Chemical cross-linking and mass spectrometry enabled systems-level structural biology.

Structural information on protein-protein interactions (PPIs) is essential for improved understanding of regulatory interactome networks that confer various physiological and pathological responses. Additionally, maladaptive PPIs constitute desirable therapeutic targets due to inherently high disease state specificity. Recent advances in chemical cross-linking strategies coupled with mass spectrometry (XL-MS) have positioned XL-MS as a promising technology to not only elucidate the molecular architecture of individual protein assemblies, but also to characterize proteome-wide PPI networks. Moreover, quantitative in vivo XL-MS provides a new capability for the visualization of cellular interactome dynamics elicited by drug treatments, disease states, or aging effects. The emerging field of XL-MS based complexomics enables unique insights on protein moonlighting and protein complex remodeling. These techniques provide complimentary information necessary for in-depth structural interactome studies to better comprehend how PPIs mediate function in living systems.

Stereotactic Ablative Radiotherapy for Gynecological Oligometastatic and Oligoprogessive Tumors.

Importance: The role of stereotactic ablative radiotherapy (SABR) for gynecologic malignant tumors has yet to be clearly defined despite recent clinical uptake. Objective: To evaluate the outcomes of SABR in patients with oligometastatic and oligoprogressive gynecologic cancers. Design, Setting, and Participants: In this retrospective pooled analysis, patients with oligometastatic and oligoprogressive gynecologic cancers receiving SABR at 5 institutions from Canada and the US were studied. Data were collected from January 2011 to December 2020, and data were analyzed from January to December 2023. Exposure: Stereotactic ablative radiotherapy. Main Outcomes and Measures: Cumulative incidence of local and distant recurrence, chemotherapy-free survival (CFS), and overall survival (OS) probabilities after SABR were calculated using Kaplan-Meier methods. Univariable and multivariable analysis was conducted using Cox regression methods. Results: A total of 215 patients with 320 lesions meeting criteria were included in the analysis; the median (range) age at primary diagnosis was 59 (23-86) years. The median (range) follow-up from SABR was 18.5 (0.1-124.5) months. The primary site included the endometrium (n = 107), ovary (n = 64), cervix (n = 30), and vulva or vagina (n = 14). Local cumulative incidence of recurrence was 13.7% (95% CI, 9.4-18.9) and 18.5% (95% CI, 13.2-24.5) at 1 and 5 years, respectively. Distant cumulative incidence of recurrence was 48.5% (95% CI, 41.4-55.1) and 73.1% (95% CI, 66.0-79.0) at 1 and 5 years, respectively. OS was 75.7% (95% CI, 69.2-81.1) and 33.1% (95% CI, 25.3-41.1) at 1 and 5 years, respectively. The median CFS was 21.7 months (95% CI, 15.4-29.9). On multivariable analysis, local recurrence was significantly associated with nodal metastasis, lesion size, biologically effective dose, treatment indication, institution, and primary disease type. Distant progression-free survival was associated with nodal targets and lesion size. OS and CFS were significantly associated with lesion size. Conclusions and Relevance: In this study, SABR appeared to have excellent local control with minimal toxic effects in this large patient group, and certain patients may achieve durable distant control and OS as well. It may be possible to delay time to chemotherapy in select patient subtypes and therefore reduce associated toxic effects. Prospective multicenter trials will be critical to establish which characteristics procure the greatest benefit from SABR use and to define the ideal time to implement SABR with other oncologic treatments.

Neoadjuvant chemotherapy with bevacizumab for locally advanced vulvar cancer.

