CD39+ tumor infiltrating T cells from colorectal cancers exhibit dysfunctional phenotype.

Recent studies revealed that CD39 was highly expressed in tumor-specific CD4+ tumor infiltrating lymphocytes (TILs). However, the divergent function of CD39+ T cells remains to be elucidated in colorectal cancer (CRC). In this study, T cells from CRC patients and tumor-bearing mice were isolated to evaluate the function of CD39 in T cells. We found that CD39 was elevated in intratumoral T cells from CRC patients, and negatively correlated with cytokine secretion capacity. T cell activation induced CD39 expression, and CD39+ T cells produced more IFN-γ in response to CRC tumor antigens. In addition, CD39+ T cells in the spleens of tumor-bearing mice exhibited a stronger anti-tumor activity in vitro than CD39- T cells, but there was no significant difference in the anti-tumor activities between CD39- TILs and CD39+ TILs. Moreover, we found that CD39+ T cells expressed higher checkpoint molecules and contained a higher proportion of Treg cells than CD39- T cells, suggesting that CD39+ T cells may be correlated with an immunosuppressive phenotype. And CD39 expression on T cells could convert pro-inflammatory eATP to immunosuppressive eADO. However, both T cells from the vaccinated-wild-type mice and CD39-/- mice could recognize and eliminate tumor cells in vitro, and adoptive transfer of these T cells resulted in tumor growth inhibition in tumor-bearing mice. In conclusion, our study revealed the divergent functions of CD39+ T cells, which were reactive to tumor antigen but exhibited a dysfunctional phenotype.

Open group cognitive behaviour therapy on acute in-patient units.

BACKGROUND: Adult mental health in-patient units primarily provide a service for people deemed to be at significant risk to themselves or others, where treatment cannot be provided safely in the community. Whilst psychological interventions are indicated during episodes of acute mental distress, they are often psychoeducational and skills-based in nature. A common complaint amongst those admitted is the lack of psychological provision at a time of crisis when they most need to make sense of their difficulties. AIMS: This article reports on service users' experiences of open group cognitive behavioural therapy where participants choose the therapeutic targets. METHOD: A total of 75 patients admitted to acute in-patient wards over a 6-month period accessed open group cognitive therapy as part of routine care. Participants completed an evaluation questionnaire that measured their experiences of the group and the usefulness of them within an in-patient setting. RESULTS: A total of 27 participants completed anonymous questionnaires (36%) and the results indicated that participants felt understood, respected and accepted within the group and felt that the group setting was helpful for sharing experiences. In addition, all participants reported that following the group they would be more likely to access psychological therapies in the future. CONCLUSIONS: Open group therapy where participants define the therapeutic targets each session is feasible and achievable on acute in-patient units and patients report finding this useful.

Sesquiterpene lactone Bigelovin induces apoptosis of colon cancer cells through inducing IKK-ß degradation and suppressing nuclear factor kappa B activation.

Bigelovin, a sesquiterpene lactone extracted from plant Inula helianthus aquatica, exhibited multiple interesting biological activities, including anti-inflammation, antiangiogenesis and cytotoxic action against cancer cells. In the present study, we found that Bigelovin reduced the viability of human colon cancer cells and induced their apoptosis in a time- and dose-dependent manner, with an IC50-5 µM. RNAseq and luciferase reporter analyses revealed that the nuclear factor kappa B (NF-κB) signaling was one of the most significantly inhibited pathways after Bigelovin treatment. Further systemic examination showed that exposure to Bigelovin resulted in ubiquitination and degradation of inhibitor of kappa-B kinase-beta (IKK-ß) and decrease of IκB-α and p65 phosphorylation, which led to the downregulation of NF-κB-regulated genes expression. Moreover, enforced expression of exogenous IKK-ß attenuated Bigelovin-induced NF-κB suppression and cell viability reduction. These results indicated that Bigelovin exerts a cytotoxic action against colon cancer cells through the induction of IKK-ß degradation and consequently the inhibition of NF-κB signaling. Given the abnormal activation of NF-κB signaling in colorectal cancer (CRC) cells and the critical role of chronic inflammation in CRC development, it is conceivable that at least some colorectal cancer cells are addictive to NF-κB activation and targeting the pathway is an effective anti-CRC strategy.

A functionally defined high-density NRF2 interactome reveals new conditional regulators of ARE transactivation.

