Deubiquitinating enzymes at the crossroads of lipid metabolism and cancer

Deregulated lipid metabolism has been recognized as a critical alteration that supports the development and growth of various types of tumors. Changes in lipid metabolism enable reprogramming of energetics and availability of synthetic intermediates and signaling mediators that can have pleiotropic effects in cellular physiology. The identification of critical factors that reshape lipid metabolism during oncogenesis can provide targets for the development of novel therapeutic protocols. Enzymes are an attractive class of molecules for the development of therapeutic compounds. This review focuses on deubiquitinating enzymes (DUBs) that have been implicated both in lipid and cellular homeostatic processes. In certain cases, a causative link between the two processes is mediated by the deubiquitinating enzyme whereas in other cases we present evidence that support a possible role for the DUB as the underlying linker of lipid content and cell growth deregulation. Collectively, our report highlights critical nodes of deubiquitination-dependent metabolic and growth regulatory processes that can be interrogated further for a detailed understanding of cancer promoting mechanisms and therapeutic exploitation

Mimicry of a constitutively active pre–B cell receptor in acute lymphoblastic leukemia cells

Pre–B cells undergo apoptosis unless they are rescued by pre–B cell receptor–dependent survival signals. We previously showed that the BCR-ABL1 kinase that is expressed in pre–B lymphoblastic leukemia bypasses selection for pre–B cell receptor–dependent survival signals. Investigating possible interference of BCR-ABL1 with pre–B cell receptor signaling, we found that neither SYK nor SLP65 can be phosphorylated in response to pre–B cell receptor engagement. Instead, Bruton's tyrosine kinase (BTK) is constitutively phosphorylated by BCR-ABL1. Activated BTK is essential for survival signals that otherwise would arise from the pre–B cell receptor, including activation of PLCγ1, autonomous Ca(2+) signaling, STAT5-phosphorylation, and up-regulation of BCLX (L). Inhibition of BTK activity specifically induces apoptosis in BCR-ABL1 (+) leukemia cells to a similar extent as inhibition of BCR-ABL1 kinase activity itself. However, BCR-ABL1 cannot directly bind to full-length BTK. Instead, BCR-ABL1 induces the expression of a truncated splice variant of BTK that acts as a linker between the two kinases. As opposed to full-length BTK, truncated BTK lacks kinase activity yet can bind to BCR-ABL1 through its SRC-homology domain 3. Acting as a linker, truncated BTK enables BCR-ABL1–dependent activation of full-length BTK, which initiates downstream survival signals and mimics a constitutively active pre–B cell receptor

