The mitotic (or spindle assembly) checkpoint system ensures accurate chromosome segregation in mitosis by preventing the onset of anaphase until correct bipolar attachment of sister chromosomes to the mitotic spindle is attained. It acts by promoting the assembly of a mitotic checkpoint complex (MCC), composed of mitotic checkpoint proteins BubR1, Bub3, Mad2, and Cdc20. MCC binds to and inhibits the action of ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome), which targets for degradation regulators of anaphase initiation. When the checkpoint system is satisfied, MCCs are disassembled, allowing the recovery of APC/C activity and initiation of anaphase. Many of the pathways of the disassembly of the different MCCs have been elucidated, but the mode of their regulation remained unknown. We find that UBR5 (ubiquitin-protein ligase N-recognin 5) is associated with the APC/C*MCC complex immunopurified from extracts of nocodazole-arrested HeLa cells. UBR5 binds to mitotic checkpoint proteins BubR1, Bub3, and Cdc20 and promotes their polyubiquitylation in vitro. The dissociation of a Bub3*BubR1 subcomplex of MCC is stimulated by UBR5-dependent ubiquitylation, as suggested by observations that this process in mitotic extracts requires UBR5 and α-β bond hydrolysis of adenosine triphosphate. Furthermore, a system reconstituted from purified recombinant components carries out UBR5- and ubiquitylation-dependent dissociation of Bub3*BubR1. Immunodepletion of UBR5 from mitotic extracts slows down the release of MCC components from APC/C and prolongs the lag period in the recovery of APC/C activity in the exit from mitotic checkpoint arrest. We suggest that UBR5 may be involved in the regulation of the inactivation of the mitotic checkpoint.

The infectious life cycle of the human immunodeficiency virus type 1 (HIV-1) is characterized by an ongoing battle between a compendium of cellular proteins that either promote or oppose viral replication. On the one hand, HIV-1 utilizes dependency factors to support and sustain infection and complete the viral life cycle. On the other hand, both inducible and constitutively expressed host factors mediate efficient and functionally diverse antiviral processes that counteract an infection. To shed light into the complex interplay between HIV-1 and cellular proteins, we previously performed a targeted siRNA screen to identify and characterize novel regulators of viral replication and identified Cullin 3 (Cul3) as a previously undescribed factor that negatively regulates HIV-1 replication. Cul3 is a component of E3-ubiquitin ligase complexes that target substrates for ubiquitin-dependent proteasomal degradation. In the present study, we show that Cul3 is expressed in HIV-1 target cells, such as CD4+ T cells, monocytes, and macrophages and depletion of Cul3 using siRNA or CRISPR/Cas9 increases HIV-1 infection in immortalized cells and primary CD4+ T cells. Conversely, overexpression of Cul3 reduces HIV-1 infection in single replication cycle assays. Importantly, the antiviral effect of Cul3 was mapped to the transcriptional stage of the viral life cycle, an effect which is independent of its role in regulating the G1/S cell cycle transition. Using isogenic viruses that only differ in their promotor region, we find that the NF-κB/NFAT transcription factor binding sites in the LTR are essential for Cul3-dependent regulation of viral gene expression. Although Cul3 effectively suppresses viral gene expression, HIV-1 does not appear to antagonize the antiviral function of Cul3 by targeting it for degradation. Taken together, these results indicate that Cul3 is a negative regulator of HIV-1 transcription which governs productive viral replication in infected cells.

