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Trametinib is a MEK1/2 inhibitor and exerts anticancer activity against a variety of cancers. However, the effect of Trametinib on colorectal cancer (CRC) is not well understood. In the current study, our results demonstrate the ability of sub‐toxic doses of Trametinib to enhance TRAIL‐mediated apoptosis in CRC cells. Our findings also indicate that Trametinib and TRAIL activate caspase‐dependent apoptosis in CRC cells. Moreover, Mcl‐1 overexpression can reduce apoptosis in CRC cells treated with Trametinib with or without TRAIL. We further demonstrate that Trametinib degrades Mcl‐1 through the proteasome pathway. In addition, GSK‐3β phosphorylates Mcl‐1 at S159 and promotes Mcl‐1 degradation. The E3 ligase FBW7, known to polyubiquitinate Mcl‐1, is involved in Trametinib‐induced Mcl‐1 degradation. Taken together, these results provide the first evidence that Trametinib enhances TRAIL‐mediated apoptosis through FBW7‐dependent Mcl‐1 ubiquitination and degradation.

Günümüzde kanser tedavisi için en umut vaat eden uygulama halen kemoterapi olmasına rağmen kanser ilaçlarına karşı ilaç direnci, medikal onkolojideki en büyük engel teşkil etmeye devam etmektedir. Kemoterapide meydana gelen başarısızlık sebeplerinin %90’ı, kanserde olan veya oluşan ilaç direnci ile meydana gelen invazyon ve metastazdan kaynaklanmaktadır. Tüm dünyada prostat kanseri, erkeklerde akciğer kanserinden (%14,3) sonra ikinci (%14,1) en sık görülen kanser tipidir. Ubiquitin ve proteozom yolağı (UPY) proteinler için bir kalite kontrol sistemidir. Hasarlı ve yanlış katlanmış proteinlerin yıkımından sorumludur. UPY; transkripsiyonda, hücre döngüsünde, hücre içi sinyallerde, antijen sunumunda ve epigenetik mekanizmalarda görev almaktadır. Proteozom inhibitörleri hematolojik malignitelerde kullanılmaktadır. Bortezomib bir proteozom inhibitörüdür. Proteozom inhibitörlerinin solid tümörlerde kullanılması umut vadetmektedir. Proteozom inhibitörlerinin otoimmün hastalıklarda da kullanılabileceği düşünülmektedir. Hücrelerin stres kaynaklarına verdiği yanıtlardan biri, hücrelerdeki moleküler değişiklikler ile kalıcı bir şekilde hücre döngüsü tutuklanması olan senesenstir. Senesent hücreler genellikle biyoaktif proteinler salgılarlar ve bunlar senesent olmayan komşu hücreleri uyararak tümör gelişimine sebep de olabilmektedir ve aynı zamanda yaşlanma sürecindeki kronik enflamasyondan sorumlu olduğu düşünülmektedir. Bu tezde; bir prostat kanseri hücre hattı olan PC3 hücrelerinde [PC3-P, parental; PC3-R, dirençli], klinikte prostat tedavi protokollerinde henüz kullanılmayan ve solid tümörlerde kullanılması umut vadeden bortezomib ilacına karşı oluşmuş ilaç direnci mekanizmaları ile bortezomib’in PC3 hücreleri üzerindeki senesens rolü araştırılmıştır. Dirençli PC3-R ve parental PC3-P hücrelerinde yaptığımız bortezomib sitotoksisitesi, hücre ölüm modu, AO/EB çift boyaması ve 3 boyutlu hücre kültürü çalışmalarında; PC3-R hücrelerinin PC3-P hücrelerine göre ilaca çok daha dirençli oldukları, apoptoza daha dirençli oldukları ve ilaç varlığında bile 3 boyutlu kompleks yapıları oluşturarak kendilerini destekleyebildikleri ve direnç mekanizmasının geri dönüşümlü olmadığı gösterilmiştir. Direnç mekanizmasını aydınlatmaya yönelik MAPK ve otofajik aktivasyon, ısı şok proteinlerin ekspresyonu gibi incelenen stratejik yolaklarda; stres koşullarında aktive olan ve dirence sebebiyet veren bu yolakların ve ilgili moleküllerin dirençli PC3-R hücrelerinde değil, parental PC3-P hücrelerinde bir sağkalım mekanizması olarak aktive oldukları gözlenmiştir. PC3-R hücrelerindeki bortezomib direncinin ilacın bağlandığı PSMB5 enziminde meydana gelen bir mutasyondan kaynaklanabileceği düşünülmektedir. PC3-P ve PC3-R hücrelerinde yapılan senesens deneyleri sonucunda, senesens markerlerinin bortezomib muamelesi sonucunda ekseriyetle azaldığı gözlenmiştir ve bu sebeple bortezomibin bir senolitik etkisinin olabileceği öne sürülmektedir. Buna ek olarak PC3-R hücrelerinde senesens, PC3-P hücrelerine göre daha az görülmektedir ve bunun ilaç direncine bir katkısı olabilir. Son olarak, literatüre göre ERK1 ve ERK2 proteinlerinin fonksiyonel olarak işlevlerinin aynı olup olmadığı hakkında tartışmalar vardır. Tez çalışmamızda bortezomib muamelesinden sonra ERK1 fosforilasyonlarının artarken ERK2 fosforilasyonlarının azaldığı görülmüştür. Bu olay, ERK1 ve ERK2’nin farklı regüle edilebildiğini ve bu sebeple farklı işlevler görebileceklerine işaret etmektedir. Chemotherapy is still the most promising application for cancer treatment today. However, drug resistance to cancer drugs remains the biggest obstacle in medical oncology as 90% of the causes of failure in chemotherapy are due to invasion and metastasis caused by drug resistance in cancer. Prostate cancer is the second most common type of cancer in men (14.1%) following lung cancer (14.3%) in the world. Ubiquitin and the proteasome pathway (UPP) is a quality control system for proteins. It is responsible for the destruction of damaged and misfolded proteins. UPP is involved in transcription, cell cycle, intracellular signals, antigen presentation, and epigenetic mechanisms. Proteasome inhibitors are used in hematological malignancies. Bortezomib is a proteasome inhibitor. The use of proteasome inhibitors in solid tumors is promising. It is thought that proteasome inhibitors can also be used in autoimmune diseases. One of the cells' responses to stressors is senescence, which is a permanent cell cycle arrest by molecular changes in the cells. Senescent cells often secrete bioactive proteins, which can stimulate neighboring non-senescent cells to cause tumorigenesis and are also thought to be responsible for chronic inflammation in the aging process. Bortezomib is not yet used in prostate treatment protocols in clinics and bortezomib is promising to be used in solid tumors. In this thesis; drug resistance mechanisms against bortezomib resistance and the senescence role of bortezomib on PC3 cells were investigated in a prostate cancer cell line PC3 cells [PC3-P, parental; PC3-R, resistant]. In the studies of bortezomib cytotoxicity, cell death mode, AO/EB double staining, and 3D cell culture performed in resistant PC3-R and parental PC3-P cells; PC3-R cells were found to be much more resistant to bortezomib and apoptosis than PC3-P cells. We have shown that the PC3-R cells can support themselves by forming 3-dimensional complex structures even in the presence of bortezomib and the resistance mechanism in resistant PC3-R cells is not reversible. In the strategic pathways we examined, such as MAPK and autophagic activation, the expression of heat shock proteins for elucidating the resistance mechanism; it has been demonstrated that these pathways and their related molecules, which are activated under stress conditions and cause resistance, are more strongly activated in parental PC3-P cells and not in resistant PC3-R cells due to cell survival mechanisms. We think that bortezomib resistance may be due to a mutation in the PSMB5 enzyme to which bortezomib drug binds in the PC3-R cells. As a result of the senescence experiments performed in PC3-P and PC3-R cells, senescence markers were observed to be decreased to bortezomib treatment in general, and we, therefore, suggest that bortezomib may have a senolytic effect. Moreover, it has been observed that senescence is less common in PC3-R cells than in PC3-P cells, and this may contribute to drug resistance. Finally, according to the literature, there are debates about whether the ERK1 and ERK2 proteins are functionally the same. In this thesis study, it was observed that ERK1 phosphorylations increased while ERK2 phosphorylations decreased after bortezomib treatment. This indicates that ERK1 and ERK2 can be regulated differently and hence they may possess distinct functions.