Regulation mechanisms of genomic expression through the Epstein-Barr virus

The Epstein-Barr virus (EBV) is a widely spread human herpes virus that has the ability to infect epithelial cells as well as B-lymphocytes following either a lytic cycle or a latent one.The protein LMP1 is the major oncogenic protein of EBV as it persists the ability to transform fibroblasts. It is fully necessary for the immortalization procedure of B-lymphocytes through EBV and is detected in the majority of the neoplasia linked to EBV. LMP1 contains in its carboxy terminal end 2 regions (CTAR1 and CTAR2) that bind various cytoplasmic factors that are implicated in the different pathways triggered by LMP1. Among those factors is the TRAF protein family, from which 5 members (TRAF1, -2, -3, -5 and -6) are implicated in the signaling through LMP1 that induce the NFκB pathway as well as the pathways of JNK and p38 (MAP kinases).Besides protein phosphorylation the procedure of dephosphorylation is equally important to bring back the proteins to their initial state. Protein complexes PP2 and PP4 are from the best known phosphatases. The regulatory subunit of the PP4 complex interacts with the RING domain of TRAF2.During this PhD thesis it was discovered that TRAF5 seems to be of great importance for the viability of two different cell lines infected with EBV using the RNA interference method. In addition stable cell lines, originating from the cell line HEK293T, were produced expressing the viral protein LMP1 and simultaneously having either TRAF1, TRAF2 or TRAF3 downregulated. Using these stable line it was found that TRAF3 probably is a negative regulator to the expression of two target genes of NFκB, ΙκΒα and MCP1.Finally the interaction between PP4R1 and the RING domain of both TRAF2 and TRAF6 was confirmed. In addition the role of PP4R1 in the inhibition of the NFκB pathway was also confirmed. Also it was found that PP4R1 is responsible for the dephosphorylation of TRAF2 at the amino acid serine 11 but not serine 55, whose phosphorylation seems to improve the binding between TRAF2 and PP4R1.Ο ιός Epstein-Barr (EBV) είναι ένας ευρύτατα διαδεδομένος ανθρώπινος ερπητοϊός που έχει την ικανότητα να μολύνει επιθηλιακά κύτταρα και Β λεμφοκύτταρα ακολουθώντας είτε μια λυτική είτε μία λανθάνουσα πορεία.Η πρωτεΐνη LMP1 είναι η κύρια ογκογονική πρωτεΐνη του EBV καθώς έχει αυτόνομη ικανότητα μετασχηματισμού ινοβλαστών, είναι απόλυτα απαραίτητη για την διαδικασία της αθανατοποίησης των Β λεμφοκυττάρων από τον EBV και εκφράζεται στις περισσότερες νεοπλασίες που σχετίζονται με τον EBV. Η LMP1 στο καρβοξυτελικό της άκρο περιέχει 2 περιοχές (CTAR1 και CTAR2) που αποτελούν θέσεις πρόσδεσης διάφορων κυτταροπλασματικών παραγόντων που λαμβάνουν μέρος στα διάφορα σηματοδοτικά μονοπάτια που ενεργοποιεί η πρωτεΐνη LMP1. Ανάμεσα στους κυτταροπλασματικούς παράγοντες που έχουν εμπλακεί στη σηματοδότηση από την LMP1 είναι η οικογένεια των πρωτεϊνών TRAF εκ της οποίας τα 5 μέλη (TRAF1, -2, -3, -5 και -6) έχουν εμπλακεί στη σηματοδότηση της LMP1 που οδηγεί στην ενεργοποίηση του μεταγραφικού παράγοντα NF-κB και των κινασών JNK και p38 που ενεργοποιούνται από μιτογόνα (MAP κινάσες).Εκτός από την φωσφορυλίωση των πρωτεϊνών εξίσου σημαντική διαδικασία είναι η αποφωσφορυλίωση και η επαναφορά τους στην αρχική τους κατάσταση. Από της πιο γνωστές φωσφατάσες είναι τα σύμπλοκα πρωτεϊνών PP2 και PP4. Η ρυθμιστική υπομονάδα της φωσφατάσης PP4 αλληλεπιδρά με την επικράτεια RING της πρωτεΐνης TRAF2.Στα πλαίσια της διδακτορικής διατριβής βρέθηκε πως η πρωτεΐνη TRAF5 δείχνει να είναι σημαντική για την επιβίωση δύο κυτταρικών σειρών Β-κυττάρων μολυσμένων με τον ιό EBV με τη χρήση της μεθόδου της παρεμβολής του RNA. Επίσης δημιουργήθηκαν σταθερές κυτταρικές σειρές από την σειρά HEK293T που εκφράζουν την ιική πρωτεΐνη LMP1 και ταυτόχρονα έχουν μια εκ των πρωτεϊνών TRAF1, TRAF2 ή TRAF3 να εκφράζεται σε κατεσταλμένα επίπεδα. Με τη βοήθεια των κυττάρων αυτών βρέθηκε επίσης πως η πρωτεΐνη TRAF3 έχει αρνητικό ρόλο στην ενεργοποίηση των γονιδίων στόχων του NFκB IκBα και MCP1.Τέλος επιβεβαιώθηκε η αλληλεπίδραση της πρωτεΐνης PP4R1 με την περιοχή RING των πρωτεϊνών TRAF2 και TRAF6. Βρέθηκε επίσης ο ρόλος που έχει στην καταστολή του μονοπατιού του μεταγραφικού παράγοντα NFκB. Όπως και βρέθηκε πως η PP4R1 ευθύνεται για την αποφωσφορυλίωση της TRAF2 στην σερίνη 11 αλλά όχι στην σερίνη 55, η οποία όμως φωσφορυλίωση ενισχύει την αλληλεπίδραση μεταξύ των πρωτεϊνών TRAF2 και PP4R1

Evolutionary relationships among Chlamydophila abortus variant strains inferred by rRNA secondary structure-based phylogeny.

