Cancer-derived mutation in the OGA stalk domain promotes cell malignancy through dysregulating PDLIM7 and p53.

O-GlcNAcase (OGA) is the sole enzyme that hydrolyzes O-GlcNAcylation from thousands of proteins and is dysregulated in many diseases including cancer. However, the substrate recognition and pathogenic mechanisms of OGA remain largely unknown. Here we report the first discovery of a cancer-derived point mutation on the OGA's non-catalytic stalk domain that aberrantly regulated a small set of OGA-protein interactions and O-GlcNAc hydrolysis in critical cellular processes. We uncovered a novel cancer-promoting mechanism in which the OGA mutant preferentially hydrolyzed the O-GlcNAcylation from modified PDLIM7 and promoted cell malignancy by down-regulating p53 tumor suppressor in different types of cells through transcription inhibition and MDM2-mediated ubiquitination. Our study revealed the OGA deglycosylated PDLIM7 as a novel regulator of p53-MDM2 pathway, offered the first set of direct evidence on OGA substrate recognition beyond its catalytic site, and illuminated new directions to interrogate OGA's precise role without perturbing global O-GlcNAc homeostasis for biomedical applications.

Elucidating the protein substrate recognition of O-GlcNAc transferase (OGT) toward O-GlcNAcase (OGA) using a GlcNAc electrophilic probe.

The essential human O-linked ß-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is the sole enzyme responsible for modifying thousands of intracellular proteins with the monosaccharide O-GlcNAc. This unique modification plays crucial roles in human health and disease, but the substrate recognition of OGT remains poorly understood. Intriguingly, the only human enzyme reported to remove this modification, O-GlcNAcase (OGA), is O-GlcNAc modified. Here, we exploited a GlcNAc electrophilic probe (GEP1A) to rapidly screen OGT mutants in a fluorescence assay that can discriminate between altered OGT-sugar and -protein substrate binding to help elucidate the binding mode of OGT toward OGA protein substrate. Since OGT tetratricopeptide repeat (TPR) domain plays a key role in OGT-OGA binding, we screened 30 OGT TPR mutants, which revealed 15 "ladder like" asparagine or aspartate residues spanning TPRs 3-7 and 10-13.5 that affect OGA O-GlcNAcylation. By applying a truncated OGA construct, we found that OGA's N-terminal region or pseudo histone acetyltransferase domain is not required for its O-GlcNAcylation, suggesting OGT functionally interacts with OGA through its catalytic and/or stalk domains. This work represents the first effort to systemically investigate each OGT TPR and our findings will facilitate the development of new strategies to investigate the role of substrate-specific O-GlcNAcylation.

Targeted covalent inhibition of O-GlcNAc transferase in cells.

O-GlcNAc transferase (OGT) glycosylates numerous proteins and is implicated in many diseases. To date, most OGT inhibitors lack either sufficient potency or characterized specificity in cells. We report the first targeted covalent inhibitor that predominantly reacts with OGT but does not affect other functionally similar enzymes. This study provides a new strategy to interrogate cellular OGT functions and to investigate other glycosyltransferases.

Chemical and Biochemical Strategies To Explore the Substrate Recognition of O-GlcNAc-Cycling Enzymes.

The O-linked N-acetylglucosamine (O-GlcNAc) modification is an essential component in cell regulation. A single pair of human enzymes conducts this modification dynamically on a broad variety of proteins: O-GlcNAc transferase (OGT) adds the GlcNAc residue and O-GlcNAcase (OGA) hydrolyzes it. This modification is dysregulated in many diseases, but its exact effect on particular substrates remains unclear. In addition, no apparent sequence motif has been found in the modified proteins, and the factors controlling the substrate specificity of OGT and OGA are largely unknown. In this minireview, we will discuss recent developments in chemical and biochemical methods toward addressing the challenge of OGT and OGA substrate recognition. We hope that the new concepts and knowledge from these studies will promote research in this area to advance understanding of O-GlcNAc regulation in health and disease.