Life is a non-equilibrium phenomenon. Owing to their high free energy content, the macromolecules of life tend to spontaneously react with ambient oxygen and water and turn into more stable inorganic molecules. A similar thermodynamic picture applies to the complex shapes of proteins: While a polypeptide is emerging unfolded from the ribosome, it may spontaneously acquire secondary structures and collapse into its functional native conformation. The spontaneity of this process is evidence that the free energy of the unstructured state is higher than that of the structured native state. Yet, under stress or because of mutations, complex polypeptides may fail to reach their native conformation and form instead thermodynamically stable aggregates devoid of biological activity. Cells have evolved molecular chaperones to actively counteract the misfolding of stress-labile proteins dictated by equilibrium thermodynamics. HSP60, HSP70 and HSP100 can inject energy from ATP hydrolysis into the forceful unfolding of stable misfolded structures in proteins and convert them into unstable intermediates that can collapse into the native state, even under conditions inauspicious for that state. Aggregates and misfolded proteins may also be forcefully unfolded and degraded by chaperone-gated endo-cellular proteases, and in eukaryotes also by chaperone-mediated autophagy, paving the way for their replacement by new, unaltered functional proteins. The greater energy cost of degrading and replacing a polypeptide, with respect to the cost of its chaperone-mediated repair represents a thermodynamic dilemma: some easily repairable proteins are better to be processed by chaperones, while it can be wasteful to uselessly try recover overly compromised molecules, which should instead be degraded and replaced. Evolution has solved this conundrum by creating a host of unfolding chaperones and degradation machines and by tuning their cellular amounts and activity rates.

