Improved Elution Conditions for Native Co-Immunoprecipitation

Native immunoprecipitation followed by protein A-mediated recovery of the immuno-complex is a powerful tool to study protein-protein interactions. A limitation of this technique is the concomitant recovery of large amounts of immunoglobulin, which interferes with down-stream applications such as mass spectrometric analysis and Western blotting. Here we report a detergent-based "soft" elution protocol that allows effective recovery of immunoprecipitated antigen and binding partners, yet avoids elution of the bulk of the immunoglobulin.We assessed the performance of the soft elution protocol using immunoprecipitation of Adaptor protein complex 1 (AP-1) and associated proteins as a test case. Relative to conventional elution conditions, the novel protocol substantially improved the sensitivity of mass spectrometric identification of immunoprecipitated proteins from unfractionated solution digests. Averaging over three independent experiments, Mascot scores of identified AP-1 binding partners were increased by 39%. Conversely, the estimated amount of recovered immunoglobulin was reduced by 44%. We tested the protocol with five further antibodies derived from rabbit, mouse and goat. In each case we observed a significant reduction of co-eluting immunoglobulin.The soft elution protocol presented here shows superior performance compared to standard elution conditions for subsequent protein identification by mass spectrometry from solution digests. The method was developed for rabbit polyclonal antibodies, but also performed well with the tested goat and mouse antibodies. Hence we expect the soft elution protocol to be widely applicable

Phosphorylation of the Herpes Simplex Virus Type 1 UL13 Protein Kinase and a Putative Target Protein Encoded by Gene UL49

Protein kinases (PKs) are ubiquitous enzymes which utilise phosphorylation, a reversible post-translational modification, to regulate protein activity. Protein phosphorylation is widespread, affecting nearly all aspects of growth and homeostasis in eukaryotic cells. However, over twenty years ago certain viral transforming genes were found to possess PK activity, bringing protein phosphorylation to the attention of virologists. Sequence analysis of the herpes simplex virus type 1 (HSV-1) genome identified two genes, US3 and UL13, whose products exhibit PK-specific amino acid sequence motifs. The US3 PK has been purified, the enzyme activity characterised and a number of targets for phosphorylation identified. The UL13 PK has not yet been purified, but its enzymatic characteristics and potential target proteins have been investigated. It localises in the nuclei of infected cells, utilises ATP or GTP to phosphorylate acidic but not basic substrates, and is a minor component of the virion tegument. The UL13 PK activity is stimulated by high salt concentration and is insensitive to inhibition by heparin. Several proteins have been identified as targets for the UL13 PK, one being the abundant viral tegument protein encoded by HSV-1 gene UL49. The aims of this project were to confirm that the UL13 PK targets the UL49 protein for phosphorylation, to characterise further the UL13 PK activity, and to map phosphorylated residues within the UL13 and UL49 proteins. From an analysis of three independent UL13 mutants, each with a lesion in a different region of the UL13 gene, it was confirmed using an in vitro assay that phosphorylation of the UL49 protein was dramatically reduced in the absence of a functional UL13 protein. In radiolabelled nuclear extracts of wt HSV-1 infected cells or membrane-stripped wt virions, the UL13 protein was detected as a highly radiolabelled 57 kDa phosphoprotein. Phosphorylation of this protein was stimulated by increasing salt concentrations. Thus, HSV-1 UL13 PK differs significantly from the previously characterised HSV-2 UL13 PK in both its salt sensitivity and optimum pH. Radiolabelling of the UL13 PK maybe attributed to autophosphorylation. However, at least two hyperphosphorylated forms of the UL13 protein were detected in radiolabelled nuclear extracts incubated with excess unlabelled ATP or GTP. The level of hyperphosphorylation differed between cell lines, and hyperphosphorylated UL13 protein was not detected in radiolabelled membrane-stripped virions. Hyperphosphorylation was insensitive to heparin, but was completely abolished in the presence of a potent inhibitor of cellular casein kinase II. The most obvious target for the UL13 PK identified during this study was a 38 kDa virion protein, which was confirmed by mass spectrometry and by use of a mutant virus expressing a C-terminally truncated form of the UL49 protein to be encoded by HSV-1 gene UL49. The UL49 protein was still phosphorylated, albeit to a greatly reduced level, in nuclear extracts prepared from UL13 mutant-infected cells. The additional PK or PKs targeting the UL49 were inhibited by heparin and sensitive to increasing salt concentrations. This suggests that a cellular PK targets the UL49 protein. However, an inhibitor of casein kinase II had little effect on phosphorylation of the UL49 protein. Initially, the UL49 protein was found to cycle phosphate, and did not appear to be hyperphosphorylated beyond a basal level. However, under altered buffer conditions, it was hyperphosphorylated and also possibly nucleotidylylated, these modifications seeming to require sequences near the C-terminus of the UL49 protein. Phosphopeptide mapping experiments performed on the UL49 protein phosphorylated at low salt concentrations led to detection of a 2 kDa tryptic phosphopeptide by SDS- PAGE, and mass spectrometric analysis identified a phosphorylated 1703.91 Da tryptic peptide which was phosphorylated at high salt concentrations. It is likely that these two peptides represent cellular and viral PK target sites. Mapping of the UL13 protein suggested that phosphopeptide fragments are located in the serine-rich, N-terminal 100 residues of the protein, a region which lies outside the established catalytic domain of the UL13PK. As well as providing data in support of UL13 encoding a PK which targets the UL49 protein for phosphorylation, the data presented in this thesis represents a further characterisation of the UL13 PK activity and provides new insights into phosphorylation of the UL49 and UL13 proteins

