Ubiquitylation is a highly controlled post-translational modification of proteins, in which proteins are conjugated either with monoubiquitin or polyubiquitin chains. Ubiquitin modifications on target proteins are recognized by ubiquitin-binding domains, which are found in several effector proteins. In this study, we describe for the first time how ubiquitin controls the function of the Toll-interacting protein (Tollip), which is an effector protein in the innate immune signaling pathway and an adaptor protein for endosomal trafficking. We have demonstrated that the central C2 domain of Tollip preferentially interacts with phosphoinositides with moderate affinity. Remarkably, we found that ubiquitin modulates Tollip's lipid binding. We have observed an ubiquitin dose-dependent inhibition of binding of Tollip to phosphoinositides and it does so specifically by blocking Tollip C2 domain-phosphoinositide interactions. This led us to discover that the Tollip C2 domain is a novel ubiquitin-binding domain. In addition, we have biophysically characterized the association of the Tollip CUE domain to ubiquitin and compared it with Tollip C2 domain-ubiquitin binding. The Tollip CUE domain reversibly binds ubiquitin with affinity higher than C2 domain and at a site that overlaps with that corresponding to the Tollip C2 domain. We have also found that ubiquitin binding to dimeric Tollip CUE domain induces a drastic conformational change in the protein, leading to the formation of a heterodimeric Tollip CUE-ubiquitin complex. These data suggest that ubiquitin binding to the Tollip C2 and CUE domains and ubiquitin-mediated dissociation of CUE dimer reduces the affinity of the Tollip protein for endosomal phosphoinositides, allowing Tollip's cytoplasmic sequestration. Overall, our findings will provide the structural and molecular basis to understand how Tollip works inside the cell and commit itself to cytosolic signalling or endosomal trafficking in a ligand dependent manner. / Ph. D.

Fear memory formation requires a coordination of molecular events, including protein synthesis, protein degradation, and epigenetic regulation of gene expression, throughout a circuit of brain regions. One mechanism highly studied for its involvement in protein degradation during fear memory is the ubiquitin-proteasome system (UPS), which utilizes the small protein ubiquitin to label proteins. Ubiquitin contains eight linkage sites that each lead to a unique outcome for the protein being labeled and a protein can gain one (monoubiquitination) or multiple (polyubiquitination) ubiquitins. The 26S proteasome is the catalytic component of the UPS and is comprised of a 20S catalytic core surrounded by two 19S regulatory caps. Phosphorylation of 19S cap regulatory subunit RPT6 at serine 120 (pRPT6-S120) has been widely implicated in controlling activity-dependent increases in proteasome activity. Interestingly, sex differences have been observed in proteasome-mediated protein degradation in the amygdala and hippocampus during fear memory formation. However, female subjects have only recently been regularly included in rodent behavioral studies so the majority of data on mechanisms of fear memory apply solely to the male brain. Considering post-traumatic stress disorder (PTSD) is two to three times more prevalent in females compared to males, understanding the mechanisms involved in fear memory in both sexes is important for understanding sex-specific development of fear-based disorders, such as PTSD. Importantly, the UPS also has non-proteolytic functions independent of proteasome-mediated protein degradation. For example, monoubiquitination and some forms of polyubiquitination do not lead to protein degradation. Additionally, 19S cap regulatory subunit RPT6 has been found to function independently of its role in the proteasome, where it has a transcription-like role in the hippocampus of male rats during fear memory formation. Unfortunately, proteasome-independent functions of the UPS have not been extensively studied in terms of different forms of ubiquitination. Additionally, it is unclear whether phosphorylation of RPT6 is necessary for its non-proteolytic roles in memory formation and the role of proteasome-independent RPT6 in general has not been investigated in female subjects. Here, we address these gaps in knowledge by 1) investigating sex-differences in the role of lysine 63 (K63-) polyubiquitination, a proteasome-independent ubiquitin linkage, in the amygdala during fear memory formation, 2) studying the role of proteasome-independent RPT6 in the hippocampus of female rats during fear memory formation, and 3) identifying proteasome-independent RPT6 target genes as well as the role of phosphorylation status of RPT6 at Serine-120 for its transcriptional activity during memory formation in the hippocampus of male rats. We first found that levels of K63-polyubiquitination targeting in the amygdala were increased in female, but not male, rats during fear memory formation. Interestingly, K63-polyubiquitination targeted proteins involved in ATP synthesis and proteasome functions in the amygdala of females and genetic manipulation of the K63 codon in the ubiquitin coding gene led to decreased ATP levels and proteasome activity. Additionally, this manipulation only led to impaired fear memory in females, suggesting that K63-polyubiquitination has a sex-selective role in the amygdala, where it regulates fear