Background: Collagen type IV (COL4)-related nephropathy includes a variety of kidney diseases that occur with or without extra-renal manifestations caused by COL4A3-5 mutations. Previous studies revealed several novel mutations, including three COL4A3 missense mutations (G619R, G801R, and C1616Y) and the COL4A3 chr:228172489delA c.4317delA p.Thr1440ProfsX87 frameshift mutation that resulted in a truncated NC1 domain (hereafter named COL4A3 c.4317delA); however, the mutation mechanisms that lead to podocyte injury remain unclear. This study aimed to further explore the mutation mechanisms that lead to podocyte injury. Methods: Wild-type (WT) and four mutant COL4A3 segments were constructed into a lentiviral plasmid, then stably transfected into human podocytes. Real-time polymerase chain reaction and Western blotting were applied to detect endoplasmic reticulum stress (ERS)- and apoptosis-related mRNA and protein levels. Then, human podocytes were treated with MG132 (a proteasome inhibitor) and brefeldin A (a transport protein inhibitor). The human podocyte findings were verified by the establishment of a mus-Col4a3 knockout mouse monoclonal podocyte using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technology. Results: Our data showed that COL4A3 mRNA was significantly overexpressed in the lentivirus stably transfected podocytes. Moreover, the COL4A3 protein level was significantly increased in all groups except the COL4A3 c.4317delA group. Compared to the other test groups, the COL4A3 c.4317delA group showed excessive ERS and apoptosis. Podocytes treated with MG132 showed remarkably increased intra-cellular expression of the COL4A3 c.4317delA mutation. MG132 intervention improved higher ERS and apoptosis levels in the COL4A3 c.4317delA group. Mouse monoclonal podocytes with COL4A3 chr:82717932insA c.4852insA p.Arg1618ThrfsX4 were successfully acquired; this NC1-truncated mutation suggested a higher level of ERS and relatively remarkable level of apoptosis compared to that of the WT group. Conclusions: We demonstrated that excessive ERS and ERS-induced apoptosis were involved in the podocyte injury caused by the NC1-truncated COL4A3 mutation. Furthermore, proteasome pathway intervention might become a potential treatment for collagen type IV-related nephropathy caused by a severely truncated COL4A3 mutation. Key words: Collagen type IV-related nephropathy; COL4A3 mutation; Podocyte injury; Proteasome pathway; MG132; Endoplasmic reticulum stress

Clinical reports have found that with the improvement of treatment, most septic patients are able to survive the severe systemic inflammatory response and to enter the immunoparalysis stage. Considering that immunoparalysis leads to numerous deaths of clinical sepsis patients, alleviation of the occurrence and development of immunoparalysis has become a top priority in the treatment of sepsis. In our study, we investigate the effects of oroxylin A on sepsis in cecal ligation and puncture (CLP) mice. We find that the 60 h + 84 h (30 mg/kg) injection scheme of oroxylin A induce the production of pro-inflammatory factors, and further significantly improves the survival of CLP mice during the middle or late stages of sepsis. Mechanistically, C/EBP-homologous protein (CHOP) is upregulated and plays anti-inflammatory roles to facilitate the development of immunoparalysis in CLP mice. Oroxylin A induces the transcription of E3 ligase F-box only protein 15 gene (fbxo15), and activated FBXO15 protein binds to CHOP and further mediates the degradation of CHOP through the proteasome pathway, which eventually relieves the immunoparalysis of CLP mice. Taken together, these findings suggest oroxylin A relieves the immunoparalysis of CLP mice by degrading CHOP through interacting with FBXO15.

Multi-factorial diseases are illnesses that exploit multiple cellular processes, or stages within one process, and thus highly targeted therapies often succumb to the disease, losing efficacy as resistance sets in. Combination therapies have become a mainstay to battle these diseases, however these regimens are plagued with caveats. An emerging avenue to treat multi-factorial diseases is polypharmacology, wherein a single drug is rationally designed to bind multiple targets, and is widely touted to be superior to combination therapy by inherently addressing the latter's shortcomings, which include poor patient compliance, narrow therapeutic windows and spiraling healthcare costs. Through its roles in intracellular trafficking, cell motility, mitosis, protein folding and as a back-up to the proteasome pathway, HDAC6 has rapidly become an exciting new target for therapeutics, particularly in the discovery of new drugs to treat Alzheimer's disease and cancer. Herein, we describe recent efforts to marry together HDAC pharmacophores, with a particular emphasis on HDAC6 selectivity, with those of other targets towards the discovery of potent therapeutics to treat these evasive diseases. Such polypharmacological agents may supercede combination therapies through inherent synergism, permitting reduced dosing, wider therapeutic windows and improved compliance.

The ubiquitin proteasome system (UPS) is a highly conserved pathway that tightly regulates protein turnover in cells. This process is integral to neuronal development, differentiation, and function. Several members of the UPS are disrupted in neuropsychiatric disorders, highlighting the importance of this pathway in brain development and function. In this review, we discuss some of these pathway members, the molecular processes they regulate, and the potential for targeting the UPS in an effort to develop therapeutic strategies in neuropsychiatric and neurodevelopmental disorders.