Role of the AP-5 adaptor protein complex in late endosome-to-Golgi retrieval

The AP-5 adaptor protein complex is presumed to function in membrane traffic, but so far nothing is known about its pathway or its cargo. We have used CRISPR-Cas9 to knock out the AP-5 ζ subunit gene, AP5Z1, in HeLa cells, and then analysed the phenotype by subcellular fractionation profiling and quantitative mass spectrometry. The retromer complex had an altered steady-state distribution in the knockout cells, and several Golgi proteins, including GOLIM4 and GOLM1, were depleted from vesicle-enriched fractions. Immunolocalisation showed that loss of AP-5 led to impaired retrieval of the cation-independent mannose 6-phosphate receptor (CIMPR), GOLIM4, and GOLM1 from endosomes back to the Golgi region. Knocking down the retromer complex exacerbated this phenotype. Both the CIMPR and sortilin interacted with the AP-5–associated protein SPG15 in pull-down assays, and we propose that sortilin may act as a link between Golgi proteins and the AP-5/SPG11/SPG15 complex. Together, our findings suggest that AP-5 functions in a novel sorting step out of late endosomes, acting as a backup pathway for retromer. This provides a mechanistic explanation for why mutations in AP-5/SPG11/SPG15 cause cells to accumulate aberrant endolysosomes, and highlights the role of endosome/lysosome dysfunction in the pathology of hereditary spastic paraplegia and other neurodegenerative disorders.The Wellcome Trust https://wellcome.ac.uk/ (grant number 086598). Received by MSR. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. German Research Foundation http://www.dfg.de/ (grant number DFG/Gottfried Wilhelm Leibniz Prize MA 1764/2-1). Contributed to GHHB's research. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The Wellcome Trust https://wellcome.ac.uk/ (grant number 100140). Strategic Award to the CIMR. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

ATF7IP-Mediated Stabilization of the Histone Methyltransferase SETDB1 Is Essential for Heterochromatin Formation by the HUSH Complex

The histone methyltransferase SETDB1 plays a central role in repressive chromatin processes, but the functional requirement for its binding partner ATF7IP has remained enigmatic. Here, we show that ATF7IP is essential for SETDB1 stability: nuclear SETDB1 protein is degraded by the proteasome upon ablation of ATF7IP. As a result, ATF7IP is critical for repression that requires H3K9 trimethylation by SETDB1, including transgene silencing by the HUSH complex. Furthermore, we show that loss of ATF7IP phenocopies loss of SETDB1 in genome-wide assays. ATF7IP and SETDB1 knockout cells exhibit near-identical defects in the global deposition of H3K9me3, which results in similar dysregulation of the transcriptome. Overall, these data identify a critical functional role for ATF7IP in heterochromatin formation by regulating SETDB1 abundance in the nucleus.This work was supported by the Wellcome Trust through a Principal Research Fellowship to P.J.L. (101835/Z/13/Z) and a Ph.D. studentship to I.A.T. The CIMR is in receipt of a Wellcome Trust strategic award.This is the final version of the article. It first appeared from Elsevier (Cell Press) via https://doi.org/10.1016/j.celrep.2016.09.05

ATF7IP-Mediated Stabilization of the Histone Methyltransferase SETDB1 Is Essential for Heterochromatin Formation by the HUSH Complex.