memory in females, but not males. We then investigated the role of proteasome-independent RPT6 in the hippocampus of females and males during fear memory formation. In females, we found RPT6 did not bind to DNA regions in the c-fos gene, a previously identified RPT6 target gene in males. However, RPT6 did bind to monoubiquitination of histone H2B at lysine-120 (H2BubiK120), an epigenetic modification identified as an RPT6 binding partner in males, suggesting a potential role for proteasome-independent RPT6 in transcriptional regulation in the hippocampus of female rats. In males, we identified RPT6 targets genes during memory formation, found that RPT6 DNA binding alone altered gene expression, and lastly observed that pRPT6-S120 was necessary for RPT6 to bind DNA and regulate transcription during memory formation. Collectively, these data reveal sex-differences in proteasome-independent roles of the UPS through ubiquitination and proteasomal subunits in both the amygdala and hippocampus during fear memory formation. Considering males and females have differences in PTSD prevalence, understanding proteasome-independent roles of the UPS in both sexes may lead to a better understanding of PTSD development as well as potential therapeutic targets in each sex. / Doctor of Philosophy / In order to remember a fear-provoking memory, or any memory, it must be stored in the brain after the event. To store a fear memory, cells in specific areas of the brain have to destroy some proteins and activate or shut off certain genes using epigenetic mechanisms. Although the DNA itself never changes, epigenetic mechanisms recruit proteins to sit on the DNA to make it more (activate) or less (shut off) accessible. For protein destruction, it has been shown that brain cells use a mechanism called the ubiquitin-proteasome system (UPS) during fear memory storage. The UPS uses a small protein called ubiquitin to label proteins in the cell. Ubiquitin is versatile in its ability to label proteins due to it having eight different binding sites that can be used as a label. Some proteins only gain one ubiquitin (monoubiquitination), while other proteins can gain multiple ubiquitin proteins (polyubiquitination) and both the number of ubiquitins and the label used determines what happens to the protein. When a protein is labeled to be destroyed, the UPS uses a large complex of proteins called the 26S proteasome, which contains a section called the 20S catalytic core and two 19S regulatory caps that sit above and below the 20S core. It has been shown that when one of the proteins in the 19S regulatory cap called RPT6 gains a phosphate molecule at the 120th amino acid, which is a serine amino acid, it increases the number of proteins destroyed by the 26S proteasome. Interestingly, the UPS does not destroy proteins in the same way in male and female brains during storage of a fear memory. This is important because females are 2-3 times more likely to develop post-traumatic stress disorder (PTSD) than males, but it is unclear why, making the study of mechanisms involved in fear memory storage in both males and females important. The UPS also functions in ways that do not involve destroying proteins. For example, proteins with a monoubiquitination label are often not destroyed. Additionally, it has been observed that RPT6, a protein in the 19S regulatory cap, can work outside of the 19S regulatory cap of the 26S proteasome to activate genes in brain cells of male rats. Sadly, the ubiquitin labels that do not cause protein destruction have not been well studied. It is also unclear whether RPT6 must gain a phosphate group to activate genes and if it activates genes in both male and female brains during fear memory storage. In the present study, we investigated the role of a common ubiquitin linkage that does not cause protein destruction called lysine 63- (K63-) polyubiquitination during fear memory storage in the amygdala, the emotional control center of the brain, in male and female rats. We found that K63-polyubiquitination is increased in female, but not male, rats during fear memory storage. In females, K63-polyubiquitination was involved in making new ATP as well as controlling the number of proteins destroyed by the 26S proteasome. Lastly, we found that female, but not male, rats required K63-polyubiquitination in the amygdala for fear memory storage, suggesting a female-specific use of this ubiquitin label. In this study, we also studied the role of RPT6 in the hippocampus, another key area of the brain for memory storage, of male and female rats. In females, we found RPT6 did not activate the same gene we previously identified in male rats, but it did bind with a monoubiquitination label on a protein that DNA is wrapped around, known as a histone. Due to this finding, it appears that RPT6 may act freely of the 19S regulatory cap to alter accessibility of genes in the hippocampus of females. On the other hand, in males we found that RPT6 activates some genes and shuts off other genes and can do so by sitting on DNA by itself. We also found that the addition of phosphate to RPT6 was necessary for it to sit on a gene and activate it during fear memory storage. These results show differences in UPS mechanisms during fear memory storage between males and females, which will be important for understanding why females are more likely to develop PTSD than males and for identifying potential treatment options.