Ribonucleoprotein (RNP) complexes form around nascent RNA during transcription to facilitate proper transcriptional elongation, RNA processing, and nuclear export. RNPs are highly heterogeneous, and different types of RNPs tend to package functionally related transcripts. These observations have inspired the hypothesis that RNP mediated mechanisms help specify coordinated gene expression. This hypothesis is supported by the observation that mutations in RNP components can cause defects in specific developmental pathways. How RNP biogenesis itself is regulated, however, is not well understood. The evolutionarily conserved THO RNP complex functions early during transcription to package nascent transcripts and facilitate subsequent RNP biogenesis. THO deficiency compromises transcriptional elongation as well as RNP mediated events like 3' end formation and nuclear export for some transcripts. Using molecularly manipulated cells and in vitro reconstituted biochemical reactions, we demonstrate that the essential THO protein component encoded by the Thoc1 gene is poly-ubiquitinated by the NEDD4-1 E3 ubiquitin ligase. Poly-ubiquitinated pThoc1 is degraded by the proteasome. These results indicate THO activity is regulated by the ubiquitin-proteasome pathway, and that this regulation is evolutionarily conserved between yeast and mammals. Manipulation of NEDD4-1 levels has modest effects on Thoc1 protein levels under steady state conditions, but destabilization of Thoc1 protein upon treatment with a transcriptional elongation inhibitor is dependent on NEDD4-1. This suggests NEDD4-1 functions in conjunction with other post-translational mechanisms to regulate Thoc1 protein and THO activity.

In eukaryotes, many proteins function in multi-subunit complexes that require proper assembly. To maintain complex stoichiometry, cells use the endoplasmic reticulum-associated degradation system to degrade unassembled membrane subunits, but how unassembled soluble proteins are eliminated is undefined. Here we show that degradation of unassembled soluble proteins (referred to as unassembled soluble protein degradation, USPD) requires the ubiquitin selective chaperone p97, its co-factor nuclear protein localization protein 4 (Npl4), and the proteasome. At the ubiquitin ligase level, the previously identified protein quality control ligase UBR1 (ubiquitin protein ligase E3 component n-recognin 1) and the related enzymes only process a subset of unassembled soluble proteins. We identify the homologous to the E6-AP carboxyl terminus (homologous to the E6-AP carboxyl terminus) domain-containing protein HUWE1 as a ubiquitin ligase for substrates bearing unshielded, hydrophobic segments. We used a stable isotope labeling with amino acids-based proteomic approach to identify endogenous HUWE1 substrates. Interestingly, many HUWE1 substrates form multi-protein complexes that function in the nucleus although HUWE1 itself is cytoplasmically localized. Inhibition of nuclear entry enhances HUWE1-mediated ubiquitination and degradation, suggesting that USPD occurs primarily in the cytoplasm. Altogether, these findings establish a new branch of the cytosolic protein quality control network, which removes surplus polypeptides to control protein homeostasis and nuclear complex assembly.

HACE1 is a HECT family E3 ubiquitin-protein ligase with broad but incompletely understood tumor suppressor activity. Here, we report a previously unrecognized link between HACE1 and signaling complexes containing mammalian target of rapamycin (mTOR). HACE1 blocks mTORC1 and mTORC2 activities by reducing mTOR stability in an E3 ligase-dependent manner. Mechanistically, HACE1 binds to and ubiquitylates Ras-related C3 botulinum toxin substrate 1 (RAC1) when RAC1 is associated with mTOR complexes, including at focal adhesions, leading to proteasomal degradation of RAC1. This in turn decreases the stability of mTOR to reduce mTORC1 and mTORC2 activity. HACE1 deficient cells show enhanced mTORC1/2 activity, which is reversed by chemical or genetic RAC1 inactivation but not in cells expressing the HACE1-insensitive mutant, RAC1K147R . In vivo, Rac1 deletion reverses enhanced mTOR expression in KRasG12D -driven lung tumors of Hace1-/- mice. HACE1 co-localizes with mTOR and RAC1, resulting in RAC1-dependent loss of mTOR protein stability. Together, our data demonstrate that HACE1 destabilizes mTOR by targeting RAC1 within mTOR-associated complexes, revealing a unique ubiquitin-dependent process to control the activity of mTOR signaling complexes.