Understanding the interplay between the proteasome pathway and autophagy in response to dual PI3K/mTOR inhibition in myeloma cells is essential for their effective clinical application

We have previously demonstrated that the E3 ligase Human Constitutive Photomorphogenic Protein (huCOP1) is expressed in human keratinocytes and negatively regulates p53. The MutS homolog 2 (MSH2) protein plays a central role in DNA MMR mechanism and is implicated in the cellular response to anticancer agents, such as cisplatin. Our aim was to clarify whether huCOP1 plays a role in DNA MMR by affecting MSH2 protein level in human keratinocytes. To define the role of huCOP1 in DNA mismatch repair, we determined whether huCOP1 affects MSH2 abundance. MSH2 protein level was detected by immunocytochemical staining using a keratinocyte cell line in which the expression level of huCOP1 was stably decreased (siCOP1). To investigate whether huCOP1 silencing influences cisplatin-induced cell death, control and siCOP1 keratinocyte cells were treated with increasing concentrations of cisplatin and cell viability was recorded after 48 and 96 h. Stable silencing of huCOP1 in human keratinocytes resulted in a reduced level of MSH2 protein. huCOP1 silencing also sensitized keratinocytes to the interstrand crosslinking inducer cisplatin. Our results indicate that decreased huCOP1 correlates with lower MSH2 levels. These protein level changes lead to increased sensitivity toward cisplatin treatment, implicating that huCOP1 plays a positive role in maintaining genome integrity in human keratinocytes.

Background: The ubiquitin–proteasome pathway functions as a main pathway in intracellular protein degradation and plays a vital role in almost all cellular events. Various inhibitors of this pathway have been developed for research purposes. The recent approval of bortezomib (PS-341, Velcade®), a proteasome inhibitor, for the treatment of multiple myeloma has opened the way to the discovery of drugs targeting the proteasome and other components of the ubiquitin–proteasome pathway. Objectives: We review the current understanding of the ubiquitin–proteasome pathway and inhibitors targeting this pathway, including proteasome inhibitors, as candidate drugs for chemical therapy. Methods: Preclinical and clinical data for inhibitors of the proteasome and the ubiquitin–proteasome pathway are discussed. Conclusions: The proteasome and other members in the ubiquitin–proteasome pathway have emerged as novel therapeutic targets.

The transcription factor PU.1 regulates erythrocyte differentiation. We previously reported that F5-5 erythroblasts differentiate into erythrocytes in response to activin by degrading PU.1, and that inhibiting PU.1- degradation suppresses F5-5 cell differentiation into erythrocytes. These findings suggest that regulating PU.1 degradation is critical for terminal differentiation of erythrocytes. Here, we investigate the mechanism underlying PU.1 degradation during successive differentiation of erythrocytes. Using 2D-MS proteomic analysis, we show that proteasome subunits and proteins required for degradation by proteasomes immunoprecipitate with PU.1 in response to activin. Furthermore, a proteasome inhibitor, lactacystin, partially suppresses differentiation of F5-5 cells into erythrocytes in response to activin, and partially inhibits PU.1 degradation. Our results indicate that degradation of PU.1 necessary for erythrocyte differentiation occurs, in part, through the proteasome pathway.

Cryptochrome1 and 2 play a critical role in the molecular oscillations of the circadian clocks of central and peripheral tissues in mammals. Mouse Cryptochrome2 (mCRY2) is phosphorylated at Ser557 in the liver, in which the Ser557-phosphorylated form accumulates during the night in parallel with mCRY2 protein. Phosphorylation of mCRY2 at Ser557 allows subsequent phosphorylation at Ser553 by glycogen synthase kinase-3beta (GSK-3beta), resulting in efficient degradation of mCRY2 by a proteasome pathway. In the present study, we found that mCRY2 is phosphorylated at Ser557 also in the region of the mouse brain containing the suprachiasmatic nucleus (SCN), the central circadian clock tissue. Daily fluctuation of the Ser557-phosphorylation level in the SCN region suggests an important role of sequential phosphorylation of Ser557 and Ser553 in the rhythmic degradation of mCRY2 in both central and peripheral clocks of mice.

The K48R mutant ubiquitin can exert profound in vivo protective effects against a variety of insults, including agents of direct clinical relevance. The manipulation of the ubiquitin/proteasome pathway has enormous potential for clinical benefit, and it is not unreasonable to expect that such benefits will include diseases of aging.

Two papers published in 1984 by the Varshavsky laboratory revealed that the ubiquitin/proteasome pathway is the principal system for degradation of short-lived proteins in mammalian cells, setting the stage for future demonstrations of this pathway's many regulatory roles. This perspective discusses the impact of those papers and highlights some of the subsequent insights that have led to our current appreciation of the breadth of ubiquitin-mediated signaling.