NRF2 (NFE2L2) is a cytoprotective transcription factor associated with >60 human diseases, adverse drug reactions and therapeutic resistance. To provide insight into the complex regulation of NRF2 responses, 1962 predicted NRF2-partner interactions were systematically tested to generate an experimentally defined high-density human NRF2 interactome. Verification and conditional stratification of 46 new NRF2 partners was achieved by co-immunoprecipitation and the novel integration of quantitative data from dual luminescence-based co-immunoprecipitation (DULIP) assays and live-cell fluorescence cross-correlation spectroscopy (FCCS). The functional impact of new partners was then assessed in genetically edited loss-of-function (NRF2-/-) and disease-related gain-of-function (NRF2T80K and KEAP1-/-) cell-lines. Of the new partners investigated >77% (17/22) modified NRF2 responses, including partners that only exhibited effects under disease-related conditions. This experimentally defined binary NRF2 interactome provides a new vision of the complex molecular networks that govern the modulation and consequence of NRF2 activity in health and disease.

Glucocorticoids rapidly inhibit cell migration through a novel, non-transcriptional HDAC6 pathway.

Glucocorticoids (GCs) act through the glucocorticoid receptor (GR, also known as NR3C1) to regulate immunity, energy metabolism and tissue repair. Upon ligand binding, activated GR mediates cellular effects by regulating gene expression, but some GR effects can occur rapidly without new transcription. Here, we show that GCs rapidly inhibit cell migration, in response to both GR agonist and antagonist ligand binding. The inhibitory effect on migration is prevented by GR knockdown with siRNA, confirming GR specificity, but not by actinomycin D treatment, suggesting a non-transcriptional mechanism. We identified a rapid onset increase in microtubule polymerisation following GC treatment, identifying cytoskeletal stabilisation as the likely mechanism of action. HDAC6 overexpression, but not knockdown of αTAT1, rescued the GC effect, implicating HDAC6 as the GR effector. Consistent with this hypothesis, ligand-dependent cytoplasmic interaction between GR and HDAC6 was demonstrated by quantitative imaging. Taken together, we propose that activated GR inhibits HDAC6 function, and thereby increases the stability of the microtubule network to reduce cell motility. We therefore report a novel, non-transcriptional mechanism whereby GCs impair cell motility through inhibition of HDAC6 and rapid reorganization of the cell architecture.This article has an associated First Person interview with the first author of the paper.

Quantitative single-cell live imaging links HES5 dynamics with cell-state and fate in murine neurogenesis.

During embryogenesis cells make fate decisions within complex tissue environments. The levels and dynamics of transcription factor expression regulate these decisions. Here, we use single cell live imaging of an endogenous HES5 reporter and absolute protein quantification to gain a dynamic view of neurogenesis in the embryonic mammalian spinal cord. We report that dividing neural progenitors show both aperiodic and periodic HES5 protein fluctuations. Mathematical modelling suggests that in progenitor cells the HES5 oscillator operates close to its bifurcation boundary where stochastic conversions between dynamics are possible. HES5 expression becomes more frequently periodic as cells transition to differentiation which, coupled with an overall decline in HES5 expression, creates a transient period of oscillations with higher fold expression change. This increases the decoding capacity of HES5 oscillations and correlates with interneuron versus motor neuron cell fate. Thus, HES5 undergoes complex changes in gene expression dynamics as cells differentiate.

Integrative Omic Profiling Reveals Unique Hypoxia Induced Signatures in Gastric Cancer Associated Myofibroblasts.

Although hypoxia is known to contribute to several aspects of tumour progression, relatively little is known about the effects of hypoxia on cancer-associated myofibroblasts (CAMs), or the consequences that conditional changes in CAM function may have on tumour development and metastasis. To investigate this issue in the context of gastric cancer, a comparative multiomic analysis was performed on populations of patient-derived myofibroblasts, cultured under normoxic or hypoxic conditions. Data from this study reveal a novel set of CAM-specific hypoxia-induced changes in gene expression and secreted proteins. Significantly, these signatures are not observed in either patient matched adjacent tissue myofibroblasts (ATMs) or non-cancer associated normal tissue myofibroblasts (NTMs). Functional characterisation of different myofibroblast populations shows that hypoxia-induced changes in gene expression not only enhance the ability of CAMs to induce cancer cell migration, but also confer pro-tumorigenic (CAM-like) properties in NTMs. This study provides the first global mechanistic insight into the molecular changes that contribute to hypoxia-induced pro-tumorigenic changes in gastric stromal myofibroblasts.