<p>A molecular depiction of the assembly, interaction and regulation of protein complexes is essential to the understanding of biological functions of protein complexes. Structural analysis of protein complexes by Nuclear Magnetic Resonance (NMR) has relied heavily on the detection and assignment of intermolecular Nuclear Overhauser Effects (NOEs) that define the interactions of protons at the molecular interface. Intermolecular NOEs have traditionally been detected from 3-D half-filtered NOE experiments by suppressing intramolecular NOEs prior to NOE transfer. However, due to insufficient suppression of undesirable signals and a lack of dispersion in the H dimension, data analysis is complicated by the interference of residual intramolecular NOEs and assignment ambiguity, both of which can lead to distorted or even erroneously packed protein complex structures. Leveraging the recent development of fast NMR technology based on sparse sampling in our lab, we developed a strategy for reliable identification and assignment of intermolecular NOEs using high resolution 4-D NOE difference spectroscopy. Spectral subtraction of individually labeled components from a uniformly labeled protein complex yields an "omit" spectrum containing only intermolecular NOEs with little signal degeneracy. </p><p>The benefit of such a strategy is first demonstrated in structural analysis of a homooligomeric protein complexes, the foldon trimer. We show that intermolecular NOEs collected from the 4-D omit NOE spectrum can be directly utilized for automated structural analysis of the foldon trimer by CYANA, whereas intermolecular NOEs derived from 3-D half-filtered NOE experiments failed to generate a converged structure under the same condition. </p><p>Such a strategy was further demonstrated on a heterodimeric protein complex in translesion sysnthesis (TLS), a DNA damage tolerance pathway. The TLS machinery consists of several translesion DNA polymerases that are recruited to the stalled replication fork in response to monoubiquitinated proliferating cell nuclear antigen (PCNA) in order to bypass DNA lesions encountered during genomic replication. The recruitment and assembly of translesion machinery is heavily dependent on ubiquitin-binding domains, including ubiquitin-binding motifs (UBMs) and ubiquitin-binding zinc fingers (UBZs) that are found in translesion DNA polymerases. Two conserved ubiquitin-binding motifs (UBM1 and UBM2) are found in the Y-family polymerase (Pol) &iota, both of which contribute to ubiquitin-mediated accumulation of Pol &iota during TLS. Although the Pol&iota UBM2-ubiquitin complex has been previous reported by our lab and others, the Pol &iota UBM1-ubiquitin complex has remained a challenge due to significant signal overlap in conventional 3-D NOE spectroscopy. In order to determine the molecular basis for ubiquitin recognition of Pol &iota, we solved the structures of human Pol &iota UBM1 and its complex with ubiquitin by 4-D fast NMR, revealing a signature helix-turn-helix motif that recognizes ubiquitin through an unconventional surface centered at L8 of ubiquitin. Importantly, the use of 4-D omit NOE spectroscopy unambiguously revealed an augmented ubiquitin binding interface that encompasses the C-terminal tail of UBM1.</p><p>4-D omit NOE spectroscopy was also used to study the Fanconi anemia associated protein 20 (FAAP20)-ubiquitin complex within the Fanconi Anemia (FA) complexes required for efficient repair of DNA interstrand crosslinks (ICLs), a process that is mediated by the ubiquitin-binding zinc finger (UBZ) domain of FAAP20. Unexpectedly, we show that the FAAP20-ubiquitin interaction extends beyond the compact UBZ module and is accompanied by transforming the disordered C-terminal tail of FAAP20 into a rigid &beta-loop, with the invariant C-terminal tryptophan (W180 of human FAAP20) emanating toward I44 of ubiquitin for enhanced binding. Accordingly, alanine substitution of the absolutely conserved C-terminal tryptophan residue of FAAP20 abolishes ubiquitin binding and impairs FA core complex-mediated ICL repair <italic>in vivo<italic>.</p><p>Reliable detection and unambiguous assignment of intermolecular NOEs is essential to NMR-based structure determination of protein complexes. The development of 4-D omit NOE spectroscopy in this thesis overcomes many limitations of conventional 3-D half-filtered experiments to allow for reliable detection and unambiguous assignment of intermolecular NOEs of heterodimeric complexes and homooligomeric complexes. These advantages render such a strategy particularly attractive for structural studies of protein complexes by biomolecular NMR.</p> / Dissertation

