Protein splicing is a post-translational process by which an intervening polypeptide, or intein, catalyzes its own removal from the flanking polypeptides, or exteins, concomitant with extein ligation. Although inteins are highly abundant in the microbial world, including within several human pathogens, they are absent in the genomes of metazoans. As protein splicing is required to permit function of essential proteins within pathogens, inteins represent attractive antimicrobial targets. Here we review key proteins interrupted by inteins in pathogenic mycobacteria and fungi, exciting discoveries that provide proof of concept that intein activity can be inhibited and that this inhibition has an effect on the host organism’s fitness, and bioanalytical methods that have been used to screen for intein activity. We also consider potential off-target inhibition of hedgehog signaling, given the similarity in structure and function of inteins and hedgehog autoprocessing domains.
e23012 Background: Reddit is an anonymous, international, social media platform with 430 million active users; most are age 18-40 and a majority are male. Its discussion-based format is well-suited to healthcare, allowing patients, loved ones, and healthcare providers (HCPs) to interact, and numerous medical-focused “subreddit” (SR) communities exist. Previous work has analyzed SR topics including breast cancer, radiation therapy, COVID-19, and depression. To date, no comprehensive list of cancer-related SRs has been compiled or analyzed. Methods: Between 1/7/21 and 2/10/21, a list of cancer-related keywords were queried on reddit.com, SRs were identified, and information was coded from their homepages; final membership numbers were updated on 2/10/21. Tumor subtypes included those listed on the SEER website (seer.cancer.gov/statfacts) where data on prevalence, annual incidence, death, and 5-year survival were pulled. Historical SR membership data was determined using subredditstats.com. Descriptive stats, Pearson’s correlation, and regression were performed using R software and Excel. Results: 36 cancer-related SRs were identified: 31 (86.1%) active in last 6 months, 17 (47.2%) with daily activity, 24 (66.7%) focused on tumor subtypes, 9 (25.0%) for general support and discussion, and 3 (8.3%) focused on treatment modalities. Median time since inception of SRs was 6 years (range 1 – 12), median number of members was 739 (range 42 – 36519), and median number of moderators was 2 (range 1 – 8). 16 (44.4%) had formal rules for members. Only 1 (2.7%) prohibited those without cancer from posting. Among the groups with > 500 members (25, 69.4%), median growth since 1/1/20 was 66% (range 10 – 370%). 16 (59.3%) of the 27 SEER tumor sub-types were represented by a SR, 5 (18.5%) of which had more than one SR, and 14 (51.8%) had > 500 members. Correlation and R Square via regression between tumor subtype prevalence, annual incidence, deaths, and 5-year survival and associated SR members was 0.61 and 0.368 (p = 0.02), 0.45 and 0.199 (p = 0.11), -0.07 and 0.005 (p = 0.82), 0.33 and 0.107 (p = 0.26), respectively. Based on the prevalence regression line, over-represented tumor subtypes include brain, testicular, and lymphoma. Under-represented sub-types include cervical, ovarian, colon/rectal, prostate, and lung. Conclusions: SRs represent rapidly growing online communities for those with cancer to seek information and support. Many tumor subtypes are represented; however, some common cancers are either not represented (renal, liver, stomach, endometrial) or underrepresented (colon, prostate, lung). The demographic makeup of Reddit users may explain the over-representation of brain, testicular, and lymphoma, as they can arise in younger patients, particularly men. Future work should assess the content of specific SRs and investigate the role of anonymous HCP input.
Halophiles are organisms that thrive in very high salt concentrations. Enzymes from halophiles tend to have a negative surface charge and a low isoelectric point to allow for stable folding. We are interested in converting a halophilic enzyme that requires high salt for activity and proper folding into one that is functional at more moderate salt concentrations. Our model enzyme is DNA polymerase II from Halobacterium salinarum, which contains an intein. Inteins are intervening peptide sequences within proteins that are spliced out from their flanking polypeptides, or exteins, which then ligate to form a functional protein. We have induced mutations to the intein that facilitate a splicing reaction at lower salt concentrations. Changing negatively charged amino acids on the surface of the intein to corresponding neutral or positive charged residues observed in homologous inteins in non‐halophilic extremophiles induces the splicing reaction at lower salt concentrations, especially when coupled with a change of the C‐terminal Gln to the more highly conserved Asn. As few as two residue changes can dramatically alter the salt‐dependence of the activity, showing that subtle changes to surface residues can result in conditional folding and activity.
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