OBJECTIVES: External beam radiation with sensitizing platinum is the recommended therapy for locally advanced vulvar cancers not amenable to curative surgery and is associated with considerable acute and chronic side effects. Radical vulvectomy post-radiation for persistent disease is often compromised with poor wound healing. We describe clinical outcomes for patients who received neoadjuvant chemotherapy plus bevacizumab followed by radical vulvectomy for locally advanced vulvar cancer. METHODS: We performed retrospective analyses of all patients at our institution who underwent radical vulvectomy from January 2015 to November 2023. Of 113 patients, 13 patients underwent neoadjuvant chemotherapy. Demographics and clinicopathologic data were extracted, and descriptive statistical analyses were performed. Cases with neoadjuvant chemotherapy plus bevacizumab were further evaluated for response, adverse effects, and survival. RESULTS: Neoadjuvant chemotherapy was administered to 13 patients with stage II-IV disease that involved the urethra, vagina, or anus. Lesion sizes ranged from 4 to 20 cm (median 7 cm). Patients received 2-6 cycles of carboplatin or cisplatin, paclitaxel, and bevacizumab. Nine (69.2%) patients had partial pathologic responses, and four patients had complete responses. All patients had negative surgical margins. Ten (76.9%) patients had radiographic evidence of inguinal lymph node metastasis prior to neoadjuvant chemotherapy, and four had residual nodal disease. Only one patient developed a superficial groin seroma. Three patients developed recurrence, two locally and one distant, and there was one death. The median follow-up was 23 months (range 6-84 months). CONCLUSIONS: Neoadjuvant chemotherapy using combination platinum/paclitaxel/bevacizumab was efficacious for locally advanced vulvar cancer, resulting in complete resections, negative margins, and excellent wound healing. A multi-institutional phase II trial is warranted to validate these findings.

Interactome dynamics during heat stress signal transmission and reception.

Among evolved molecular mechanisms, cellular stress response to altered environmental conditions to promote survival is among the most fundamental. The presence of stress-induced unfolded or misfolded proteins and molecular registration of these events constitute early steps in cellular stress response. However, what stress-induced changes in protein conformations and protein-protein interactions within cells initiate stress response and how these features are recognized by cellular systems are questions that have remained difficult to answer, requiring new approaches. Quantitative in vivo chemical cross-linking coupled with mass spectrometry (qXL-MS) is an emerging technology that provides new insight on protein conformations, protein-protein interactions and how the interactome changes during perturbation within cells, organelles, and even tissues. In this work, qXL-MS and quantitative proteome analyses were applied to identify significant time-dependent interactome changes that occur prior to large-scale proteome abundance remodeling within cells subjected to heat stress. Interactome changes were identified within minutes of applied heat stress, including stress-induced changes in chaperone systems as expected due to altered functional demand. However, global analysis of all interactome changes revealed the largest significant enrichment in the gene ontology molecular function term of RNA binding. This group included more than 100 proteins among multiple components of protein synthesis machinery, including mRNA binding, spliceosomes, and ribosomes. These interactome data provide new conformational insight on the complex relationship that exists between transcription, translation and cellular stress response mechanisms. Moreover, stress-dependent interactome changes suggest that in addition to conformational stabilization of RNA-binding proteins, adaptation of RNA as interacting ligands offers an additional fitness benefit resultant from generally lower RNA thermal stability. As such, RNA ligands also serve as fundamental temperature sensors that signal stress through decreased conformational regulation of their protein partners as was observed in these interactome dynamics.

Cs-131 prostate brachytherapy boost and effect of hydrogel rectal spacer on long-term patient-reported rectal bleeding and bowel quality of life.