Poly-ubiquitylation is a common post-translational modification that can impart various functions to a target protein. Several distinct mechanisms have been reported for the assembly of poly-ubiquitin chains, involving either stepwise transfer of ubiquitin monomers or attachment of a preformed poly-ubiquitin chain and requiring either a single pair of ubiquitin-conjugating enzyme (E2) and ubiquitin ligase (E3), or alternatively combinations of different E2s and E3s. We have analysed the mechanism of poly-ubiquitylation of the replication clamp PCNA by two cooperating E2-E3 pairs, Rad6-Rad18 and Ubc13-Mms2-Rad5. We find that the two complexes act sequentially and independently in chain initiation and stepwise elongation, respectively. While loading of PCNA onto DNA is essential for recognition by Rad6-Rad18, chain extension by Ubc13-Mms2-Rad5 is only slightly enhanced by loading. Moreover, in contrast to initiation, chain extension is tolerant to variations in the attachment site of the proximal ubiquitin moiety. Our results provide information about a unique conjugation mechanism that appears to be specialised for a regulatable pattern of dual modification.

In normal and transformed cells, the F-box protein p45(SKP2) is required for S phase and forms stable complexes with p19(SKP1) and cyclin A-cyclin-dependent kinase (CDK)2. Here we identify human CUL-1, a member of the cullin family, and the ubiquitin-conjugating enzyme CDC34 as additional partners of p45(SKP2) in vivo. CUL-1 also associates with cyclin A and p19(SKP1) in vivo and, with p45(SKP2), they assemble into a large multiprotein complex. In Saccharomyces cerevisiae, a complex of similar molecular composition (an F-box protein, a member of the cullin family and a homolog of p19(SKP1)) forms a functional E3 ubiquitin protein ligase complex, designated SCFCDC4, that facilitates ubiquitination of a CDK inhibitor by CDC34. The data presented here imply that the p45(SKP2)-CUL-1-p19(SKP1) complex may be a human representative of an SCF-type E3 ubiquitin protein ligase. We propose that all eukaryotic cells may use a common ubiquitin conjugation apparatus to promote S phase. Finally, we show that multiprotein complex formation involving p45(SKP2)-CUL-1 and p19(SKP1) is governed, in part, by periodic, S phase-specific accumulation of the p45(SKP2) subunit and by the p45(SKP2)-bound cyclin A-CDK2. The dependency of p45(SKP2)-p19(SKP1) complex formation on cyclin A-CDK2 may ensure tight coordination of the activities of the cell cycle clock with those of a potential ubiquitin conjugation pathway.

Cardiac Kir2.1 and Nav1.5 channels generate the inward rectifier K+ (IK1) and the Na+ (INa) currents, respectively. There is a mutual interplay between the ventricular INa and IK1 densities, because Nav1.5 and Kir2.1 channels exhibit positive reciprocal modulation. Here we compared some of the biological properties of Nav1.5 and Kir2.1 channels when they are expressed together or separately to get further insights regarding their putative interaction. First we demonstrated by proximity ligation assays (PLAs) that in the membrane of ventricular myocytes Nav1.5 and Kir2.1 proteins are in close proximity to each other (<40 nm apart). Furthermore, intracellular dialysis with anti-Nav1.5 and anti-Kir2.1 antibodies suggested that these channels form complexes. Patch-clamp experiments in heterologous transfection systems demonstrated that the inhibition of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) decreased the INa and the IK1 generated by Nav1.5 and Kir2.1 channels when they were coexpressed, but not the IK1 generated by Kir2.1 channels alone, suggesting that complexes, but not Kir2.1 channels, are a substrate of CaMKII. Furthermore, inhibition of CaMKII precluded the interaction between Nav1.5 and Kir2.1 channels. Inhibition of 14-3-3 proteins did not modify the INa and IK1 densities generated by each channel separately, whereas it decreased the INa and IK1 generated when they were coexpressed. However, inhibition of 14-3-3 proteins did not abolish the Nav1.5-Kir2.1 interaction. Inhibition of dynamin-dependent endocytosis reduced the internalization of Kir2.1 but not of Nav1.5 or Kir2.1-Nav1.5 complexes. Inhibition of cytoskeleton-dependent vesicular trafficking via the dynein/dynactin motor increased the IK1, but reduced the INa, thus suggesting that the dynein/dynactin motor is preferentially involved in the backward and forward traffic of Kir2.1 and Nav1.5, respectively. Conversely, the dynein/dynactin motor participated in the forward movement of Kir2.1-Nav1.5 complexes. Ubiquitination by Nedd4-2 ubiquitin-protein ligase promoted the Nav1.5 degradation by the proteasome, but not that of Kir2.1 channels. Importantly, the Kir2.1-Nav1.5 complexes were degraded following this route as demonstrated by the overexpression of Nedd4-2 and the inhibition of the proteasome with MG132. These results suggested that Kir2.1 and Nav1.5 channels closely interact with each other leading to the formation of a pool of complexed channels whose biology is similar to that of the Nav1.5 channels.