Integrated omics profiling reveals novel patterns of epigenetic programming in cancer-associated myofibroblasts.

There is increasing evidence that stromal myofibroblasts play a key role in the tumour development however, the mechanisms by which they become reprogrammed to assist in cancer progression remain unclear. As cultured cancer-associated myofibroblasts (CAMs) retain an ability to enhance the proliferation and migration of cancer cells in vitro, it is possible that epigenetic reprogramming of CAMs within the tumour microenvironment may confer long-term pro-tumourigenic changes in gene expression. This study reports the first comparative multi-omics analysis of cancer-related changes in gene expression and DNA methylation in primary myofibroblasts derived from gastric and oesophageal tumours. In addition, we identify novel CAM-specific DNA methylation signatures, which are not observed in patient-matched adjacent tissue-derived myofibroblasts, or corresponding normal tissue-derived myofibroblasts. Analysis of correlated changes in DNA methylation and gene expression shows that different patterns of gene-specific DNA methylation have the potential to confer pro-tumourigenic changes in metabolism, cell signalling and differential responses to hypoxia. These molecular signatures provide new insights into potential mechanisms of stromal reprogramming in gastric and oesophageal cancer, while also providing a new resource to facilitate biomarker identification and future hypothesis-driven studies into mechanisms of stromal reprogramming and tumour progression in solid tumours.

KSHV SOX mediated host shutoff: the molecular mechanism underlying mRNA transcript processing.

Onset of the lytic phase in the KSHV life cycle is accompanied by the rapid, global degradation of host (and viral) mRNA transcripts in a process termed host shutoff. Key to this destruction is the virally encoded alkaline exonuclease SOX. While SOX has been shown to possess an intrinsic RNase activity and a potential consensus sequence for endonucleolytic cleavage identified, the structures of the RNA substrates targeted remained unclear. Based on an analysis of three reported target transcripts, we were able to identify common structures and confirm that these are indeed degraded by SOX in vitro as well as predict the presence of such elements in the KSHV pre-microRNA transcript K12-2. From these studies, we were able to determine the crystal structure of SOX productively bound to a 31 nucleotide K12-2 fragment. This complex not only reveals the structural determinants required for RNA recognition and degradation but, together with biochemical and biophysical studies, reveals distinct roles for residues implicated in host shutoff. Our results further confirm that SOX and the host exoribonuclease Xrn1 act in concert to elicit the rapid degradation of mRNA substrates observed in vivo, and that the activities of the two ribonucleases are co-ordinated.

Ubiquitin-Dependent Modification of Skeletal Muscle by the Parasitic Nematode, Trichinella spiralis.

Trichinella spiralis is a muscle-specific parasitic worm that is uniquely intracellular. T. spiralis reprograms terminally differentiated skeletal muscle cells causing them to de-differentiate and re-enter the cell cycle, a process that cannot occur naturally in mammalian skeletal muscle cells, but one that holds great therapeutic potential. Although the host ubiquitin pathway is a common target for viruses and bacteria during infection, its role in parasite pathogenesis has been largely overlooked. Here we demonstrate that the secreted proteins of T. spiralis contain E2 Ub-conjugating and E3 Ub-ligase activity. The E2 activity is attributed to TsUBE2L3, a novel and conserved T. spiralis enzyme located in the secretory organ of the parasite during the muscle stages of infection. TsUBE2L3 cannot function with any T.spiralis secreted E3, but specifically binds to a panel of human RING E3 ligases, including the RBR E3 ARIH2 with which it interacts with a higher affinity than the mammalian ortholog UbcH7/UBE2L3. Expression of TsUBE2L3 in skeletal muscle cells causes a global downregulation in protein ubiquitination, most predominantly affecting motor, sarcomeric and extracellular matrix proteins, thus mediating their stabilization with regards to proteasomal degradation. This effect is not observed in the presence of the mammalian ortholog, suggesting functional divergence in the evolution of the parasite protein. These findings demonstrate the first example of host-parasite interactions via a parasite-derived Ub conjugating enzyme; an E2 that demonstrates a novel muscle protein stabilization function.