PURPOSE: This study evaluates long-term patient-reported bowel quality of life (QOL), rectal bleeding, and bleeding bother in patients with prostate cancer treated with external beam radiation therapy (EBRT) and Cesium-131 LDR brachytherapy (LDR-BT) boost with and without hydrogel rectal spacer. METHODS AND MATERIALS: This is a retrospective analysis of prostate cancer patients treated between 2007 and 2022 with 45 Gy EBRT followed by 85 Gy Cs-131 LDR-BT boost with or without hydrogel rectal spacer. Expanded Prostate Cancer Index Composite (EPIC) QOL questionnaires pre-treatment and at each follow-up were collected. Patient-reported rectal bleeding occurring more than "rarely" and bother from rectal bleeding occurring more than a "very small problem" were deemed clinically significant. Fisher's exact test was used to test the association of rectal spacer use and the incidence of clinically significant rectal bleeding and bleeding bother. Paired samples t-test was used to analyze mean bowel scores at each time point. RESULTS: Three hundred and forty-one patients were included in the analysis. The rectal spacer was used in 108 patients. Overall median follow-up was 48 months (IQR, 24-72), with a median follow-up of 24 months (IQR, 12-37.5) for the hydrogel group and 60 months (IQR, 36-84) for the non-hydrogel group. EPIC questionnaire response rates at median follow-up were 33% and 37% for the hydrogel and non-hydrogel groups, respectively. A clinically significant decrease in mean bowel domain scores was seen in the bowel bother domain at 6 and 12 months for patients who did not receive a rectal spacer. At the last follow-up of 60 months, the prevalence of clinically significant rectal bleeding and bleeding bother were 2.2% and 2.2%, respectively. The cumulative incidence of clinically significant long-term rectal bleeding was 2.8% and 18.9% in the hydrogel group and non-hydrogel group, respectively (Fisher's exact test, p < 0.0001). The cumulative incidence of clinically significant long-term bowel bother was 4.6% and 19.7% in the hydrogel group and non-hydrogel group, respectively (Fisher's exact test, p < 0.001). CONCLUSIONS: Use of hydrogel rectal spacer with EBRT and Cs-131 LDR-BT boost was significantly associated with a lower incidence of patient-reported rectal bleeding and bother from rectal bleeding, and better long-term bowel QOL. Cumulative incidence was 2.8% (hydrogel group) versus 18.9% (non-hydrogel group) and 4.6% (hydrogel group) versus 19.7% (non-hydrogel group) for clinically significant long-term rectal bleeding and long-term bleeding bother, respectively.

Integrated Analysis of Cross-Links and Dead-End Peptides for Enhanced Interpretation of Quantitative XL-MS.

Chemical cross-linking with mass spectrometry provides low-resolution structural information on proteins in cells and tissues. Combined with quantitation, it can identify changes in the interactome between samples, for example, control and drug-treated cells or young and old mice. A difference can originate from protein conformational changes that alter the solvent-accessible distance separating the cross-linked residues. Alternatively, a difference can result from conformational changes localized to the cross-linked residues, for example, altering the solvent exposure or reactivity of those residues or post-translational modifications of the cross-linked peptides. In this manner, cross-linking is sensitive to a variety of protein conformational features. Dead-end peptides are cross-links attached only at one end to a protein with the other terminus being hydrolyzed. As a result, changes in their abundance reflect only conformational changes localized to the attached residue. For this reason, analyzing both quantified cross-links and their corresponding dead-end peptides can help elucidate the likely conformational changes giving rise to observed differences in cross-link abundance. We describe analysis of dead-end peptides in the XLinkDB public cross-link database and, with quantified mitochondrial data isolated from failing heart versus healthy mice, show how a comparison of abundance ratios between cross-links and their corresponding dead-end peptides can be leveraged to reveal possible conformational explanations.

Integrated Analysis of Cross-Links and Dead-End Peptides for Enhanced Interpretation of Quantitative XL-MS.