During aging, the production of free radicals increases. This can result in damage to protein, the accumulation of which is characteristic of the aging process. This questions the efficacy of proteolytic systems. Among these systems, the proteasome and the adenosine triphosphate-ubiquitin-dependent pathway have been shown to play an important role in the elimination of abnormal proteins. There are two major steps in the ubiquitin-proteasome pathway: the conjugation of a polyubiquitin degradation signal to the substrate and the subsequent degradation of the tagged protein by the 26S proteasome. The 26S proteasome is build-up from the 20S proteasome, which is a cylinder-shaped multimeric complex, and two additional 19S complexes. The 20S proteasome can also bind to 11S regulator and is then implicated in antigen presentation. These regulators confer a high adaptability on proteasome. With advancing age, predisposition to neurodegenerative diseases increases. These diseases are also characterized by protein aggregation. Several findings such as the presence of ubiquinated proteins, usually broken down by proteasomes, and genetic anomalies involving the ubiquitin-proteasome system (parkin, UCH-L1) suggest a link between the ubiquitin-proteasome pathway and the genesis of these diseases.

The present study was conducted to explore the potential role of proteasome pathway in NSAIDs-induced apoptosis. We employed sulindac as a NSAID, and chose the lactacystin for inhibition of proteasome activity. Assessment of apoptosis and proteasome activity assay were undertaken. We demonstrated that sulindac treatment resulted in a decrease of proteasome activity, and that the co-treatment of a proteasome inhibitor lactacystin potentiated the extent of sulindac-induced apoptosis in HT-29 cells by augmentation of the decrease in proteasome activity. Elucidation of the mechanism underlying the regression of colon cancers by combinations of sulindac and lactacystin seems to be an immediate challenge for the near future.

The Vif (viral infectivity factor) protein of human immunodeficiency virus type-1 (HIV-1) is critical for HIV-1 infectivity. CBF-β is required for HIV-1 Vif function, as it increases the steady-state level of the HIV-1 Vif protein to promote host restriction factor APOBEC3 degradation. However, the precise mechanism by which CBF-β promotes HIV-1 Vif levels remains unclear. In the present study, we provided evidences that CBF-β promoted steady-state levels of HIV-1 Vif by inhibiting the degradation of HIV-1 Vif through the proteasome pathway. Our results reveal a new mechanism by which a cellular protein supports viral infectivity by inhibiting viral protein degradation.

The identification of genetic and epigenetic factors that are associated with an increased risk of developing amyotrophic lateral sclerosis (ALS), or that modify the age of onset or rate of progression, requires a multimodal research strategy, facilitated through international collaboration. The discovery of several ALS genes strongly linked to RNA biology, the proteasome pathway, and axonal transport suggest they have an important role in pathogenesis, but the immense complexity of these processes is also apparent. The increasing rate of genetic discoveries brings the hope of designing more targeted and efficacious therapies.

International audience; Heavy metals are known to generate reactive oxygen species that lead to the oxidation and fragmentation of proteins, which become toxic when accumulated in the cell. In this study, we investigated the role of the proteasome during cadmium stress in the leaves of Arabidopsis thaliana plants. Using biochemical and proteomics approaches, we present the first evidence of an active proteasome pathway in plants. We identified and characterized the peptidases acting sequentially downstream from the proteasome in animal cells as follows: tripeptidyl-peptidase II, thimet oligopeptidase, and leucine aminopeptidase. We investigated the proteasome proteolytic pathway response in the leaves of 6-week-old A. thaliana plants grown hydroponically for 24, 48, and 144 h in the presence or absence of 50 mum cadmium. The gene expression and proteolytic activity of the proteasome and the different proteases of the pathway were found to be up-regulated in response to cadmium. In an in vitro assay, oxidized bovine serum albumin and lysozyme were more readily degraded in the presence of 20 S proteasome and tripeptidyl-peptidase II than their nonoxidized form, suggesting that oxidized proteins are preferentially degraded by the Arabidopsis 20 S proteasome pathway. These results show that, in response to cadmium, the 20 S proteasome proteolytic pathway is up-regulated at both RNA and activity levels in Arabidopsis leaves and may play a role in degrading oxidized proteins generated by the stress.

The binding of phosphorylated peptides to the receptor plays a major role in many basic cellular processes in a variety of pathological states. Human β-TrCP is a key component of a recently characterized E3 ubiquitin ligase complex that regulates protein degradation through the ubiquitin-dependent proteasome pathway. Docking studies were carried out to explore the structural requirements for the β-TrCP substrates. Docking studies were performed on the bound conformation of the phosphorylated peptides determined by NMR, whereas the β-TrCP structure was derived by X-ray from Protein Data Bank. After the docking calculation, during which the peptides were conformationally restrained, the complex presented herein was analyzed in terms of ligand-protein interactions and properties of contacting surfaces. The structural requirements for phosphorylated substrates in interaction with β-TrCP were explored and compared with experimental data from TRNOESY and STD NMR results. The analysis revealed that the bend of t...