The histone methyltransferase SETDB1 plays a central role in repressive chromatin processes, but the functional requirement for its binding partner ATF7IP has remained enigmatic. Here, we show that ATF7IP is essential for SETDB1 stability: nuclear SETDB1 protein is degraded by the proteasome upon ablation of ATF7IP. As a result, ATF7IP is critical for repression that requires H3K9 trimethylation by SETDB1, including transgene silencing by the HUSH complex. Furthermore, we show that loss of ATF7IP phenocopies loss of SETDB1 in genome-wide assays. ATF7IP and SETDB1 knockout cells exhibit near-identical defects in the global deposition of H3K9me3, which results in similar dysregulation of the transcriptome. Overall, these data identify a critical functional role for ATF7IP in heterochromatin formation by regulating SETDB1 abundance in the nucleus.This work was supported by the Wellcome Trust through a Principal Research Fellowship to P.J.L. (101835/Z/13/Z) and a Ph.D. studentship to I.A.T. The CIMR is in receipt of a Wellcome Trust strategic award.This is the final version of the article. It first appeared from Elsevier (Cell Press) via https://doi.org/10.1016/j.celrep.2016.09.05

The WDR11 complex facilitates the tethering of AP-1-derived vesicles.

Vesicluar transport of proteins from endosomes to the trans-Golgi network (TGN) is an essential cellular pathway, but much of its machinery is still unknown. A screen for genes involved in endosome-to-TGN trafficking produced two hits, the adaptor protein-1 (AP-1 complex), which facilitates vesicle budding, and WDR11. Here we demonstrate that WDR11 forms a stable complex with two other proteins, which localises to the TGN region and does not appear to be associated with AP-1, suggesting it may act downstream from budding. In a vesicle tethering assay, capture of vesicles by golgin-245 was substantially reduced in WDR11-knockout cells. Moreover, structured illumination microscopy and relocation assays indicate that the WDR11 complex is initially recruited onto vesicles rather than the TGN, where it may in turn recruit the golgin binding partner TBC1D23. We propose that the complex acts together with TBC1D23 to facilitate the golgin-mediated capture of vesicles that were generated using AP-1

Fat mass and obesity-related (FTO) shuttles between the nucleus and cytoplasm.

SNPs (single nucleotide polymorphisms) on a chromosome 16 locus encompassing FTO, as well as IRX3, 5, 6, FTM and FTL are robustly associated with human obesity. FTO catalyses the Fe(II)- and 2OG-dependent demethylation of RNA and is an AA (amino acid) sensor that couples AA levels to mTORC1 (mammalian target of rapamycin complex 1) signalling, thereby playing a key role in regulating growth and translation. However, the cellular compartment in which FTO primarily resides to perform its biochemical role is unclear. Here, we undertake live cell imaging of GFP (green fluorescent protein)-FTO, and demonstrate that FTO resides in both the nucleus and cytoplasm. We show using 'FLIP' (fluorescence loss in photobleaching) that a mobile FTO fraction shuttles between both compartments. We performed a proteomic study and identified XPO2 (Exportin 2), one of a family of proteins that mediates the shuttling of proteins between the nucleus and the cytoplasm, as a binding partner of FTO. Finally, using deletion studies, we show that the N-terminus of FTO is required for its ability to shuttle between the nucleus and cytoplasm. In conclusion, FTO is present in both the nucleus and cytoplasm, with a mobile fraction that shuttles between both cellular compartments, possibly by interaction with XPO2.This is the final published version. It first appeared at http://www.bioscirep.org/bsr/034/bsr034e144.htm

Temporal Proteomic Analysis of BK Polyomavirus Infection Reveals Virus-Induced G2 Arrest and Highly Effective Evasion of Innate Immune Sensing.