XL-MS provides low-resolution structural information of proteins in cells and tissues. Combined with quantitation, it can identify changes in the interactome between samples, for example, control and drug-treated cells, or young and old mice. A difference can originate from protein conformational changes altering the solvent-accessible distance separating the cross-linked residues. Alternatively, a difference can result from conformational changes localized to the cross-linked residues, for example, altering the solvent exposure or reactivity of those residues or post-translational modifications on the cross-linked peptides. In this manner, cross-linking is sensitive to a variety of protein conformational features. Dead-end peptides are cross-links attached only at one end to a protein, the other terminus being hydrolyzed. As a result, changes in their abundance reflect only conformational changes localized to the attached residue. For this reason, analyzing both quantified cross-links and their corresponding dead-end peptides can help elucidate the likely conformational changes giving rise to observed differences of cross-link abundance. We describe analysis of dead-end peptides in the XLinkDB public cross-link database and, with quantified mitochondrial data isolated from failing heart versus healthy mice, show how a comparison of abundance ratios between cross-links and their corresponding dead-end peptides can be leveraged to reveal possible conformational explanations.

Studying Protein-Ligand Interactions by Protein Denaturation and Quantitative Cross-Linking Mass Spectrometry.

Recently, several mass spectrometry methods have utilized protein structural stability for the quantitative study of protein-ligand engagement. These protein-denaturation approaches, which include thermal proteome profiling (TPP) and stability of proteins from rates of oxidation (SPROX), evaluate ligand-induced denaturation susceptibility changes with a MS-based readout. The different techniques of bottom-up protein-denaturation methods each have their own advantages and challenges. Here, we report the combination of protein-denaturation principles with quantitative cross-linking mass spectrometry using isobaric quantitative protein interaction reporter technologies. This method enables the evaluation of ligand-induced protein engagement through analysis of cross-link relative ratios across chemical denaturation. As a proof of concept, we found ligand-stabilized cross-linked lysine pairs in well-studied bovine serum albumin and ligand bilirubin. These links map to the known binding sites Sudlow Site I and subdomain IB. We propose that protein denaturation and qXL-MS can be combined with similar peptide-level quantification approaches, like SPROX, to increase the coverage information profiled for facilitating protein-ligand engagement efforts.

Skeletal muscle mitochondrial interactome remodeling is linked to functional decline in aged female mice.

Genomic, transcriptomic and proteomic approaches have been used to gain insight into molecular underpinnings of aging in laboratory animals and in humans. However, protein function in biological systems is under complex regulation and includes factors besides abundance levels, such as modifications, localization, conformation and protein-protein interactions. By making use of quantitative chemical cross-linking technologies, we show that changes in the muscle mitochondrial interactome contribute to mitochondrial functional decline in aging in female mice. Specifically, we identify age-related changes in protein cross-links relating to assembly of electron transport system complexes I and IV, activity of glutamate dehydrogenase, and coenzyme-A binding in fatty acid ß-oxidation and tricarboxylic acid cycle enzymes. These changes show a remarkable correlation with complex I respiration differences within the same young-old animal pairs. Each observed cross-link can serve as a protein conformational or protein-protein interaction probe in future studies, which will provide further molecular insights into commonly observed age-related phenotypic differences. Therefore, this data set could become a valuable resource for additional in-depth molecular studies that are needed to better understand complex age-related molecular changes.

Is Substantial Lymphovascular Space Invasion Prognostic in Patients With Pathologically Lymph Node-Negative Endometrial Cancer?