The evolutionary relationships among known Chlamydophila abortus variant strains including the LLG and POS, previously identified as being highly distinct, were investigated based on rRNA secondary structure information. PCR-amplified overlapping fragments of the 16S, 16S-23S intergenic spacer (IS), and 23S domain I rRNAs were subjected to cloning and sequencing. Secondary structure analysis revealed the presence of transitional single nucleotide variations (SNVs), two of which occurred in loops, while seven in stem regions that did not result in compensatory substitutions. Notably, only two SNVs, in 16S and 23S, occurred within evolutionary variable regions. Maximum likelihood and Bayesian phylogeny reconstructions revealed that C. abortus strains could be regarded as representing two distinct lineages, one including the "classical" C. abortus strains and the other the "LLG/POS variant", with the type strain B577(T) possibly representing an intermediate of the two lineages. The two C. abortus lineages shared three unique (apomorphic) characters in the 23S domain I and 16S-23S IS, but interestingly lacked synapomorphies in the 16S rRNA. The two lineages could be distinguished on the basis of eight positions; four of these comprised residues that appeared to be signature or unique for the "classical" lineage, while three were unique for the "LLG/POS variant". The U277 (E. coli numbering) signature character, corresponding to a highly conserved residue of the 16S molecule, and the unique G681 residue, conserved in a functionally strategic region also of 16S, are the most pronounced attributes (autapomorphies) of the "classical" and the "LLG/POS variant" lineages, respectively. Both lineages were found to be descendants of a common ancestor with the Prk/Daruma C. psittaci variant. Compared with the "classical", the "LLG/POS variant" lineage has retained more ancestral features. The current rRNA secondary structure-based analysis and phylogenetic inference reveal new insights into how these two C. abortus lineages have differentiated during their evolution

Characterization of tumor suppressor CYLD expression in clear cell renal cell carcinoma

Cyld is a tumor suppressor gene that has attracted particular interest recently, due to its involvement in many types of neo-plasia, including head and neck squamous cell carcinoma, multiple myeloma, melanoma, hepatocellular carcinoma and colon cancer. Cyld encodes a predominantly cytoplasmic protein (CYLD), which is a deubiquitinating enzyme that regulates primarily cell survival and cell division pathways. Studies on the molecular function of CYLD have shown that it can modulate NF-κΒ, JNK, p38, TGF-beta, Wnt and Notch signaling. The present study aimed to investigate whether CYLD expression can be correlated with the development of clear cell renal cell carcinoma (ccRCC). Towards this goal, immunohistochemistry and Real Time PCR experiments were performed in order to analyze CYLD expression in clear cell renal cell carcinoma and matched normal tissue specimens. In addition, a clonogenic assay was performed to analyze the effect of CYLD wt and a catalytically inactive mutant CYLD on the growth of human embryonic kidney cells. The results of the present study show that CYLD is downregulated at protein and mRNA level in patients with ccRCC. This is further corroborated by the results of a clonogenic assay, which showed a deubiquitinating activity-dependent growth inhibitory role of CYLD in human embryonic kidney cells. Our results support the notion that CYLD can have a tumor suppressing role, at least in a subset of clear cell renal cell carcinoma, suggesting that it can be incorporated in the future in the development of targeted therapeutic approaches

Functional analysis of the <i>C</i>. <i>elegans cyld-1</i> gene reveals extensive similarity with its human homolog