Electrophilic probes for deciphering substrate recognition by O-GlcNAc transferase.

O-linked ß-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is an essential human glycosyltransferase that adds O-GlcNAc modifications to numerous proteins. However, little is known about the mechanism with which OGT recognizes various protein substrates. Here we report on GlcNAc electrophilic probes (GEPs) to expedite the characterization of OGT-substrate recognition. Data from mass spectrometry, X-ray crystallization, and biochemical and radiolabeled kinetic assays support the application of GEPs to rapidly report the impacts of OGT mutations on protein substrate or sugar binding and to discover OGT residues crucial for protein recognition. Interestingly, we found that the same residues on the inner surface of the N-terminal domain contribute to OGT interactions with different protein substrates. By tuning reaction conditions, a GEP enables crosslinking of OGT with acceptor substrates in situ, affording a unique method to discover genuine substrates that weakly or transiently interact with OGT. Hence, GEPs provide new strategies to dissect OGT-substrate binding and recognition.

Deciphering the Functions of Protein O-GlcNAcylation with Chemistry.

O-GlcNAcylation is the modification of serine and threonine residues with ß-N-acetylglucosamine (O-GlcNAc) on intracellular proteins. This dynamic modification is attached by O-GlcNAc transferase (OGT) and removed by O-GlcNAcase (OGA) and is a critical regulator of various cellular processes. Furthermore, O-GlcNAcylation is dysregulated in many diseases, such as diabetes, cancer, and Alzheimer's disease. However, the precise role of this modification and its cycling enzymes (OGT and OGA) in normal and disease states remains elusive. This is partially due to the difficulty in studying O-GlcNAcylation with traditional genetic and biochemical techniques. In this review, we will summarize recent progress in chemical approaches to overcome these obstacles. We will cover new inhibitors of OGT and OGA, advances in metabolic labeling and cellular imaging, synthetic approaches to access homogeneous O-GlcNAcylated proteins, and cross-linking methods to identify O-GlcNAc-protein interactions. We will also discuss remaining gaps in our toolbox for studying O-GlcNAcylation and questions of high interest that are yet to be answered.

Distributive O-GlcNAcylation on the Highly Repetitive C-Terminal Domain of RNA Polymerase II.

O-GlcNAcylation is a nutrient-responsive glycosylation that plays a pivotal role in transcriptional regulation. Human RNA polymerase II (Pol II) is extensively modified by O-linked N-acetylglucosamine (O-GlcNAc) on its unique C-terminal domain (CTD), which consists of 52 heptad repeats. One approach to understanding the function of glycosylated Pol II is to determine the mechanism of dynamic O-GlcNAcylation on the CTD. Here, we discovered that the Pol II CTD can be extensively O-GlcNAcylated in vitro and in cells. Efficient glycosylation requires a minimum of 20 heptad repeats of the CTD and more than half of the N-terminal domain of O-GlcNAc transferase (OGT). Under conditions of saturated sugar donor, we monitored the attachment of more than 20 residues of O-GlcNAc to the full-length CTD. Surprisingly, glycosylation on the periodic CTD follows a distributive mechanism, resulting in highly heterogeneous glycoforms. Our data suggest that initial O-GlcNAcylation can take place either on the proximal or on the distal region of the CTD, and subsequent glycosylation occurs similarly over the entire CTD with nonuniform distributions. Moreover, removal of O-GlcNAc from glycosylated CTD is also distributive and is independent of O-GlcNAcylation level. Our results suggest that O-GlcNAc cycling enzymes can employ a similar mechanism to react with other protein substrates on multiple sites. Distributive O-GlcNAcylation on Pol II provides another regulatory mechanism of transcription in response to fluctuating cellular conditions.

A novel perineal shield for low-dose-rate prostate brachytherapy.