PURPOSE: Lymphovascular space invasion (LVSI) predicts for higher rates of recurrence and increased mortality in endometrial cancer. Using 3-tier LVSI scoring, a PORTEC-1 and -2 trials analysis demonstrated that substantial LVSI was associated with worse locoregional (LR-DFS) and distant metastasis disease-free survival (DM-DFS), and these patients possibly benefited from external beam radiation therapy (EBRT). Furthermore, LVSI is a predictor for lymph node (LN) involvement, but the significance of substantial LVSI is unknown in patients with a pathologically negative LN assessment. We aimed to evaluate clinical outcomes of these patients in relation to the 3-tier LVSI scoring system. METHODS AND MATERIALS: We performed a single-institutional retrospective review of patients with stage I endometrioid-type endometrial cancer who underwent surgical staging with pathologically negative LN evaluation from 2017 to 2019 with 3-tier LVSI scoring (none, focal, or substantial). Clinical outcomes (LR-DFS, DM-DFS, and overall survival) were analyzed using the Kaplan-Meier method. RESULTS: A total of 335 patients with pathologically LN-negative stage I endometrioid-type endometrial carcinoma were identified. Substantial LVSI was present in 17.6% of patients; 39.7% of patients received adjuvant vaginal brachytherapy and 6.9% of patients received EBRT. Adjuvant radiation treatment varied by LVSI status. In patients with focal LVSI, 81.0% received vaginal brachytherapy. Among patients with substantial LVSI, 57.9% received vaginal brachytherapy alone, and 31.6% of patients received EBRT. The 2-year LR-DFS rates were 92.5%, 98.0%, and 91.4% for no LVSI, focal LVSI, and substantial LVSI, respectively. The 2-year DM-DFS rates were 95.5%, 93.3%, and 93.8% for no LVSI, focal LVSI, and substantial LVSI, respectively. CONCLUSIONS: In our institutional study, patients with pathologically LN-negative stage I endometrial cancer with substantial LVSI had similar rates of LR-DFS and DM-DFS compared with patients with none or focal LVSI. These findings highlight the need for multi-institutional studies to validate the prognostic value of substantial LVSI in this patient population.

The Impact of Racial Disparities on Outcome in Patients With Stage IIIC Endometrial Carcinoma: A Pooled Data Analysis.

OBJECTIVE: To report the impact of race on clinical outcomes in patients with stage IIIC endometrial carcinoma. MATERIALS AND METHODS: A retrospective multi-institutional study included 90 black and 568 non-black patients with stage IIIC endometrial carcinoma who received adjuvant chemotherapy and radiation treatments. Overall survival (OS) and recurrence-free survival (RFS) were calculated by the Kaplan-Meier method. Propensity score matching (PSM) was conducted. Statistical analyses were conducted using SPSS version 27. RESULTS: The Median follow-up was 45.3 months. black patients were significantly older, had more nonendometrioid histology, grade 3 tumors, and were more likely to have >1 positive paraaortic lymph nodes compared with non-black patients (all P <0.0001). The 5-year estimated OS and RFS rates were 45% and 47% compared with 77% and 68% for black patients versus non-black patients, respectively ( P <0.001). After PSM, the 2 groups were well-balanced for all prognostic covariates. The estimated hazard ratios of black versus non-black patients were 1.613 ( P value=0.045) for OS and 1.487 ( P value=0.116) for RFS. After PSM, black patients were more likely to receive the "Sandwich" approach and concurrent chemoradiotherapy compared with non-black ( P =0.013) patients. CONCLUSIONS: Black patients have higher rates of nonendometrioid histology, grade 3 tumors, and number of involved paraaortic lymph nodes, worse OS, and RFS, and were more likely to receive the "Sandwich" approach compared with non-black patients. After PSM, black patients had worse OS with a nonsignificant trend in RFS. Access to care, equitable inclusion on randomized trials, and identification of genomic differences are warranted to help mitigate disparities.

Towards a structurally resolved human protein interaction network.

Cellular functions are governed by molecular machines that assemble through protein-protein interactions. Their atomic details are critical to studying their molecular mechanisms. However, fewer than 5% of hundreds of thousands of human protein interactions have been structurally characterized. Here we test the potential and limitations of recent progress in deep-learning methods using AlphaFold2 to predict structures for 65,484 human protein interactions. We show that experiments can orthogonally confirm higher-confidence models. We identify 3,137 high-confidence models, of which 1,371 have no homology to a known structure. We identify interface residues harboring disease mutations, suggesting potential mechanisms for pathogenic variants. Groups of interface phosphorylation sites show patterns of co-regulation across conditions, suggestive of coordinated tuning of multiple protein interactions as signaling responses. Finally, we provide examples of how the predicted binary complexes can be used to build larger assemblies helping to expand our understanding of human cell biology.