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

A cotton fiber cDNA, GhRING1, and its corresponding gene have been cloned and characterized. The GhRING1 gene encodes a RING-type ubiquitin ligase (E3) containing 337 amino acids (aa). The GhRING1 protein contains a RING finger motif with conserved cysteine and histine residues at the C-terminus and is classified as a C3H2C3-type RING protein. Blast searches show that GhRING1 has the highest homology to At3g19950 from Arabidopsis. Real time RT-PCR analysis indicates that the GhRING1 gene is highly expressed in cotton fiber in a developmental manner. The transcript level of the GhRING1 gene reaches a maximum in elongating fibers at 15 DPA. In vitro auto-ubiquitination assays using wheat germ extract and a reconstitution system demonstrate that GhRING1 has the ubiquitin E3 ligase activity. A fiber specific lipid transfer protein 4 (FSltp4) is identified as the target substrate of GhRING1 by using a bacterial two-hybrid system. The binding of GhRING1 and FSltp4 is confirmed by using an in vitro pull down assay and a yeast two-hybrid system. The histochemical GUS assay was performed to analyze tissue specificity of the GhRING1 and At3g19950 promoters in transgenic Arabidopsis plants. The GUS assay shows that the promoter of At3g19950 is highly activated in leaves, roots, trichomes and also in anthers and stigma of flowers. In contrast, the GUS expression directed by the promoter of GhRING1 is only located at stipules and anthers and stigma of flowers. The GhRING1 is the first ubiquitin E3 gene isolated and studied from cotton. Based on the expression pattern of GhRING1, FSltp4, and GhUBC E2s and the identification of a fiber-specific target protein, FSltp4, we propose that protein ubiquitination occurs in fiber and the ubiquitin-proteasome pathway regulates fiber development.

No description available.

No description available.

Integral membrane proteins are targeted to discrete compartments through the action of a number of transport pathways. The post-translational modification of cargo with ubiquitin is a key regulator of protein sorting. Ubiquitinated cargo are bound by specific cargo sorting machinery and directed towards the appropriate destination. Therefore, the identification and characterization of the proteins involved in cargo ubiquitination is critical to understanding the regulation of protein sorting. In the work presented here, we characterize the role of the E3 ubiquitin ligases, RNF126 and Rabring7, in two distinct membrane trafficking pathways. First, we show that RNF126 and Rabring7 are involved in the ligand induced downregulation of cell surface receptors. RNF126 and Rabring7 associate with the EGFR, amongst other RTKs, and promotes its ubiquitination. RNF126 and Rabring7 are required for the efficient sorting of the EGFR through the late endocytic compartment. We also show that the depletion of Rabring7 attenuates the degradation of MET and that both RNF126 and Rabring7 regulate the sorting of CXCR4 from an early endocytic compartment. In addition, the depletion of RNF126 or Rabring7 destabilizes ESCRT-II and reduces the number of multivesicular bodies formed after EGF stimulation. Second, we found that RNF126 regulates the sorting of the CI-MPR. In cells transiently depleted of RNF126, the CI-MPR is dispersed into a transferrin receptor positive endocytic compartment. This effect is specific to the CI-MPR as other cargos that are sorted between the endosome at the Golgi remain unaffected. We found that RNF126 physically associates with the clathrin adaptor GGA3 and promotes its ubiquitination, suggesting that RNF126 regulates GGA3 mediated CI-MPR sorting. Together, this work furthers our understanding regarding the role of ubiquitin in membrane traffic.

