SETD2 transcriptional control of ATG14L/S isoforms regulates autophagosome-lysosome fusion

Macroautophagy/autophagy is an evolutionarily conserved and tightly regulated catabolic process involved in the maintenance of cellular homeostasis whose dysregulation is implicated in several pathological processes. Autophagy begins with the formation of phagophores that engulf cytoplasmic cargo and mature into double-membrane autophagosomes; the latter fuse with lysosomes/vacuoles for cargo degradation and recycling. Here, we report that yeast Set2, a histone lysine methyltransferase, and its mammalian homolog, SETD2, both act as positive transcriptional regulators of autophagy. However, whereas Set2 regulates the expression of several autophagy-related (Atg) genes upon nitrogen starvation, SETD2 effects in mammals were found to be more restricted. In fact, SETD2 appears to primarily regulate the differential expression of protein isoforms encoded by the ATG14 gene. SETD2 promotes the expression of a long ATG14 isoform, ATG14L, that contains an N-terminal cysteine repeats domain, essential for the efficient fusion of the autophagosome with the lysosome, that is absent in the short ATG14 isoform, ATG14S. Accordingly, SETD2 loss of function decreases autophagic flux, as well as the turnover of aggregation-prone proteins such as mutant HTT (huntingtin) leading to increased cellular toxicity. Hence, our findings bring evidence to the emerging concept that the production of autophagy-related protein isoforms can differentially affect core autophagy machinery bringing an additional level of complexity to the regulation of this biological process in more complex organisms.Peer reviewe

TAp73 is a marker of glutamine addiction in medulloblastoma

Children with Cancer UK fellowship (reference no. 2014/178); Medical Research Council UK project grant (MR/N000528/1); Medical Research Council UK Programme grant

Phosphorylcholine Allows for Evasion of Bactericidal Antibody by Haemophilus influenzae

The human pathogen Haemophilus influenzae has the ability to quickly adapt to different host environments through phase variation of multiple structures on its lipooligosaccharide (LPS), including phosphorylcholine (ChoP). During colonization with H. influenzae, there is a selection for ChoP+ phase variants. In a murine model of nasopharyngeal colonization, this selection is lost in the absence of adaptive immunity. Based on previous data highlighting the importance of natural antibody in limiting H. influenzae colonization, the effect of ChoP expression on antibody binding and its bactericidal activity was investigated. Flow cytometric analysis revealed that ChoP+ phase variants had decreased binding of antibody to LPS epitopes compared to ChoP− phase variants. This difference in antibody binding correlated with increased survival of ChoP+ phase variants in the presence of antibody-dependent, complement-mediated killing. ChoP+ phase variants were also more resistant to trypsin digestion, suggesting a general effect on the physical properties of the outer membrane. Moreover, ChoP-mediated protection against antibody binding correlated with increased resilience of outer membrane integrity. Collectively, these data suggest that ChoP expression provides a selective advantage during colonization through ChoP-mediated effects on the accessibility of bactericidal antibody to the cell surface

Structural and genetic characterization of lipopolysaccharides extracted from disease causing non-typeable Haemophilus influenzae strains