Little progresses have been made in the treatment of glioblastoma (GBM), the most aggressive and lethal among brain tumors. Recently we have demonstrated that Chloride Intracellular Channel-1 (CLIC1) is overexpressed in GBM compared to normal tissues, with highest expression in patients with poor prognosis. Moreover, CLIC1-silencing in cancer stem cells (CSCs) isolated from human GBM patients negatively influences proliferative capacity and self-renewal properties in vitro and impairs the in vivo tumorigenic potential. Here we show that CLIC1 exists also as a circulating protein, secreted via extracellular vesicles (EVs) released by either cell lines or GBM-derived CSCs. Extracellular vesicles (EVs), comprising exosomes and microvesicles based on their composition and biophysical properties, have been shown to sustain tumor growth in a variety of model systems, including GBM. Interestingly, treatment of GBM cells with CLIC1-containing EVs stimulates cell growth both in vitro and in vivo in a CLIC1-dose dependent manner. EVs derived from CLIC1-overexpressing GBM cells are strong inducers of proliferation in vitro and tumor engraftment in vivo. These stimulations are significantly attenuated by treatment of GBM cells with EVs derived from CLIC1-silenced cells. However, CLIC1 modulation appears to have no direct role in EV structure, biogenesis and secretion. These findings reveal that, apart from the function of CLIC1 cellular reservoir, CLIC1 contained in EVs is a novel regulator of GBM growth.

Ubiquitination is an important post-translational modification involved in diverse biological processes. Therefore, genomewide representation of the ubiquitination system for a species is important.

Pathogens commonly utilize endocytic pathways to gain cellular access. The endosomal pattern recognition receptors TLR7 and TLR9 detect pathogen-encoded nucleic acids to initiate MyD88-dependent proinflammatory responses to microbial infection. Using genome-wide RNAi screening and integrative systems-based analysis, we identify 190 cofactors required for TLR7- and TLR9-directed signaling responses. A set of cofactors were crossprofiled for their activities downstream of several immunoreceptors and then functionally mapped based on the known architecture of NF-κB signaling pathways. Protein complexes and pathways involved in ubiquitin-protein ligase activities, sphingolipid metabolism, chromatin modifications, and ancient stress responses were found to modulate innate recognition of endosomal nucleic acids. Additionally, hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) was characterized as necessary for ubiquitin-dependent TLR9 targeting to the endolysosome. Proteins and pathways identified here should prove useful in delineating strategies to manipulate innate responses for treatment of autoimmune disorders and microbial infection.