Die essenzielle, Ubiquitin-selektive ATPase p97 reguliert eine Vielzahl unterschiedlicher Prozesse in Eukaryoten. Dazu zählen Proteinqualitätskontrolle, DNA-Reparatur, Signaltransduktion, Zellzykluskontrolle, Autophagie sowie das endolysosomale System. Diese unterschiedlichen Funktionen von p97 werden durch die Bindung von Kofaktoren engmaschig gesteuert und kontrolliert. Die größte und am besten untersuchte Gruppe von p97-Kofaktoren sind die Proteine der UBX Familie. Diese zeichnen sich durch den Besitz einer UBX-Domäne aus, welche die Bindung an p97 vermittelt. Das in höheren Eukaryoten konservierte Familienmitglied UBXD1 besitzt darüber hinaus mit einer PUB-Domäne und einem VIM-Motiv noch mindestens zwei weitere p97-Bindemodule. UBXD1 kann an Vesikel des endolysosomalen Degradationssytems lokalisieren, seine genauen zellulären Funktionen sind jedoch noch weitgehend unbekannt. Ziel dieser Arbeit war die funktionelle Charakterisierung von humanem UBXD1. Dafür wurden Kandidaten eines zuvor durchgeführten Yeast-Two-Hybrid-Screens auf ihre Two Hybrid-Interaktion mit unterschiedlichen UBXD1-Varianten getestet. Darüber hinaus wurde durch Immunpräzipitationsexperimente untersucht, ob die Kandidatenproteine auch in Säugerzellen mit UBXD1 interagieren. Als vielversprechende neue Bindungspartner von UBXD1 wurden so die Ubiquitin-Ligase TRIAD3A und das Ubiquitin-editierende Protein A20 identifiziert. Desweiteren konnte gezeigt werden, dass die Interaktion zwischen UBXD1 und A20 von einer funktionellen PUB Domäne und dem siebten Zinkfinger Motiv von A20 abhängig ist. Da sowohl TRIAD3A als auch A20 negative Regulatoren des NF B Signalweges sind, wurde daraufhin untersucht, ob auch UBXD1 eine Funktion in diesem Signalweg besitzt. Tatsächlich war in UBXD1-depletierten HeLa 57A-Zellen die NF B-abhängige Expression eines Reportgens nach Aktivierung des Signalweges durch TNF, IL-1, Doxorubicin und H2O2 stark reduziert. Dabei spricht die verringerte Aktivierung nach unterschiedlichen Stimuli für eine generelle Rolle von UBXD1 im NF B Signalweg. Durch quantitative Echtzeit-PCR konnte gezeigt werden, dass in HeLa- und HEK293T-Zellen nach UBXD1-Depletion auch die Expression endogener NF B Zielgene verringert ist. Da in UBXD1-depletierten Zellen nach Stimulation mit TNF oder IL-1 bereits die Kerntranslokation des NF B-Transkriptionsfaktor p65 reduziert ist, ist davon auszugehen, dass UBXD1 an einer früheren Phase der Aktivierung des Signalweges beteiligt ist. Möglicherweise ist dies darauf zurückzuführen, dass UBXD1 bekannte Funktionen von A20 reguliert und etwa die Bindung von A20 an Vesikel des endolysosomalen Systems oder an lineare Ubiquitinketten beeinflusst. Diese Arbeit beschreibt somit eine neue Funktion des p97-Kofaktors UBXD1 im NF B-Signalweg. / The essential, ubiquitin-selective ATPase p97 regulates a variety of cellular processes in eukaryotes. Among others, these include protein quality control, DNA repair, signal-transduction, cell cycle control, autophagy and the endolysosomal system. The distinct functions of p97 are tightly controlled by regulatory cofactors. UBX domain-containing proteins are the largest and best studied group of p97 cofactors . They are characterized by a UBX domain, which mediates binding to p97. The family-member UBXD1 is highly conserved in higher eukaryotes and possesses at least two additional p97 binding modules, a PUB domain and a VIM motif. While UBXD1 can localize to vesicles of the endolysosomal degradation system, its exact cellular function is still poorly understood. The aim of this study was the functional characterisation of human UBXD1. To that end, candidates of a previous yeast two-hybrid screen were tested for their two-hybrid interaction with different UBXD1 variants. Immunoprecipitation experiments were used to analyse if the candidates also