Outcomes of 3D MRI based HDR brachytherapy with hybrid multichannel vaginal cylinder applicator and freehand needles for treatment of vaginal disease.

Freehand needles can be used with multichannel vaginal cylinders (MCVC) to cover vaginal cancer >7 mm thick or with supra-vaginal extension. We report our institutional outcomes using this novel hybrid technique. Patients with vaginal malignancies treated with HDR BT using MCVC plus freehand needles from 2014-2021 at our institution were identified. Clinical characteristics, details of brachytherapy, initial response, and overall local control (LC) outcomes were recorded. LC was analyzed via Kaplan-Meier method. 34 patients were identified with median follow-up 1.9 years. 19 patients had primary endometrial cancer with vaginal recurrence/disease, and remaining had primary vaginal cancer or other primaries. 7 patients had recurrence after previous RT course. 25 patients received EBRT with median dose 45 Gy in 25 fractions, and rest received BT alone. Median HR-CTV D90 for patients treated with EBRT plus BT was 77.4 Gy. 30 patients had complete local response to BT on initial examination and/or follow-up imaging. 1 and 2-year LC rates in patients without prior RT treated with EBRT + BT were 94.1% and 94.1%, respectively. 1 and 2-year LC rates for those without prior RT were 88.1% and 76.4%, respectively. 1 and 2-year LC rates for those with prior RT were 68.6% and 34.3%, respectively. 1 patient had vaginal laceration requiring surgical repair, and 1 patient developed small bowel obstruction 1 month after BT, with no additional acute grade 3+ toxicities identified. Our approach with MCVC plus freehand needles with MRI-based planning was feasible and safe, with excellent initial local response and low rate of serious toxicities.

Factors associated with ductal carcinoma in situ (DCIS) treatment patterns and patient-reported outcomes across a large integrated health network.

PURPOSE: To examine associations between ductal carcinoma in situ (DCIS) patients' characteristics, treating locations and DCIS treatments received and to pilot assessing quality-of-life (QoL) values among DCIS patients with diverse backgrounds. METHODS: We performed a retrospective tumor registry review of all patients diagnosed and treated with DCIS from 2018 to 2019 in the UPMC-integrated network throughout central and western Pennsylvania. Demographics, clinical information, and administered treatments were compiled from tumor registry records. We categorized contextual factors such as different hospital setting (academic vs. community), socioeconomic status based on the neighborhood deprivation index (NDI) as well as age and race. QoL survey was administered to DCIS patients with diverse backgrounds via QoL questionnaire breast cancer module 23 and qualitative assessment questions. RESULTS: A total of 912 patients were reviewed. There were no treatment differences noted for age, race, or NDI. Mastectomy rate was higher in academic sites than community sites (29 vs. 20.4%; p = 0.0045), while hormone therapy (HT) utilization rate was higher in community sites (74 vs. 62%; p = 0.0012). QoL survey response rate was 32%. Only HT side effects negatively affected in QoL scores and there was no significant difference in QoL domains and decision-making process between races, age, NDI, treatment groups, and treatment locations. CONCLUSION: Our integrated health network did not show chronically noted disparities arising from social determinates of health for DCIS treatments by implementing clinical pathways and system-wide peer review. Also, we demonstrated feasibility in collecting QoL for DCIS women with diverse backgrounds and different socioeconomic statuses.

Mitochondrial interactome quantitation reveals structural changes in metabolic machinery in the failing murine heart.