There is a body of evidence that the aging immune system is linked to cancer. In this study, with aging- and immune-related DNA methylation data, we investigated the DNA methylation regulation changes in promoters with other regions of genes during aging and their association with the immune-cell proportion in the circulating whole blood of individuals. The analyses for aging- and CD4+ T cell proportion-derived differential genes showed that ubiquitination plays an important role in the aging immune system and tumorigenesis. Therefore, starting from a set of pre-annotated ubiquitination genes, we found that among the differentially ubiquitinated genes, DZIP3, an E3 ubiquitin ligase with no reports on its function in immune cells and tumorigenesis, was significantly associated with both aging (P-value = 3.86e-06) and CD4+ T cell proportion (P-value = 1.97e-05) in circulating blood. By collecting a cohort of 100 colon cancer patients and 50 healthy individuals, we validated that the 1st exon DNA methylation of DZIP3 could predict the onset of early stage (AUC = 0.833, OR = 8.82) and all pTNM stages of colorectal cancer (AUC = 0.782, OR = 5.70). Thus, the epigenetically regulated ubiquitination machine plays an important role in immune aging and tumorigenesis.

Sequence variants in vacuolar protein sorting 35 (VPS35) have been reported to be associated with Parkinson's disease (PD). To investigate if the genetic variants in VPS35 contribute to Taiwanese PD, VPS35 cDNA fragments from 62 patients with PD were sequenced. A cohort of PD (n = 560) and ethnically matched controls (n = 506) were further examined for the identified mutation. The effects of the mutation on cation-independent mannose-6-phosphate receptor (CI-MPR) sorting and mitochondrial morphology were further examined in 293T cells expressing the mutant VPS35. Here, a novel heterozygous A320V in the VPS35 gene was identified in two late-onset PD (LOPD) patients, which was absent in 506 normal controls. Expression of the A320V mutant in 293T cells demonstrated increased colocalization of VPS35 with CI-MPR and decreased CI-MPR and lysosomal-associated membrane protein 2 (LAMP2) levels. Decreased CI-MPR manifested in missorting of cathepsin D and decreased proteolysis of α-synuclein. A320V mutation also increased mitochondrial E3 ubiquitin protein ligase 1 (MUL1) and thus led to mitofusin 2 (MFN2) degradation. The results suggest that the expression of VPS35 A320V leads to disrupted CI-MPR sorting and impaired mitochondrial morphology, which may partly explain its action in PD.

Cytokines, such as interferons, erythropoietin, leptin and most interleukins, signal through type 1 cytokine receptors and activate the canonical JAK-STAT pathway. Aberrant cytokine signalling underlies numerous pathologies and adequate, temporary receptor activation is therefore under tight control. Negative-feedback mechanisms are very well studied, but cellular sensitivity also depends on the number of receptors exposed at the cell surface. This is determined by the equilibrium between receptor synthesis and transport to the plasma membrane, internalisation and recycling, degradation and ectodomain shedding, but the molecular basis of how cells establish steady state receptor levels is poorly understood. Here, we report that ring finger protein 41 (RNF41, also known as E3 ubiquitin-protein ligase Nrdp1) interacts with JAK2-associated cytokine receptor complexes and modulates their cell surface exposure and signalling. Moreover, ectopic expression of RNF41 affected turnover of leptin, leukaemia inhibitory factor and interleukin-6 receptor in a dual way: it blocked intracellular cathepsin-L-dependent receptor cleavage and concomitantly enhanced receptor shedding by metalloproteases of the ADAM family. Receptor degradation and shedding are thus interconnected phenomena with a single protein, RNF41, determining the balance.