interact with UBXD1 in mammalian cells. This led to the identification of the ubiquitin-ligase TRIAD3A and the ubiquitin-editing protein A20 as promising new binding partners of UBXD1. Moreover, it could be demonstrated that the interaction between UBXD1 and A20 depends on a functional PUB domain and the seventh zinc finger motif of A20. Because both TRIAD3A and A20 are negative regulators of the NF-B signaling pathway, it was subsequently tested if UBXD1 also has a function in NF-B signaling. Indeed, UBXD1-depleted HeLa 57A cells showed a strongly reduced NF B dependent expression of a reporter gene after activation of the signaling pathway by TNF, IL-1, Doxorubicin and H2O2. The reduced activity observed after various stimuli argues for a general role of UBXD1 in the NF-B signaling pathway. Quantitative real-time PCR demonstrated that the expression of endogenous NF-B target genes in HeLa and HEK293T cells was also reduced upon UBXD1-depletion. Since the nuclear translocation of the NF-B subunit p65 upon stimulation with TNF or IL-1was also reduced in UBXD1-depleted cells, UBXD1 is likely to participate in an earlier phase of NF-B activation. It is possible that UBXD1 regulates a known function of A20 and influences for example the binding of A20 to endocytic vesicles or to linear ubiquitin chains. In summary, this work describes a novel function of the p97 cofactor UBXD1 as a positive regulator of the NF-B signaling pathway.

Induction of type I interferons by the transcription factors IRF3 and IRF7 is essential in the initiation of antiviral innate immunity. Activation of IRF3/7 requires C-terminal phosphorylation by the upstream kinases TBK1/IKKi, where IRF3/7 phosphorylation promotes dimerization, and subsequent nuclear translocation to the IFN-beta promoter. Recent studies have described the ubiquitin-editing enzyme A20 as a negative regulator of IRF3 signaling by associating with TBK1/IKKi, however the regulatory mechanism of A20 inhibition remains unclear. Here we describe the adaptor protein, TAX1BP1, as a key regulator of A20 function in terminating signaling to IRF3. Murine embryonic fibroblasts (MEFs) deficient in TAX1BP1 displayed increased amounts of IFN-beta production upon viral challenge compared to WT MEFs. TAX1BP1 inhibited virus-mediated activation of IRF3 at the level of TBK1/IKKi. TAX1BP1 and A20 blocked antiviral signaling by disrupting K63-linked polyubiquitination of TBK1/IKKi independently of the A20 deubiquitination (DUB) domain. Furthermore, TAX1BP1 was required for A20 effector function as A20 was defective for the targeting and inactivation of TBK1 and IKKi in Tax1bp1–/– MEFs. Additionally, we found the E3 ubiquitin ligase TRAF3 to play a critical role in promoting TBK1/IKKi ubiquitination. Collectively, our results demonstrate TBK1/IKKi to be novel substrates for A20 and further identifies a novel mechanism whereby A20 and TAX1BP1 restrict antiviral signaling by disrupting a TRAF3/TBK1/IKKi signaling complex. Several viruses utilize a number of strategies to evade the host innate immune response by inhibiting the production of type I interferons. The Human T-cell leukemia virus type 1 (HTLV-1) has been shown to block interferon signaling, however the mechanism of inhibition is poorly understood. We show here that the HTLV-1 encoded protein, Tax plays a critical role in blunting the activation of type I interferons. Tax expression rendered MEFs hyper-permissive in supporting virus replication. Correspondingly, Tax blocked the production of IFN-beta. Interestingly, Tax did not require NEMO interaction to inhibit antiviral signaling to IRF3/7. Instead, Tax targeted RIP1 and further blocked IRF7 K63-linked polyubiquitination. Altogether, we show that Tax inhibits IFN activation by disrupting the ubiquitin dependent activation of IRF7 mediated by RIP1.