This thesis deals with the structure and genetic blueprint of lipopolysaccharides (LPS) expressed by the Gram-negative bacterium Haemophilus influenzae (H.influenzae). H. influenzae is an opportunistic pathogen that regularly colonizes the upper respiratory tract and exists in encapsulated (typeable) or nonencapsulated (non-typeable) forms (NTHi). Prior research has indicated that the surface expressed lipopolysaccharides (LPS) is a major virulence factor of H. influenzae. Pathogenic behavior can for example result in respiratory tract infections, otitis media (OM) or invasive disease such as meningitis. The thesis contains detailed studies of the phase-variable glycosyltransferase lex2. This transferase was shown to act either as a glucosyltransferase or galactosyltransferase depending on one single key amino acid. This was established by elucidation of LPS expressed by genetically defined lex2 mutant strains or transformant strains in which the transferase activity was removed or altered. Moreover, two structural characterizations of non-typeable clinical isolates, strains 2019 and R2866 are investigated. NTHi R2866 is an atypical non-typeable strain as itwas isolated from a child with meningitis. Our data indicate that R2866 produces an extremely heterogeneous population of glycoforms with expression of L-glycero-Dmanno-heptose (L,D-Hep) in its outer core. This residue was evidenced to carry a phoshocholine (PCho) residue in O-7 position, a substitution which is novel. Moreover, this study includes data obtained for genetically defined mutant strains R2866lpsA and R2866losB2 as these demonstrated detailed LPS structures not seen in wild-type. The characterization of LPS expressed by 2019 wild-type strain and mutant strains 2019lex2, 2019lpt3 and 2019pgmB add to the previously published structure of 2019. In 1992 it was established that lactose is linked to the proximal heptose (Hep I) of the conserved triheptosyl inner-core moiety, no other structures were reported. We show that the middle heptose (Hep II) can express a beta-D-Galp-(1--->4)-beta-D-Glcp-(1--->4)-alpha-DGlcp-(1--->3 epitope. Interestingly, the lex2 mutant was indicated to be substituted at O-2 at Hep III by beta-D-Glcp which, in turn, can be further extended. Such elongations have never been reported for NTHi 2019 before. NTHi 2019 belongs to a small subset of strains that express one additonal phosphoethanol amine (PEtn) residue in its outer core. Here, we establish that PEtn substitutes O-3 of the distal heptose (Hep III). This PEtn substituent was absent in the lpt3 mutant indicating Lpt3 to be the transferase required to add PEtn to Hep III

Microglia: agents of the CNS pro-inflammatory response

The pro-inflammatory immune response driven by microglia is a key contributor to the pathogenesis of several neurodegenerative diseases. Though the research of microglia spans over a century, the last two decades have increased our understanding exponentially. Here, we discuss the phenotypic transformation from homeostatic microglia towards reactive microglia, initiated by specific ligand binding to pattern recognition receptors including toll-like receptor-4 (TLR4) or triggering receptors expressed on myeloid cells-2 (TREM2), as well as pro-inflammatory signaling pathways triggered such as the caspase-mediated immune response. Additionally, new research disciplines such as epigenetics and immunometabolism have provided us with a more holistic view of how changes in DNA methylation, microRNAs, and the metabolome may influence the pro-inflammatory response. This review aimed to discuss our current knowledge of pro-inflammatory microglia from different angles, including recent research highlights such as the role of exosomes in spreading neuroinflammation and emerging techniques in microglia research including positron emission tomography (PET) scanning and the use of human microglia generated from induced pluripotent stem cells (iPSCs). Finally, we also discuss current thoughts on the impact of pro-inflammatory microglia in neurodegenerative diseases

A dual role for the lex2 locus: identification of galactosyltransferase activity in non-typeable Haemophilus influenzae strains 1124 and 2019.

Lipopolysaccharide (LPS) of Haemophilus influenzae comprises a conserved tri-l-glycero-d-manno-heptosyl inner-core moiety (l-alpha-d-Hepp-(1-->2)-[PEtn-->6]-l-alpha-d-Hepp-(1-->3)-[beta-d-GlcIp-(1-->4)]-l-alpha-d-Hepp-(1-->5)-alpha-Kdop) to which addition of beta-d-Glcp to O-4 of GlcI in serotype b strains is controlled by the gene lex2B. In non-typeable H. influenzae strains 1124 and 2019, however, a beta-d-Galp is linked to O-4 of GlcI. In order to test the hypothesis that the lex2 locus is involved in the expression of beta-d-Galp-(1-->4-beta-d-Glcp-(1--> from HepI, lex2B was inactivated in strains 1124 and 2019, and LPS glycoform populations from the resulting mutant strains were investigated. Detailed structural analyses using NMR techniques and electrospray-ionisation mass spectrometry (ESIMS) on O-deacylated LPS and core oligosaccharide material (OS), as well as ESIMS(n) on permethylated dephosphorylated OS, indicated both lex2B mutant strains to express only beta-d-Glcp extensions from HepI. This provides strong evidence that Lex2B functions as a galactosyltransferase adding a beta-d-Galp to O-4 of GlcI in these strains, indicating that allelic polymorphisms in the lex2B sequence direct alternative functions of the gene product

Detailed structural features of lipopolysaccharide glycoforms in nontypeable Haemophilus influenzae strain 2019.

