Functional Defects From Endocrine Disease-Associated Mutations in HLXB9 and Its Interacting Partner, NONO.

The insulin-secreting pancreatic neuroendocrine tumors, insulinomas, characterized by increased pancreatic islet ß-cell proliferation, express the phosphorylated isoform of the ß-cell differentiation factor HLXB9 that interacts with NONO/p54NRB, a survival factor. Interestingly, two different homozygous germline mutations in HLXB9, p.F248L and p.F272L, were reported in neonatal diabetes, a condition with functional ß-cell deficiency. Also, two somatic heterozygous NONO mutations were found in endocrine-related tumors, p.H146R (parathyroid) and p.R293H (small intestine neuroendocrine tumor). However, the biological consequence of the mutations, and the role of HLXB9-NONO interaction in normal or abnormal ß cells, is not known. Expression, localization, and functional analysis of the clinically relevant HLXB9 and NONO mutants showed that HLXB9/p.F248L mutant localized in the nucleus but lacked phosphorylation, and NONO/p.R293H mutant was structurally impaired. The HLXB9 and NONO mutants retained the ability to interact, and overexpression of wild-type or mutant HXLB9 in MIN6 cells suppressed cell proliferation. To further understand the biological consequence of the HLXB9-NONO interaction, we mapped the NONO-interacting region in HLXB9. An 80-amino acid conserved region of HLXB9 could compete with full-length HLXB9 to interact with NONO; however, in functional assays, nuclear expression of this HLXB9-conserved region in MIN6 cells did not interfere with cell proliferation. Overall, our results highlight the importance of HLXB9 in conditions of ß-cell excess (insulinomas) and in conditions of ß-cell loss or dysfunction (diabetes). Our studies implicate therapeutic strategies for either reducing ß-cell proliferation in insulinomas or alleviating normal ß-cell deficiency in diabetes through the modulation of HLXB9 phosphorylation.

A polysaccharide utilization locus from Flavobacterium johnsoniae enables conversion of recalcitrant chitin.

BACKGROUND: Chitin is the second most abundant polysaccharide on earth and as such a great target for bioconversion applications. The phylum Bacteroidetes is one of nature's most ubiquitous bacterial lineages and is essential in the global carbon cycle with many members being highly efficient degraders of complex carbohydrates. However, despite their specialist reputation in carbohydrate conversion, mechanisms for degrading recalcitrant crystalline polysaccharides such as chitin and cellulose are hitherto unknown. RESULTS: Here we describe a complete functional analysis of a novel polysaccharide utilization locus (PUL) in the soil Bacteroidete Flavobacterium johnsoniae, tailored for conversion of chitin. The F. johnsoniae chitin utilization locus (ChiUL) consists of eleven contiguous genes encoding carbohydrate capture and transport proteins, enzymes, and a two-component sensor-regulator system. The key chitinase (ChiA) encoded by ChiUL is atypical in terms of known Bacteroidetes-affiliated PUL mechanisms as it is not anchored to the outer cell membrane and consists of multiple catalytic domains. We demonstrate how the extraordinary hydrolytic efficiency of ChiA derives from synergy between its multiple chitinolytic (endo- and exo-acting) and previously unidentified chitin-binding domains. Reverse genetics show that ChiA and PUL-encoded proteins involved in sugar binding, import, and chitin sensing are essential for efficient chitin utilization. Surprisingly, the ChiUL encodes two pairs of SusC/D-like outer membrane proteins. Ligand-binding and structural studies revealed functional differences between the two SusD-like proteins that enhance scavenging of chitin from the environment. The combined results from this study provide insight into the mechanisms employed by Bacteroidetes to degrade recalcitrant polysaccharides and reveal important novel aspects of the PUL paradigm. CONCLUSIONS: By combining reverse genetics to map essential PUL genes, structural studies on outer membrane chitin-binding proteins, and enzymology, we provide insight into the mechanisms employed by Bacteroidetes to degrade recalcitrant polysaccharides and introduce a new saccharolytic mechanism used by the phylum Bacteroidetes. The presented discovery and analysis of the ChiUL will greatly benefit future enzyme discovery efforts as well as studies regarding enzymatic intramolecular synergism.

Pro-oncogenic Roles of HLXB9 Protein in Insulinoma Cells through Interaction with Nono Protein and Down-regulation of the c-Met Inhibitor Cblb (Casitas B-lineage Lymphoma b).

Pancreatic islet ß-cells that lack the MEN1-encoded protein menin develop into tumors. Such tumors express the phosphorylated isoform of the ß-cell differentiation transcription factor HLXB9. It is not known how phospho-HLXB9 acts as an oncogenic factor in insulin-secreting ß-cell tumors (insulinomas). In this study we investigated the binding partners and target genes of phospho-HLXB9 in mouse insulinoma MIN6 ß-cells. Co-immunoprecipitation coupled with mass spectrometry showed a significant association of phospho-HLXB9 with the survival factor p54nrb/Nono (54-kDa nuclear RNA-binding protein, non-POU-domain-containing octamer). Endogenous phospho-HLXB9 co-localized with endogenous Nono in the nucleus. Overexpression of HLXB9 decreased the level of overexpressed Nono but not endogenous Nono. Anti-phospho-HLXB9 chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) identified the c-Met inhibitor, Cblb, as a direct phospho-HLXB9 target gene. Phospho-HLXB9 occupied the promoter of Cblb and reduced the expression of Cblb mRNA. Cblb overexpression or HLXB9 knockdown decreased c-Met protein and reduced cell migration. Also, increased phospho-HLXB9 coincided with reduced Cblb and increased c-Met in insulinomas of two mouse models of menin loss. These data provide mechanistic insights into the role of phospho-HLXB9 as a pro-oncogenic factor by interacting with a survival factor and by promoting the oncogenic c-Met pathway. These mechanisms have therapeutic implications for reducing ß-cell proliferation in insulinomas by inhibiting phospho-HLXB9 or its interaction with Nono and modulating the expression of its direct (Cblb) or indirect (c-Met) targets. Our data also implicate the use of pro-oncogenic activities of phospho-HLXB9 in ß-cell expansion strategies to alleviate ß-cell loss in diabetes.