The binding of phosphorylated peptides to the receptor plays a major role in many basic cellular processes in a variety of pathological states. Human beta-TrCP is a key component of a recently characterized E3 ubiquitin ligase complex that regulates protein degradation through the ubiquitin-dependent proteasome pathway. Docking studies were carried out to explore the structural requirements for the beta-TrCP substrates. Docking studies were performed on the bound conformation of the phosphorylated peptides determined by NMR, whereas the beta-TrCP structure was derived by X-ray from Protein Data Bank. After the docking calculation, during which the peptides were conformationally restrained, the complex presented herein was analyzed in terms of ligand-protein interactions and properties of contacting surfaces. The structural requirements for phosphorylated substrates in interaction with beta-TrCP were explored and compared with experimental data from TRNOESY and STD NMR results. The analysis revealed that the bend of the peptide structures, which is indispensable for beta-TrCP recognition, aligns two charged phosphate groups and a central hydrophobic group in a favorable arrangement that leads to the burial of the peptide surface in the binding cleft upon complexation. Through docking simulations, we have identified different specific binding pockets of beta-TrCP according to the ligand in interaction. These data should be valuable in the rational design of a ligand to be used in therapeutic approaches.

The Delta/Notch signal transduction pathway is an evolutionary conserved pathway involved in many diverse developmental processes. These include neurogenesis, somitogenesis, left-right development, pancreatic development and development of the sensory hair in the inner ear. Numerous genes have been identified in the last years that are part of the signal transduction pathway or can influence the pathway in a way. A model to explain the process of lateral inhibition, one of the main features of the pathway, has been established. However, it is not clear how the known genes involved in Delta/Notch signalling can account for such diverse processes. In order to get new insights into the regulation of Delta/Notch signalling and to identify targets of Dll1 the transcriptome and the proteome of E10.5 wildtype and Dll1-/- embryos was analysed using DNA-chip technology and 2D-gelelectrophoresis combined with mass spectrometry. In the transcriptome analysis 22 up- and 30 downregulated transcripts were identified. In the proteome analysis 13 proteins were up- and 37 proteins were downregulated at E10.5. Further methods to verify the data on transcriptomics side included whole mount in situ hybridisation and real-time PCR and semi-quantitative immunoblotting on the proteomics side. The most promising candidate out of this analyses is the gene Ifitm1 which shows expression in regions where Delta/Notch signalling is known to occur, namely the presomitic mesoderm and the latest formed somites. The expression was strongly reduced in Dll1, Dll3 and Jag1 mutants which indicates a direct connection between Dll1 or Delta/Notch signalling respectively, and Ifitm1. Furthermore Ifitm1 was only recently discovered as marker for primordial germ cells and has no assigned function so far. To assess the exact function of Ifitm1 in the mouse a classical knockout targeting construct has been designed and prepared. The knockout animals will provide insights into the relevance of Ifitm1 for somitogenesis and other developmental processes. Further genes have been identified which show expression patterns indicative of Delta/Notch signalling and which also were altered in Dll1 mutant mouse embryos, and some also in Dll3 and Jag1 embryos. These genes are Csk, Ddx6, Nes, Sema5b and Smarcc1. On the protein level a number of interesting proteins have been identified. These include six subunits of the 26S proteasome, four translation initiation or elongation factors, proteins involved in cell signalling such as two 14-3-3 proteins and proteins involved in intracellular transport. Taken the data from the transcriptome and proteome analyses together it seems to become clear that in the Dll1 mutant many cellular processes might be altered. Regulation on the genomic level seems to be disturbed at the process of chromatin remodelling, regulation on post-transcriptional levels seems to be disturbed at the process of translation initiation and elongation as well as at the process of protein degradation through the proteasome. Intracellular trafficking seems to be altered on the level of phosphorylation and dephosphorylation through protein phosphatase 2A (PP2A) and C-src tyrosine kinase (Csk), as well as through the 14-3-3 proteins. Further aspects of intracellular trafficking involve the cytoskeleton. Nestin and g-tubulin, two components of the cytoskeleton were deregulated in the Dll1 mutant. Furthermore intracellular membrane trafficking seems to be disrupted through the downregulation of the SNARE protein Nsf and its interaction partner Munc18-3. It also seemed to turn out that developmental processes are altered in the mutants. Four transcription factors have been identified which have not been brought into context with Delta/Notch signalling before. It is not clear yet if they might play roles on the side of the signalling (Delta expressing) or the receiving (Notch expressing) cell. Neural crest cell migration also seems to be altered since Sema5b a gene involved in axonal