Many diagnostic and therapeutic concepts require antibodies of high specificity. Recombinant binder libraries and related selection approaches allow the efficient isolation of antibodies against almost every target of interest. Nevertheless, it cannot be guaranteed that selected antibodies perform well and interact specifically enough with analytes unless an elaborate characterisation is performed. Here, we present an approach to shorten this process by combining the selection of suitable antibodies with the identification of informative target molecules by means of antibody microarrays, thereby reducing the effort of antibody characterisation by concentrating on relevant molecules. In a pilot scheme, a library of 456 single-chain variable fragment (scFv) binders to 134 antigens was used. They were arranged in a microarray format and incubated with the protein content of clinical tissue samples isolated from pancreatic ductal adenocarcinoma and healthy pancreas, as well as recurrent and non-recurrent bladder tumours. We observed significant variation in the expression of the E3 ubiquitin-protein ligase (CHFR) as well as the glutamate receptor interacting protein 2 (GRIP2), for example, always with more than one of the scFvs binding to these targets. Only the relevant antibodies were then characterised further on antigen microarrays and by surface plasmon resonance experiments so as to select the most specific and highest affinity antibodies. These binders were in turn used to confirm a microarray result by immunohistochemistry analysis.

Understanding molecular interactions and regulatory mechanisms in tumor initiation, progression, and treatment response are key requirements towards advanced cancer diagnosis and novel treatment procedures in personalized medicine. Beyond decoding the gene expression, malfunctioning and cancer-related epigenetic pathways, investigations of the spatial receptor arrangements in membranes and genome organization in cell nuclei, on the nano-scale, contribute to elucidating complex molecular mechanisms in cells and tissues. By these means, the correlation between cell function and spatial organization of molecules or molecular complexes can be studied, with respect to carcinogenesis, tumor sensitivity or tumor resistance to anticancer therapies, like radiation or antibody treatment. Here, we present several new applications for bio-molecular nano-probes and super-resolution, laser fluorescence localization microscopy and their potential in life sciences, especially in biomedical and cancer research. By means of a tool-box of fluorescent antibodies, green fluorescent protein (GFP) tagging, or specific oligonucleotides, we present tumor relevant re-arrangements of Erb-receptors in membranes, spatial organization of Smad specific ubiquitin protein ligase 2 (Smurf2) in the cytosol, tumor cell characteristic heterochromatin organization, and molecular re-arrangements induced by radiation or antibody treatment. The main purpose of this article is to demonstrate how nano-scaled distance measurements between bio-molecules, tagged by appropriate nano-probes, can be applied to elucidate structures and conformations of molecular complexes which are characteristic of tumorigenesis and treatment responses. These applications open new avenues towards a better interpretation of the spatial organization and treatment responses of functionally relevant molecules, at the single cell level, in normal and cancer cells, offering new potentials for individualized medicine.

Cancer-associated fibroblasts (CAFs) are the most abundant stromal components in the tumor microenvironment (TME) and closely involved in tumor progression. However, the precise biological functions and molecular mechanisms of CAFs in the TME have yet to be understood. Here, we demonstrate that WEE2-AS1 is highly expressed in the CAF-derived small extracellular vesicles (sEVs). Moreover, WEE2-AS1 is markedly higher in plasma sEVs of CRC patients than in healthy subjects and its high level predicts advanced pathological staging and poor survival. Then, we conducted a series of in vitro and in vivo experiments. Elevated expression of WEE2-AS1 in sEVs increases CRC cell proliferation in vitro. Importantly, aberrant CAF-sEVsWEE2-AS1 leads to tumor formation and progression in BALB/c nude mice and promotes AOM/DSS-induced tumorigenesis. Mechanistically, WEE2-AS1 functions as a modular scaffold for the MOB1A and E3 ubiquitin-protein ligase praja2 complexes, leading to MOB1A degradation via the ubiquitin-proteasome pathway. The Hippo pathway is then inhibited and more YAP are transported into the nucleus, where they activate downstream gene transcription. Together, our data reveal that CAF-sEVsWEE2-AS1 interacts with MOB1A, promotes degradation of MOB1A, inhibits the Hippo pathway, and facilitates the growth of CRC cells. Hence, exosomal WEE2-AS1 may be a promising therapeutic target and circulating biomarker for CRC diagnosis and prognosis.