Dissecting molecular cross-talk between Nrf2 and NF-κB response pathways.

In most tissues, cells are exposed to frequent changes in levels of oxidative stress and inflammation. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and nuclear factor-κB (NF-κB) are the two key transcription factors that regulate cellular responses to oxidative stress and inflammation respectively. Pharmacological and genetic studies suggest that there is functional cross-talk between these two important pathways. The absence of Nrf2 can exacerbate NF-κB activity leading to increased cytokine production, whereas NF-κB can modulate Nrf2 transcription and activity, having both positive and negative effects on the target gene expression. This review focuses on the potentially complex molecular mechanisms that link the Nrf2 and NF-κB pathways and the importance of designing more effective therapeutic strategies to prevent or treat a broad range of neurological disorders.

The melanoma-associated antigen 1 (MAGEA1) protein stimulates the E3 ubiquitin-ligase activity of TRIM31 within a TRIM31-MAGEA1-NSE4 complex.

The MAGE (Melanoma-associated antigen) protein family members are structurally related to each other by a MAGE-homology domain comprised of 2 winged helix motifs WH/A and WH/B. This family specifically evolved in placental mammals although single homologs designated NSE3 (non-SMC element) exist in most eukaryotes. NSE3, together with its partner proteins NSE1 and NSE4 form a tight subcomplex of the structural maintenance of chromosomes SMC5-6 complex. Previously, we showed that interactions of the WH/B motif of the MAGE proteins with their NSE4/EID partners are evolutionarily conserved (including the MAGEA1-NSE4 interaction). In contrast, the interaction of the WH/A motif of NSE3 with NSE1 diverged in the MAGE paralogs. We hypothesized that the MAGE paralogs acquired new RING-finger-containing partners through their evolution and form MAGE complexes reminiscent of NSE1-NSE3-NSE4 trimers. In this work, we employed the yeast 2-hybrid system to screen a human RING-finger protein library against several MAGE baits. We identified a number of potential MAGE-RING interactions and confirmed several of them (MDM4, PCGF6, RNF166, TRAF6, TRIM8, TRIM31, TRIM41) in co-immunoprecipitation experiments. Among these MAGE-RING pairs, we chose to examine MAGEA1-TRIM31 in detail and showed that both WH/A and WH/B motifs of MAGEA1 bind to the coiled-coil domain of TRIM31 and that MAGEA1 interaction stimulates TRIM31 ubiquitin-ligase activity. In addition, TRIM31 directly binds to NSE4, suggesting the existence of a TRIM31-MAGEA1-NSE4 complex reminiscent of the NSE1-NSE3-NSE4 trimer. These results suggest that MAGEA1 functions as a co-factor of TRIM31 ubiquitin-ligase and that the TRIM31-MAGEA1-NSE4 complex may have evolved from an ancestral NSE1-NSE3-NSE4 complex.

Brusatol provokes a rapid and transient inhibition of Nrf2 signaling and sensitizes mammalian cells to chemical toxicity-implications for therapeutic targeting of Nrf2.

The transcription factor Nrf2 regulates the basal and inducible expression of a battery of cytoprotective genes. Whereas numerous Nrf2-inducing small molecules have been reported, very few chemical inhibitors of Nrf2 have been identified to date. The quassinoid brusatol has recently been shown to inhibit Nrf2 and ameliorate chemoresistance in vitro and in vivo. Here, we show that brusatol provokes a rapid and transient depletion of Nrf2 protein, through a posttranscriptional mechanism, in mouse Hepa-1c1c7 hepatoma cells. Importantly, brusatol also inhibits Nrf2 in freshly isolated primary human hepatocytes. In keeping with its ability to inhibit Nrf2 signaling, brusatol sensitizes Hepa-1c1c7 cells to chemical stress provoked by 2,4-dinitrochlorobenzene, iodoacetamide, and N-acetyl-p-benzoquinone imine, the hepatotoxic metabolite of acetaminophen. The inhibitory effect of brusatol toward Nrf2 is shown to be independent of its repressor Keap1, the proteasomal and autophagic protein degradation systems, and protein kinase signaling pathways that are known to modulate Nrf2 activity, implying the involvement of a novel means of Nrf2 regulation. These findings substantiate brusatol as a useful experimental tool for the inhibition of Nrf2 signaling and highlight the potential for therapeutic inhibition of Nrf2 to alter the risk of adverse events by reducing the capacity of nontarget cells to buffer against chemical and oxidative insults. These data will inform a rational assessment of the risk:benefit ratio of inhibiting Nrf2 in relevant therapeutic contexts, which is essential if compounds such as brusatol are to be developed into efficacious and safe drugs.