<div><p>The human cylindromatosis tumor suppressor (HsCyld) has attracted extensive attention due to its association with the development of multiple types of cancer. HsCyld encodes a deubiquitinating enzyme (HsCYLD) with a broad range of functions that include the regulation of several cell growth, differentiation and death pathways. HsCyld is an evolutionarily conserved gene. Homologs of HsCyld have been identified in simple model organisms such as <i>Drosophila melanogaster</i> and <i>Caenorhabditis elegans</i> (C. <i>elegans</i>) which offer extensive possibilities for functional analyses. In the present report we have investigated and compared the functional properties of HsCYLD and its <i>C</i>. <i>elegans</i> homolog (CeCYLD). As expected from the mammalian CYLD expression pattern, the CeCyld promoter is active in multiple tissues with certain gastrointestinal epithelia and neuronal cells showing the most prominent activity. CeCYLD is a functional deubiquitinating enzyme with similar specificity to HsCYLD towards K63- and M1-linked polyubiquiting chains. CeCYLD was capable of suppressing the TRAF2-mediated activation of NF-kappaB and AP1 similarly to HsCYLD. Finally, CeCYLD could suppress the induction of TNF-dependent gene expression in mammalian cells similarly to HsCYLD. Our results demonstrate extensively overlapping functions between the HsCYLD and CeCYLD, which establish the <i>C</i>. <i>elegans</i> protein as a valuable model for the elucidation of the complex activity of the human tumor suppressor protein.</p></div

CeCyld inhibits TNF-induced IL8 expression.

<p>HeLa cells (10⁵ cells per well in 12-well plates) were transfected with 0.1μg, 1.4μg and 0.4μg of plasmids expressing HsCYLD, CeCYLD and CeCYLDC774S respectively. After 24h, each transfected cell population was split equally in two new wells and 24 h later, half of each transfected cell population was left untreated or treated with TNFα (40ng/ml, 1h). The cells were then harvested and from each cell population RNA and total protein was extracted. The overexpressed FLAG-tagged proteins were immuno-precipitated and the products of immunoprecipitation were used to assess the expression levels of each protein by immuoblotting. (A) Values are shown as the mean +/- standard error of relative IL8 expression induction from four independent experiments of RT-PCR. The values that were compared statistically are indicated by horizontal lines. (B) Immunoblot showing the expression levels of immuno-precipitated proteins with anti-FLAG antibody (IP:αFLAG) along with the immunoglobulin heavy chains (Ig-HC) and β-actin in the whole cell lysate (WCL). *p< 0.05, **p<0.01 ***p<0.001.</p

The <i>C</i>. <i>elegans cyld-1</i> expresses in the pharynx, in the nerve ring and in the intestine.

<p>Images of the head (A, B and E) and tail (C &D) region of transgenic worms that expressed the <i>cyld-1p</i>::<i>DsRed2</i> reporter. (A and B) Expression in the pharyngeal muscles pm3, pm4, pm6 & pm7. (B) The presumptive command interneurons AVB/AVD were identified using the <i>glr-1</i>::<i>GFP</i> reporter which expresses in command interneurons in the nerve ring. (C) AVB/AVD bundles run the entire length of the ventral nerve cord and terminate just before the rectum. (D) <i>cyld-1</i> expresses in the intestine. (E) Some animals show <i>cyld-1p</i>::<i>DsRed2</i> in the pharyngeal gland G2.</p

CeCYLD can inhibit the activation of AP1.

<p>HEK 293T cells (2x10⁵ cells per well in 12-well plates) were cotransfected with a AP1-dependent luciferase reporter plasmid (7xAP1), a β-galactosidase expression plasmid for transfection efficiency normalization (pGKβgal), and 0.1μg, 1μg and 0.3μg of plasmids expressing HsCYLD, CeCYLD and CeCYLDC774S respectively. After 18h, the cells were harvested and lysed in luciferase lysis buffer (Promega). The lysates were assayed for luciferase and β-galactosidase activities. (A) Values are shown as the mean +/- standard error of relative luciferase activity from five independent experiments. The values that were compared statistically are indicated by horizontal lines. (B) Representative immunoblot showing the expression levels of transfected proteins and β-actin. *p< 0.05, **p<0.01.</p