Meta-analysis revealed that Leucine Zipper Down-Regulated In Cancer 1 (LDOC1) increased methylation more in people with lung tumors than in those who were healthy and never smoked. Quantitative methylation-specific PCR revealed that cigarette smoke condensate (CSC) exposure drives LDOC1 promoter hypermethylation and silence in human bronchial cells. Immunohistochemistry studies showed that LDOC1 downregulation is associated with poor survival of patients with lung cancer. Loss and gain of LDOC1 functions enhanced and attenuated aggressive phenotypes in lung adenocarcinoma A549 and non⁻small cell lung carcinoma H1299 cell lines, respectively. We found that LDOC1 deficiency led to reinforcing a reciprocal loop of IL-6/JAK2/STAT3, through which LDOC1 mediates the cancer progression. LDOC1 knockdown considerably augmented tumorigenesis and the phosphorylation of JAK2 and STAT3 in vivo. Results from immunoprecipitation and immunofluorescent confocal microscopy indicated that LDOC1 negatively regulates JAK2 activity by forming multiple protein complexes with pJAK2 and E3 ubiquitin-protein ligase LNX1, and in turn, LDOC1 targets pJAK2 to cause ubiquitin-dependent proteasomal degradation. LDOC1 deficiency attenuates the interactions between LNX1 and pJAK2, leading to ineffective ubiquitination of pJAK2, which activates STAT3. Overall, our results elucidated a crucial role of LDOC1 in lung cancer and revealed how LDOC1 acts as a bridge between tobacco exposure and the IL-6/JAK2/STAT3 loop in this human malignancy.

Serglycin has been initially characterized as an intracellular proteoglycan expressed by hematopoietic cells. All inflammatory cells highly synthesize serglycin and store it in granules, where it interacts with numerous inflammatory mediators, such as proteases, chemokines, cytokines, and growth factors. Serglycin is implicated in their storage into the granules and their protection since they are secreted as complexes and delivered to their targets after secretion. During the last decade, numerous studies have demonstrated that serglycin is also synthesized by various non-hematopoietic cell types. It has been shown that serglycin is highly expressed by tumor cells and promotes their aggressive phenotype and confers resistance against drugs and complement system attack. Apart from its direct beneficial role to tumor cells, serglycin may promote the inflammatory process in the tumor cell microenvironment thus enhancing tumor development. In the present review, we discuss the role of serglycin in inflammation and tumor progression.

Therapeutic gene silencing in the brain is usually achieved using highly invasive intracranial administration methods and/or comparatively toxic vectors. In this work, we use a relatively biocompatible vector: poly(ethylene glycol) star-shaped polymer capped with amine groups (4APPA) via the nose to brain route. 4APPA complexes anti- itchy E3 ubiquitin protein ligase (anti-ITCH) siRNA to form positively charged (zeta potential +15 ± 5 mV) 150 nm nanoparticles. The siRNA-4APPA polyplexes demonstrated low cellular toxicity (IC50 = 13.92 ± 6 mg mL-1) in the A431 cell line and were three orders of magnitude less toxic than Lipofectamine 2000 (IC50 = 0.033 ± 0.04 mg mL-1) in this cell line. Cell association and uptake of fluorescently labelled siRNA bound to siRNA-4APPA nanoparticles was demonstrated using fluorescent activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), respectively. Gene silencing of the ITCH gene was observed in vitro in the A431 cell line (65% down regulation when compared to the use of anti-ITCH siRNA alone). On intranasal dosing with fluorescently labelled siRNA-4APPA polyplexes, fluorescence was seen in the cells of the olfactory bulb, cerebral cortex and mid-brain regions. Finally, down regulation of ITCH was seen in the brain cells (54 ± 13% ITCH remaining compared to untreated controls) in a healthy rat model, following intranasal dosing of siRNA-4APPA nanoparticles (0.15 mg kg-1 siRNA twice daily for 3 days). Gene silencing in the brain may be achieved by intranasal administration of siRNA- poly(ethylene glycol) based polyplexes.