BK polyomavirus (BKPyV) is a small DNA virus that establishes a life-long persistent infection in the urinary tract of most people. BKPyV is known to cause severe morbidity in renal transplant recipients and can lead to graft rejection. The simple 5.2-kbp double-stranded DNA (dsDNA) genome expresses just seven known proteins; thus, it relies heavily on the host machinery to replicate. How the host proteome changes over the course of infection is key to understanding this host-virus interplay. Here, for the first time quantitative temporal viromics has been used to quantify global changes in >9,000 host proteins in two types of primary human epithelial cells throughout 72 h of BKPyV infection. These data demonstrate the importance of cell cycle progression and pseudo-G2 arrest in effective BKPyV replication, along with a surprising lack of an innate immune response throughout the whole virus replication cycle. BKPyV thus evades pathogen recognition to prevent activation of innate immune responses in a sophisticated manner.IMPORTANCE BK polyomavirus can cause serious problems in immune-suppressed patients, in particular, kidney transplant recipients who can develop polyomavirus-associated kidney disease. In this work, we have used advanced proteomics techniques to determine the changes to protein expression caused by infection of two independent primary cell types of the human urinary tract (kidney and bladder) throughout the replication cycle of this virus. Our findings have uncovered new details of a specific form of cell cycle arrest caused by this virus, and, importantly, we have identified that this virus has a remarkable ability to evade detection by host cell defense systems. In addition, our data provide an important resource for the future study of kidney epithelial cells and their infection by urinary tract pathogens.Isaac Newton Trust (part funding of ISSF award to C.M.C.

The Proteasome Distinguishes between Heterotypic and Homotypic Lysine-11-Linked Polyubiquitin Chains.

Proteasome-mediated degradation occurs with proteins principally modified with lysine-48 polyubiquitin chains. Whether the proteasome also can bind atypical ubiquitin chains, including those linked by lysine-11, has not been well established. This is critically important, as lysine-11 polyubiquitination has been implicated in both proteasome-mediated degradation and non-degradative outcomes. Here we demonstrate that pure homotypic lysine-11-linked chains do not bind strongly to the mammalian proteasome. By contrast, heterotypic polyubiquitin chains, containing lysine-11 and lysine-48 linkages, not only bind to the proteasome but also stimulate the proteasomal degradation of the cell-cycle regulator cyclin B1. Thus, while heterotypic lysine-11-linked chains facilitate proteasomal degradation, homotypic lysine-11 linkages adopt conformations that prevent association with the proteasome. Our data demonstrate the capacity of the proteasome to bind ubiquitin chains of distinct topology, with implications for the recognition and diverse biological functions of mixed ubiquitin chains.This work is supported by a Wellcome Trust Senior Clinical Research Fellowship to JAN (102770/Z/13/Z), a Wellcome Trust Fellowship to MPW (093966/Z/10/Z) and a National Institute of Health grant (GM067945) to SPG. The Cambridge Institutefor Medical Research is in receipt of a Wellcome Trust Strategic Award [100140].This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.celrep.2015.06.06

Clathrin heavy chain 22 contributes to the control of neuropeptide degradation and secretion during neuronal development.

The repertoire of cell types in the human nervous system arises through a highly orchestrated process, the complexity of which is still being discovered. Here, we present evidence that CHC22 has a non-redundant role in an early stage of neural precursor differentiation, providing a potential explanation of why CHC22 deficient patients are unable to feel touch or pain. We show the CHC22 effect on neural differentiation is independent of the more common clathrin heavy chain CHC17, and that CHC22-dependent differentiation is mediated through an autocrine/paracrine mechanism. Using quantitative proteomics, we define the composition of clathrin-coated vesicles in SH-SY5Y cells, and determine proteome changes induced by CHC22 depletion. In the absence of CHC22 a subset of dense core granule (DCG) neuropeptides accumulated, were processed into biologically active 'mature' forms, and secreted in sufficient quantity to trigger neural differentiation. When CHC22 is present, however, these DCG neuropeptides are directed to the lysosome and degraded, thus preventing differentiation. This suggests that the brief reduction seen in CHC22 expression in sensory neural precursors may license a step in neuron precursor neurodevelopment; and that this step is mediated through control of a novel neuropeptide processing pathway