guidance was downregulated in the Dll1 mutant embryos. Using a combination of transcriptomics and the proteomics approaches it could be shown that RNA- and protein expression profiling are both versatile and powerful approaches which complement each other. Thus it was possible to gain new insights into regulatory processes which would not have been possible with one approach alone. Der Delta/Notch Signaltransduktionsweg ist eine an vielfältigen Entwicklungsprozessen beteiligte, evolutionär konservierte Signalkaskade. Bisher wurde die Wirkungsweise während der Neurogenese, Somitogenese, links-rechts Achsenbildung, Pakreasentwicklung und der Entwicklung der sensorischen Härchen im Innenohr beobachtet. In den letzten Jahren wurde eine Anzahle von Genen identifiziert, die direkt an der Signaltransduktion beteiligt sind, oder die den Wechselweg auf verschiedene Weise beeinflussen. Für den wichtigen Entwicklungsprozess der lateralen Inhibition, der von Delta/Notch Signaltransduktion gesteuert wird, wurde ein Modell etabliert. Es bleibt jedoch unklar, wie die begrenzte Anzahl an bisher identifizierten Genen eine solche Vielfalt an Entwicklungsprozessen steuern kann. Um neue Einblicke in die Regulation der Delta/Notch Signaltransduktionskaskade zu gewinnen und um Zielgene von Dll1 zu identifizieren wurde das Transkriptom, sowie das Proteom von 10.5 dpc Wildtyp und Dll1 mutanten Mausembryonen untersucht. Dazu wurde die DNA-Chip Technologie und 2D-Gelelektrophorese kombiniert mit Massenspektrometrie durchgeführt. Durch die Transkriptomanalyse wurden in der Dll1 Mutante 22 hoch- und 30 herunter regulierte Gene identifiziert. Die Proteomanalyse lieferte 13 hoch- und 37 herunter regulierte Proteine am Tag 10.5 der Embryonalentwicklung. Zur Bestätigung und weiteren Analyse der identifizierten Kandidaten wurde Real-Time PCR und Ganzkörper in situ Hybridisierung auf Nukleinsäure-Ebene und semi-quantitatives Immunoblotting auf Protein-Ebene durchgeführt. Der aussichtsreichste Kandidat der Analysen ist das Gen Ifitm1. Es ist in Regionen des Embryos exprimiert von denen man weiß, dass dort Delta/Notch Signalübertragung stattfindet, wie beispielsweise im präsomitischen Mesoderm und den jüngsten Somiten. In Dll1, Dll3 und Jag1 mutanten Mausembryonen war die Expression in diesen Regionen deutlich reduziert, was auf eine direkte Verbindung zwischen Dll1, bzw. Delta/Notch Signaltransduktion und Ifitm1 hinweist. Ifitm1 wurde erst kürzlich als Markergen für primordiale Keimzellen identifiziert, eine genaue Funktion ist bisher nicht bekannt. Um die Funktion von Ifitm1 detailliert untersuchen zu können wurde ein klassisches Targeting Konstrukt entworfen und hergestellt. Mit Hilfe der Knock-out Tiere werden neue Einblicke in die Bedeutung von Ifitm1 für die Somitogenese und andere Entwicklungsprozesse gewonnen werden. Es wurden weitere Gene gefunden, die ein bezüglich Delta/Notch Signaltransduktion bemerkenswertes Expressionsmuster zeigen, und deren Expression in Dll1 Mutanten, sowie auch in Dll3 und Jag1 Mutanten verändert war. Diese Gene sind Csk, Ddx6, Nes, Sema5b and Smarcc1. Auch auf Protein-Ebene wurde eine Reihe interessanter Proteine identifiziert. Dazu gehören beispielsweise sechs Untereinheiten des 26S Proteasoms, vier „translation initiation“ bzw. „elongation“ Faktoren, Proteine der Signalisierungskaskade der Zellen, wie zwei 14-3-3 Proteine und Proteine, die in den intrazellulären Transport involviert sind. Betrachtet man die Daten der Transkriptom- und Proteomanalyse gemeinsam so scheint klar zu werden, dass in der Dll1 Mutante eine Anzahl an zellulären Prozessen beeinträchtigt sein könnte. Auf genomischer Ebene scheint die Regulation der Chromatin Modellierung verändert zu sein. Die post-transkriptionelle Regulation scheint auf der Ebene der Initiierung sowie der Elongation der Transkription ebenso gestört zu sein, wie auch bei dem Prozess der Protein Degradation am Proteasom. Intrazelluläre und regulatorische Transportmechanismen scheinen auf der Ebene der Phosphorylierung und Dephosphorylierung durch Protein Phosphatase 2A (PP2A) und C-src Tyrosin Kinase (Csk) und auch durch 14-3-3 Proteine verändert zu sein. Intrazellulärer Transport hängt weiterhin vom Cytoskelett ab. Zwei Komponenten des Cytoskeletts, Nestin und g-Tubulin, sind reguliert in der Dll1 Mutante. Durch verminderte Expression des SNARE Proteins Nsf, wie auch dessen Interaktionspartner Munc18-3, scheinen auch Transportmechanismen, die mit Hilfe der Zellmembran ablaufen, beeinträchtigt zu sein. Weiterhin scheinen auch Entwicklungsprozesse in der Mutante betroffen zu sein. Beispielsweise wurden vier Transkriptionsfaktoren identifiziert, die noch nie mit der Delta/Notch Signaltransduktion in Zusammenhang gebracht wurden. Im Moment bleibt offen, ob diese Transkriptionsfaktoren auf der Seite der Delta exprimierenden, signalgebenden Zelle, oder auf der Seite der Notch exprimierenden, signalempfangenden Zelle eine Rolle spielen. Eine mögliche Beeinträchtigung der Wanderung der Neuralleistenzellen könnte durch die verminderte Expression von Sema5b angezeigt werden. Sema5b ist in den Prozess der Wegleitung der Neuronen involviert. Mit Hilfe einer Kombination aus Analysen des Transkriptoms und des Proteoms konnte gezeigt werden, dass RNA- und Protein-Expression Profiling zwei vielseitige und leistungsstarke Ansätze sind, die einander komplementieren. Es war auf diese Weise möglich neue Einblicke in regulatorische Mechanismen zu erhalten, die man mit einer Methode allein nicht hätte bekommen können.

