Sushi domain-containing protein 4 binds to epithelial growth factor receptor and initiates autophagy in an EGFR phosphorylation independent manner.

BACKGROUND: Sushi domain-containing protein 4 (SUSD4) is a recently discovered protein with unknown cellular functions. We previously revealed that SUSD4 can act as complement inhibitor and as a potential tumor suppressor. METHODS: In a syngeneic mouse model of breast cancer, tumors expressing SUSD4 had a smaller volume compared with the corresponding mock control tumors. Additionally, data from three different expression databases and online analysis tools confirm that for breast cancer patients, high mRNA expression of SUSD4 in the tumor tissue correlates with a better prognosis. In vitro experiments utilized triple-negative breast cancer cell lines (BT-20 and MDA-MB-468) stably expressing SUSD4. Moreover, we established a cell line based on BT-20 in which the gene for EGFR was knocked out with the CRISPR-Cas9 method. RESULTS: We discovered that the Epithelial Growth Factor Receptor (EGFR) interacts with SUSD4. Furthermore, triple-negative breast cancer cell lines stably expressing SUSD4 had higher autophagic flux. The initiation of autophagy required the expression of EGFR but not phosphorylation of the receptor. Expression of SUSD4 in the breast cancer cells led to activation of the tumor suppressor LKB1 and consequently to the activation of AMPKα1. Finally, autophagy was initiated after stimulation of the ULK1, Atg14 and Beclin-1 axis in SUSD4 expressing cells. CONCLUSIONS: In this study we provide novel insight into the molecular mechanism of action whereby SUSD4 acts as an EGFR inhibitor without affecting the phosphorylation of the receptor and may potentially influence the recycling of EGFR to the plasma membrane.

Alternative splicing encodes functional intracellular CD59 isoforms that mediate insulin secretion and are down-regulated in diabetic islets.

Human pancreatic islets highly express CD59, which is a glycosylphosphatidylinositol (GPI)-anchored cell-surface protein and is required for insulin secretion. How cell-surface CD59 could interact with intracellular exocytotic machinery has so far not been described. We now demonstrate the existence of CD59 splice variants in human pancreatic islets, which have unique C-terminal domains replacing the GPI-anchoring signal sequence. These isoforms are found in the cytosol of ß-cells, interact with SNARE proteins VAMP2 and SNAP25, colocalize with insulin granules, and rescue insulin secretion in CD59-knockout (KO) cells. We therefore named these isoforms IRIS-1 and IRIS-2 (Isoforms Rescuing Insulin Secretion 1 and 2). Antibodies raised against each isoform revealed that expression of both IRIS-1 and IRIS-2 is significantly lower in islets isolated from human type 2 diabetes (T2D) patients, as compared to healthy controls. Further, glucotoxicity induced in primary, healthy human islets led to a significant decrease of IRIS-1 expression, suggesting that hyperglycemia (raised glucose levels) and subsequent decreased IRIS-1 expression may contribute to relative insulin deficiency in T2D patients. Similar isoforms were also identified in the mouse CD59B gene, and targeted CRISPR/Cas9-mediated knockout showed that these intracellular isoforms, but not canonical CD59B, are involved in insulin secretion from mouse ß-cells. Mouse IRIS-2 is also down-regulated in diabetic db/db mouse islets. These findings establish the endogenous existence of previously undescribed non­GPI-anchored intracellular isoforms of human CD59 and mouse CD59B, which are required for normal insulin secretion.

Non-invasive 19F NMR analysis of a protein-templated N-acylhydrazone dynamic combinatorial library.

Dynamic combinatorial chemistry (DCC) is a powerful tool to identify ligands for biological targets. We used 19F NMR as an in situ, non-invasive technique for measuring the composition of a dynamic combinatorial library (DCL) of N-acylhydrazones (NAHs). An NAH DCL, constructed from a fluoro-aromatic aldehyde and a small set of hydrazides, was targetted at ecFabH, an essential enzyme in bacterial fatty acid biosynthesis. Our NMR analysis identified a tert-butyl NAH as the best binder which was confirmed by enzymatic assay.

Structural evidence for the covalent modification of FabH by 4,5-dichloro-1,2-dithiol-3-one (HR45).

We use mass spectrometry analysis and molecular modelling to show the established antimicrobial inhibitor 4,5-dichloro-1,2-dithiol-3-one (HR45) acts by forming a covalent adduct with the target ß-ketoacyl-ACP synthase III (FabH). The 5-chloro substituent directs attack of the essential active site thiol (C112) via a Michael-type addition elimination reaction mechanism.

PlantCAZyme: a database for plant carbohydrate-active enzymes.

PlantCAZyme is a database built upon dbCAN (database for automated carbohydrate active enzyme annotation), aiming to provide pre-computed sequence and annotation data of carbohydrate active enzymes (CAZymes) to plant carbohydrate and bioenergy research communities. The current version contains data of 43,790 CAZymes of 159 protein families from 35 plants (including angiosperms, gymnosperms, lycophyte and bryophyte mosses) and chlorophyte algae with fully sequenced genomes. Useful features of the database include: (i) a BLAST server and a HMMER server that allow users to search against our pre-computed sequence data for annotation purpose, (ii) a download page to allow batch downloading data of a specific CAZyme family or species and (iii) protein browse pages to provide an easy access to the most comprehensive sequence and annotation data. DATABASE URL: http://cys.bios.niu.edu/plantcazyme/

Synthesis and biological evaluation of nitric oxide-donating analogues of sulindac for prostate cancer treatment.

A series of analogues of the non-steroidal anti-inflammatory drug (NSAID) sulindac 1 were synthesised tethered to nitric oxide (NO) donating functional groups. Sulindac shows antiproliterative effects against immortal PC3 cell lines. It was previously demonstrated that the effect can be enhanced when tethered to NO releasing groups such as nitrate esters, furoxans and sydnonimines. To explore this approach further, a total of fifty-six sulindac-NO analogues were prepared and they were evaluated as NO-releasing cytotoxic agents against prostate cancer (PCa) cell lines. Compounds 1k and 1n exhibited significant cytotoxic with IC50 values of 6.1±4.1 and 12.1±3.2µM, respectively, coupled with observed nitric oxide release.