and clinical research. Data are available via ProteomeXchange (PXD000953-954) and SWATHAtlas (SAL00016-35).
doi:10.1038/sdata.2014.31
PMCID: PMC4322573  PMID: 25977788
25.  The LKB1-salt-inducible kinase pathway functions as a key gluconeogenic suppressor in the liver 
Nature Communications  2014;5:4535.
LKB1 is a master kinase that regulates metabolism and growth through adenosine monophosphate-activated protein kinase (AMPK) and 12 other closely related kinases. Liver-specific ablation of LKB1 causes increased glucose production in hepatocytes in vitro and hyperglycaemia in fasting mice in vivo. Here we report that the salt-inducible kinases (SIK1, 2 and 3), members of the AMPK-related kinase family, play a key role as gluconeogenic suppressors downstream of LKB1 in the liver. The selective SIK inhibitor HG-9-91-01 promotes dephosphorylation of transcriptional co-activators CRTC2/3 resulting in enhanced gluconeogenic gene expression and glucose production in hepatocytes, an effect that is abolished when an HG-9-91-01-insensitive mutant SIK is introduced or LKB1 is ablated. Although SIK2 was proposed as a key regulator of insulin-mediated suppression of gluconeogenesis, we provide genetic evidence that liver-specific ablation of SIK2 alone has no effect on gluconeogenesis and insulin does not modulate SIK2 phosphorylation or activity. Collectively, we demonstrate that the LKB1–SIK pathway functions as a key gluconeogenic gatekeeper in the liver.
The liver is an important regulator of glucose homeostasis. Here, the authors provide insight into the molecular signalling pathways controlling hepatic gluconeogenesis by showing that SIK protein kinases suppress gluconeogenesis, and that glucagon—but not insulin—regulates phosphorylation of SIK2.
doi:10.1038/ncomms5535
PMCID: PMC4143937  PMID: 25088745

Results 1-25 (130)