The Delta/Notch signal transduction pathway is an evolutionary conserved pathway involved in many diverse developmental processes. These include neurogenesis, somitogenesis, left-right development, pancreatic development and development of the sensory hair in the inner ear. Numerous genes have been identified in the last years that are part of the signal transduction pathway or can influence the pathway in a way. A model to explain the process of lateral inhibition, one of the main features of the pathway, has been established. However, it is not clear how the known genes involved in Delta/Notch signalling can account for such diverse processes. In order to get new insights into the regulation of Delta/Notch signalling and to identify targets of Dll1 the transcriptome and the proteome of E10.5 wildtype and Dll1-/- embryos was analysed using DNA-chip technology and 2D-gelelectrophoresis combined with mass spectrometry. In the transcriptome analysis 22 up- and 30 downregulated transcripts were identified. In the proteome analysis 13 proteins were up- and 37 proteins were downregulated at E10.5. Further methods to verify the data on transcriptomics side included whole mount in situ hybridisation and real-time PCR and semi-quantitative immunoblotting on the proteomics side. The most promising candidate out of this analyses is the gene Ifitm1 which shows expression in regions where Delta/Notch signalling is known to occur, namely the presomitic mesoderm and the latest formed somites. The expression was strongly reduced in Dll1, Dll3 and Jag1 mutants which indicates a direct connection between Dll1 or Delta/Notch signalling respectively, and Ifitm1. Furthermore Ifitm1 was only recently discovered as marker for primordial germ cells and has no assigned function so far. To assess the exact function of Ifitm1 in the mouse a classical knockout targeting construct has been designed and prepared. The knockout animals will provide insights into the relevance of Ifitm1 for somitogenesis and other developmental processes. Further genes have been identified which show expression patterns indicative of Delta/Notch signalling and which also were altered in Dll1 mutant mouse embryos, and some also in Dll3 and Jag1 embryos. These genes are Csk, Ddx6, Nes, Sema5b and Smarcc1. On the protein level a number of interesting proteins have been identified. These include six subunits of the 26S proteasome, four translation initiation or elongation factors, proteins involved in cell signalling such as two 14-3-3 proteins and proteins involved in intracellular transport. Taken the data from the transcriptome and proteome analyses together it seems to become clear that in the Dll1 mutant many cellular processes might be altered. Regulation on the genomic level seems to be disturbed at the process of chromatin remodelling, regulation on post-transcriptional levels seems to be disturbed at the process of translation initiation and elongation as well as at the process of protein degradation through the proteasome. Intracellular trafficking seems to be altered on the level of phosphorylation and dephosphorylation through protein phosphatase 2A (PP2A) and C-src tyrosine kinase (Csk), as well as through the 14-3-3 proteins. Further aspects of intracellular trafficking involve the cytoskeleton. Nestin and g-tubulin, two components of the cytoskeleton were deregulated in the Dll1 mutant. Furthermore intracellular membrane trafficking seems to be disrupted through the downregulation of the SNARE protein Nsf and its interaction partner Munc18-3. It also seemed to turn out that developmental processes are altered in the mutants. Four transcription factors have been identified which have not been brought into context with Delta/Notch signalling before. It is not clear yet if they might play roles on the side of the signalling (Delta expressing) or the receiving (Notch expressing) cell. Neural crest cell migration also seems to be altered since Sema5b a gene involved in axonal guidance was downregulated in the Dll1 mutant embryos. Using a combination of transcriptomics and the proteomics approaches it could be shown that RNA- and protein expression profiling are both versatile and powerful approaches which complement each other. Thus it was possible to gain new insights into regulatory processes which would not have been possible with one approach alone.

Our understanding of neuronal cell biology in the last 10 years has exploded. In parallel, our grasp of basic cellular processes, such as protein synthesis and protein degradation has also grown exponentially. In this review, we provide an in-depth background to details of current knowledge of the Ub/proteasome pathway. We also provide examples of recent experiments in neurobiology that suggest a central role for targeted protein degradation by the Ub/proteasome pathway to ensure proper neuronal function. From the examples provided, it is clear the activity of the proteasome is required for neuronal pathfinding during development, regulation of synaptic branching and number, and synaptic plasticity. We conclude with a discussion of how defects in proteasome pathway function may lead to neuronal dysfunction, with specific emphasis on diseases characterized by the accumulation of ubiquitin (Ub)-positive inclusions. Our goal is to excite the expert neurobiologist to the myriad ways that specific neuronal functions could be regulated (or dysregulated) by mechanisms involving the Ub/proteasome pathway.