Network based meta-analysis prediction of microenvironmental relays involved in stemness of human embryonic stem cells.

Background. Human embryonic stem cells (hESCs) are pluripotent cells derived from the inner cell mass of in vitro fertilised blastocysts, which can either be maintained in an undifferentiated state or committed into lineages under determined culture conditions. These cells offer great potential for regenerative medicine, but at present, little is known about the mechanisms that regulate hESC stemness; in particular, the role of cell-cell and cell-extracellular matrix interactions remain relatively unexplored. Methods and Results. In this study we have performed an in silico analysis of cell-microenvironment interactions to identify novel proteins that may be responsible for the maintenance of hESC stemness. A hESC transcriptome of 8,934 mRNAs was assembled using a meta-analysis approach combining the analysis of microarrays and the use of databases for annotation. The STRING database was utilised to construct a protein-protein interaction network focused on extracellular and transcription factor components contained within the assembled transcriptome. This interactome was structurally studied and filtered to identify a short list of 92 candidate proteins, which may regulate hESC stemness. Conclusion. We hypothesise that this list of proteins, either connecting extracellular components with transcriptional networks, or with hub or bottleneck properties, may contain proteins likely to be involved in determining stemness.

Direct and indirect control of mitogen-activated protein kinase pathway-associated components, BRAP/IMP E3 ubiquitin ligase and CRAF/RAF1 kinase, by the deubiquitylating enzyme USP15.

The opposing regulators of ubiquitylation status, E3 ligases and deubiquitylases, are often found to be associated in complexes. Here we report on a novel interaction between the E3 ligase BRAP (also referred to as IMP), a negative regulator of the MAPK scaffold protein KSR, and two closely related deubiquitylases, USP15 and USP4. We map the interaction to the N-terminal DUSP-UBL domain of USP15 and the coiled coil region of BRAP. USP15 as well as USP4 oppose the autoubiquitylation of BRAP, whereas BRAP promotes the ubiquitylation of USP15. Importantly, USP15 but not USP4 depletion destabilizes BRAP by promoting its proteasomal degradation, and BRAP-protein levels can be rescued by reintroducing catalytically active but not inactive mutant USP15. Unexpectedly, USP15 depletion results in a decrease in amplitude of MAPK signaling in response to EGF and PDGF. We provide evidence for a model in which the dominant effect of prolonged USP15 depletion upon signal amplitude is due to a decrease in CRAF levels while allowing for the possibility that USP15 may also function to dampen MAPK signaling through direct stabilization of a negative regulator, the E3 ligase BRAP.

Structural basis of the intracellular sorting of the SNARE VAMP7 by the AP3 adaptor complex.

VAMP7 is involved in the fusion of late endocytic compartments with other membranes. One possible mechanism of VAMP7 delivery to these late compartments is via the AP3 trafficking adaptor. We show that the linker of the δ-adaptin subunit of AP3 binds the VAMP7 longin domain and determines the structure of their complex. Mutation of residues on both partners abolishes the interaction in vitro and in vivo. The binding of VAMP7 to δ-adaptin requires the VAMP7 SNARE motif to be engaged in SNARE complex formation and hence AP3 must transport VAMP7 when VAMP7 is part of a cis-SNARE complex. The absence of δ-adaptin causes destabilization of the AP3 complex in mouse mocha fibroblasts and mislocalization of VAMP7. The mislocalization can be rescued by transfection with wild-type δ-adaptin but not by δ-adaptin containing mutations that abolish VAMP7 binding, despite in all cases intact AP3 being present and LAMP1 trafficking being rescued.

Folylpolyglutamate synthetase gene transcription is regulated by a multiprotein complex that binds the TEL-AML1 fusion in acute lymphoblastic leukemia.