We have investigated the structure of the lipopolysaccharide (LPS) of nontypeable Haemophilus influenzae (NTHi) strain 2019, a prototype strain that is used for studies of NTHi biology and disease. Analysis of LPS from wild type and lex2B, lpt3 and pgm mutant strains using NMR techniques and ESI-MS on O-deacylated LPS and core oligosaccharide material (OS), as well as ESI-MS(n) on permethylated dephosphorylated OS, confirmed the previously established structure in which lactose is linked to the proximal heptose (HepI) of the conserved triheptosyl inner-core moiety, l-α-D-Hepp-(1→2)-[PEtn→6]-l-α-D-Hepp-(1→3)-l-α-D-Hepp-(1→5)-[PPEtn→4]-α-Kdo-(2→6)-lipid A. Importantly, our data provide further structural detail whereby extensions from the middle heptose (HepII) are now characterized as β-D-Galp-(1→4)-β-D-Glcp-(1→4)-α-D-Glcp-(1→3 and truncated versions thereof. PEtn substitutes O-3 of the distal heptose (HepIII) of the inner-core moiety. This PEtn substituent was absent in the lpt3 mutant indicating that Lpt3 is the transferase required to add PEtn to the distal heptose. Interestingly, in the lex2B mutant strain HepIII was found to be substituted at O-2 by β-D-Glcp which, in turn, can be further extended. Contrary to previous findings, LPS of the pgm mutant strain contained minor glycoforms having β-D-Glcp linked to O-4 of HepI and also glycoforms with an additional PEtn which could be assigned to HepIII. Acetate groups and one glycine residue further substitute HepIII in NTHi 2019

SETD2 mutation in renal clear cell carcinoma suppress autophagy via regulation of ATG12

Inactivating mutations in the SETD2 gene, encoding for a nonredundant histone H3 methyltransferase and regulator of transcription, is a frequent molecular feature in clear cell renal cell carcinomas (ccRCC). SETD2 deficiency is associated with recurrence of ccRCC and bears low prognostic values. Targeting autophagy, a conserved catabolic process with critical functions in maintenance of cellular homeostasis and cell conservation under stress condition, is emerging as a potential therapeutic strategy to combat ccRCC. Epigenetics-based pathways are now appreciated as key components in the regulation of autophagy. However, whether loss of function in the SETD2 histone modifying enzyme occurring in ccRCC cells may impact on their ability to undergo autophagy remained to be explored. Here, we report that SETD2 deficiency in RCC cells is associated with the aberrant accumulation of both free ATG12 and of an additional ATG12-containing complex, distinct from the ATG5-ATG12 complex. Rescue of SETD2 functions in the SETD2 deficiency in RCC cells, or reduction of SETD2 expression level in RCC cells wild type for this enzyme, demonstrates that SETD2 deficiency in RCC is directly involved in the acquisition of these alterations in the autophagic process. Furthermore, we revealed that deficiency in SETD2, known regulator of alternative splicing, is associated with increased expression of a short ATG12 spliced isoform at the depend of the canonical long ATG12 isoform in RCC cells. The defect in the ATG12-dependent conjugation system was found to be associated with a decrease autophagic flux, in accord with the role for this ubiquitin-like protein conjugation system in autophagosome formation and expansion. Finally, we report that SETD2 and ATG12 gene expression levels are associated with favorable respective unfavorable prognosis in ccRCC patients. Collectively, our findings bring further argument for considering the SETD2 gene status of ccRCC tumors, when therapeutic interventions, such as targeting the autophagic process, are considered to combat these kidney cancers