Outer membrane proteins related to SusC and SusD are not required for Cytophaga hutchinsonii cellulose utilization.

Cytophaga hutchinsonii, a member of the phylum Bacteroidetes, employs a novel collection of cell-associated proteins to digest crystalline cellulose. Other Bacteroidetes rely on cell surface proteins related to the starch utilization system (Sus) proteins SusC and SusD to bind oligosaccharides and import them across the outer membrane for further digestion. These bacteria typically produce dozens of SusC-like porins and SusD-like oligosaccharide-binding proteins to facilitate utilization of diverse polysaccharides. C. hutchinsonii specializes in cellulose digestion and its genome has only two susC-like genes and two susD-like genes. Single and multiple gene deletions were constructed to determine if the susC-like and susD-like genes have roles in cellulose utilization. A mutant lacking all susC-like and all susD-like genes digested cellulose and grew on cellulose as well as wild-type cells. Further, recombinantly expressed SusD-like proteins CHU_0547 and CHU_0554 failed to bind cellulose or ß-glucan hemicellulosic polysaccharides. The results suggest that the Bacteroidetes Sus paradigm for polysaccharide utilization may not apply to the cellulolytic bacterium C. hutchinsonii.

Flavobacterium johnsoniae PorV is required for secretion of a subset of proteins targeted to the type IX secretion system.

Flavobacterium johnsoniae exhibits gliding motility and digests many polysaccharides, including chitin. A novel protein secretion system, the type IX secretion system (T9SS), is required for gliding and chitin utilization. The T9SS secretes the cell surface motility adhesins SprB and RemA and the chitinase ChiA. Proteins involved in secretion by the T9SS include GldK, GldL, GldM, GldN, SprA, SprE, and SprT. Porphyromonas gingivalis has orthologs for each of these that are required for secretion of gingipain protease virulence factors by its T9SS. P. gingivalis porU and porV have also been linked to T9SS-mediated secretion, and F. johnsoniae has orthologs of these. Mutations in F. johnsoniae porU and porV were constructed to determine if they function in secretion. Cells of a porV deletion mutant were deficient in chitin utilization and failed to secrete ChiA. They were also deficient in secretion of the motility adhesin RemA but retained the ability to secrete SprB. SprB is involved in gliding motility and is needed for formation of spreading colonies on agar, and the porV mutant exhibited gliding motility and formed spreading colonies. However, the porV mutant was partially deficient in attachment to glass, apparently because of the absence of RemA and other adhesins on the cell surface. The porV mutant also appeared to be deficient in secretion of numerous other proteins that have carboxy-terminal domains associated with targeting to the T9SS. PorU was not required for secretion of ChiA, RemA, or SprB, indicating that it does not play an essential role in the F. johnsoniae T9SS.

Flavobacterium johnsoniae chitinase ChiA is required for chitin utilization and is secreted by the type IX secretion system.

Flavobacterium johnsoniae, a member of phylum Bacteriodetes, is a gliding bacterium that digests insoluble chitin and many other polysaccharides. A novel protein secretion system, the type IX secretion system (T9SS), is required for gliding motility and for chitin utilization. Five potential chitinases were identified by genome analysis. Fjoh_4555 (ChiA), a 168.9-kDa protein with two glycoside hydrolase family 18 (GH18) domains, was targeted for analysis. Disruption of chiA by insertional mutagenesis resulted in cells that failed to digest chitin, and complementation with wild-type chiA on a plasmid restored chitin utilization. Antiserum raised against recombinant ChiA was used to detect the protein and to characterize its secretion by F. johnsoniae. ChiA was secreted in soluble form by wild-type cells but remained cell associated in strains carrying mutations in any of the T9SS genes, gldK, gldL, gldM, gldNO, sprA, sprE, and sprT. Western blot and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses suggested that ChiA was proteolytically processed into two GH18 domain-containing proteins. Proteins secreted by T9SSs typically have conserved carboxy-terminal domains (CTDs) belonging to the TIGRFAM families TIGR04131 and TIGR04183. ChiA does not exhibit strong similarity to these sequences and instead has a novel CTD. Deletion of this CTD resulted in accumulation of ChiA inside cells. Fusion of the ChiA CTD to recombinant mCherry resulted in secretion of mCherry into the medium. The results indicate that ChiA is a soluble extracellular chitinase required for chitin utilization and that it relies on a novel CTD for secretion by the F. johnsoniae T9SS.