Acute Lymphoblastic Leukemia (ALL) non-random fusions influence clinical outcome and alter the accumulation of MTX-PGs in vivo. Analysis of primary ALL samples uncovered subtype-specific patterns of folate gene expression. Using an FPGS-luciferase reporter gene assay, we determined that E2A-PBX1 and TEL-AML1 expression decreased FPGS transcription. ChIP assays uncovered HDAC1, AML1, mSin3A, E2F, and Rb interactions with the FPGS promoter region. We demonstrate that FPGS expression is epigenetically regulated through binding of selected ALL fusions to a multiprotein complex, which also controls the cell cycle dependence of FPGS expression. This study provides insights into the pharmacogenomics of MTX in ALL subtypes.

Proteomic analysis of Nrf2 deficient transgenic mice reveals cellular defence and lipid metabolism as primary Nrf2-dependent pathways in the liver.

The transcription factor Nrf2 regulates expression of multiple cellular defence proteins through the antioxidant response element (ARE). Nrf2-deficient mice (Nrf2(-/-)) are highly susceptible to xenobiotic-mediated toxicity, but the precise molecular basis of enhanced toxicity is unknown. Oligonucleotide array studies suggest that a wide range of gene products is altered constitutively, however no equivalent proteomics analyses have been conducted. To define the range of Nrf2-regulated proteins at the constitutive level, protein expression profiling of livers from Nrf2(-/-) and wild type mice was conducted using both stable isotope labelling (iTRAQ) and gel electrophoresis methods. To establish a robust reproducible list of Nrf2-dependent proteins, three independent groups of mice were analysed. Correlative network analysis (MetaCore) identified two predominant groups of Nrf2-regulated proteins. As expected, one group comprised proteins involved in phase II drug metabolism, which were down-regulated in the absence of Nrf2. Surprisingly, the most profound changes were observed amongst proteins involved in the synthesis and metabolism of fatty acids and other lipids. Importantly, we show here for the first time, that the enzyme ATP-citrate lyase, responsible for acetyl-CoA production, is negatively regulated by Nrf2. This latter finding suggests that Nrf2 is a major regulator of cellular lipid disposition in the liver.

Network analysis of primary hepatocyte dedifferentiation using a shotgun proteomics approach.

The liver is the major site of xenobiotic metabolism and detoxification. Primary cultures of hepatocytes are a vital tool in the development of new therapeutic agents but their utility is hindered by the rapid loss of phenotype. Hepatocytes cultured in a sandwich of extracellular matrix protein maintain better hepatic function compared with cells cultured as a monolayer but a wide-ranging proteomics study of the differences in cultures has never been performed. We characterize the changing phenotype of rat hepatocytes in primary culture using iTRAQ proteomics and systems biology network analysis of the identified, significantly regulated, proteins. A total of 754 unique proteins were identified from 4 independent experiments. Of these, 413 proteins were common to at least 3 experiments and 328 proteins were identified in all experiments. Both culture systems displayed altered expression of many common proteins. Network analysis showed that the primary functions of these proteins were in metabolic pathways, immune responses and cytoskeleton remodelling. Monolayer cultures uniquely regulate proteins mapping to pathways of oxidative stress and cell migration, whereas sandwich culture affected translation regulation and apoptosis pathways. These experiments provide a detailed proteomics data set to direct further work into maintaining hepatic phenotype using cultured primary hepatocytes and stem cell derived hepatocyte-like cells.

Endogenous spartin (SPG20) is recruited to endosomes and lipid droplets and interacts with the ubiquitin E3 ligases AIP4 and AIP5.

The HSPs (hereditary spastic paraplegias) are genetic conditions in which there is distal degeneration of the longest axons of the corticospinal tract, resulting in spastic paralysis of the legs. The gene encoding spartin is mutated in Troyer syndrome, an HSP in which paralysis is accompanied by additional clinical features. There has been controversy over the subcellular distribution of spartin. We show here that, at steady state, endogenous spartin exists in a cytosolic pool that can be recruited to endosomes and to lipid droplets. Cytosolic endogenous spartin is mono-ubiquitinated and we demonstrate that it interacts via a PPXY motif with the ubiquitin E3 ligases AIP4 [atrophin-interacting protein 4; ITCH (itchy E3 ubiquitin protein ligase homologue] [corrected] and AIP5 (WWP1). Surprisingly, the PPXY motif, AIP4 and AIP5 are not required for spartin's ubiquitination, and so we propose that spartin acts as an adaptor for these proteins. Our results suggest that spartin is involved in diverse cellular functions, which may be of relevance to the complex phenotype seen in Troyer syndrome.