PURPOSE: To study the impact on radiation exposure to staff through the use of an original perineal shield during low-dose-rate prostate brachytherapy. MATERIAL AND METHODS: We designed a 1 mm thick stainless steel shield that duplicates and is able to slide directly over a standard commercialized prostate brachytherapy grid. We then analyzed the post-procedure exposure in 15 consecutive patients who underwent Iodine-125 seed placement. Measurements were performed with and without the shield in place at fixed locations relative to the grid template. Endpoints were analyzed using the paired two-sample t-test, with statistical significance defined as a p-value < 0.05. RESULTS: The exposure at the midline grid template ranged from 0.144-0.768 mSv/hr without the shield, and 0.038-0.144 mSv/hr with the shield (p < 0.0001). The exposure 10 cm left of the grid template was 0.134-0.576 mSv/hr without the shield, and 0.001-0.012 mSv/hr with the shield (p < 0.0001). The exposure 10 cm right of the grid template was 0.125-0.576 mSv/hr without the shield, and 0.001-0.012 mSv/hr with the shield (p < 0.0001). The median reduction of exposure at the grid was 76% midline, 98.5% left, and 99% right. Similarly, each individual dose rate was recorded at 25 cm from the perineum, both with and without shield. The median reduction of exposure 25 cm from the perineum was 73.7% midline, 77.7% left and 81.6% right (p < 0.0001). CONCLUSIONS: Our novel shield took seconds to install and was non-restrictive during the procedure, and provided at least a four-fold reduction in radiation exposure to the brachytherapist.

Crystal structure of rac-(3a'R,9a'R)-3a'-(indol-3-yl)-1',2',3',3a',4',9a'-hexa-hydro-spiro-[cyclo-pentane-1,9'-penta-leno[1,2-b]indole] p-xylene hemisolvate.

The title compound, C26H26N2·0.5C8H10, is the first reported characterized 2:2 product from acid-catalyzed condensation of indole with cyclo-penta-none and no other 2:2 products were observed. Recrystallization from p-xylene gave the title hemisolvate with the p-xylene mol-ecule located about an inversion center. The terminal penta-lene ring is envelope-flap disordered at the C atom farthest from the skeletal indole unit, with a refined occupancy ratio of 0.819 (4):0.181 (4). The major component has this C atom bent away from the spiro-fused cyclo-pentane ring. In the crystal, mol-ecules are connected by N-H⋯π inter-actions, forming chains along [100], and N-H⋯π and C-H⋯π inter-actions, forming chains along [001], which results in the formation of slabs parallel to (010).

Assessment of risk of late rectal bleeding for patients with prostate cancer started on anticoagulation before or after radiation treatment.

AIM: To evaluate the risk of late rectal bleeding and its association with the timing and type of anticoagulation use in patients receiving dose-escalated radiation therapy (RT) (≥ 7,560 cGy) for prostate cancer. PATIENTS AND METHODS: Between 2003-2010, 465 patients were treated at our Institution with dose-escalated RT and included in this analysis. Patients were placed into the following categories: no anticoagulation use, aspirin during RT, clopidogrel/warfarin during RT, aspirin after completion of RT, clopidogrel/warfarin after completion of RT. RESULTS: The overall bleeding rate was 7.5%. For those on aspirin during RT, the 4-year freedom from rectal bleeding (FFBS) rate was 91%, compared to 94.7% for patients who were never on anticoagulation (p=0.16). For those on warfarin/clopidogrel during RT the 4-year FFBS rate was 78.2%, compared to 94.7% in those never on anticoagulation (p<0.001). On multivariate analysis, use of warfarin/clopidogrel during radiation treatment were strongly associated with an increased risk of rectal bleeding (multivariate HR=4.84, 95% CI=1.84-12.68, p=0.001). However, initiation of anticoagulation after completion of radiation treatment did not significantly increase the risk of rectal bleeding (multivariate HR=0.78, 95% CI=0.21-2.91, p=0.71). CONCLUSION: The use of clopidogrel or warfarin during radiation is associated with significantly increased risk of rectal bleeding. However, initiation of these medications after completion of radiation does not appear to impact such risk.