Advancements in cross-linking mass spectrometry (XL-MS) bridge the gap between purified systems and native tissue environments, allowing the detection of protein structural interactions in their native state. Here we use isobaric quantitative protein interaction reporter technology (iqPIR) to compare the mitochondria protein interactomes in healthy and hypertrophic murine hearts, 4 weeks post-transaortic constriction. The failing heart interactome includes 588 statistically significant cross-linked peptide pairs altered in the disease condition. We observed an increase in the assembly of ketone oxidation oligomers corresponding to an increase in ketone metabolic utilization; remodeling of NDUA4 interaction in Complex IV, likely contributing to impaired mitochondria respiration; and conformational enrichment of ADP/ATP carrier ADT1, which is non-functional for ADP/ATP translocation but likely possesses non-selective conductivity. Our application of quantitative cross-linking technology in cardiac tissue provides molecular-level insights into the complex mitochondria remodeling in heart failure while bringing forth new hypotheses for pathological mechanisms.

Upregulation of mitochondrial ATPase inhibitory factor 1 (ATPIF1) mediates increased glycolysis in mouse hearts.

In hypertrophied and failing hearts, fuel metabolism is reprogrammed to increase glucose metabolism, especially glycolysis. This metabolic shift favors biosynthetic function at the expense of ATP production. Mechanisms responsible for the switch are poorly understood. We found that inhibitory factor 1 of the mitochondrial FoF1-ATP synthase (ATPIF1), a protein known to inhibit ATP hydrolysis by the reverse function of ATP synthase during ischemia, was significantly upregulated in pathological cardiac hypertrophy induced by pressure overload, myocardial infarction, or α-adrenergic stimulation. Chemical cross-linking mass spectrometry analysis of hearts hypertrophied by pressure overload suggested that increased expression of ATPIF1 promoted the formation of FoF1-ATP synthase nonproductive tetramer. Using ATPIF1 gain- and loss-of-function cell models, we demonstrated that stalled electron flow due to impaired ATP synthase activity triggered mitochondrial ROS generation, which stabilized HIF1α, leading to transcriptional activation of glycolysis. Cardiac-specific deletion of ATPIF1 in mice prevented the metabolic switch and protected against the pathological remodeling during chronic stress. These results uncover a function of ATPIF1 in nonischemic hearts, which gives FoF1-ATP synthase a critical role in metabolic rewiring during the pathological remodeling of the heart.

Effect of short-term corticosteroid usage on acute urinary toxicity following Cs-131 prostate brachytherapy.

PURPOSE: To evaluate short-term patient reported urinary quality of life scores in patients with prostate cancer treated at our institution with and without perioperative prednisone following Cesium-131 (131Cs) prostate LDR brachytherapy. METHODS AND MATERIALS: We started routinely using a perioperative 7-day course of prednisone at a dose of 5 mg per day, beginning 1 day prior to 131Cs prostate LDR brachytherapy from 2013 with goal of improving acute urinary symptomatology. One hundred consecutive patients treated with prednisone were selected, with comparison to 100 consecutive patients who were not treated with prednisone. We analyzed for differences in mean change with standard deviation (SD) in EPIC and AUA scores at 0.5-1 month and 3 months with or without prednisone by Mann-Whitney U Test. Binary logistic regression was performed to assess for impact of prednisone on postoperative urinary catheter use. RESULTS: Pretreatment EPIC and AUA scores were available in 197 patients. Less reduction in EPIC US score was noted at 0.5-1.0 month in the group who received prednisone with mean change of -22.9 (SD 15.4) when compared to the group who did not receive prednisone with mean change of -31.7 (SD 19.3), p < 0.01, with significance lost at 3 months. There was no significant difference in acute urinary retention requiring postoperative urinary catheter placement with perioperative prednisone (OR 1.13, p = 0.71). CONCLUSIONS: A short course of perioperative low-dose prednisone was associated with less severe worsening in urinary symptoms by the EPIC questionnaire at the 0.5-1.0-month timepoint suggesting some improvement in acute urinary quality of life, although differences did not remain statistically significant at 3 months.

Differential Proteome and Interactome Analysis Reveal the Basis of Pleiotropy Associated With the Histidine Methyltransferase Hpm1p.

The methylation of histidine is a post-translational modification whose function is poorly understood. Methyltransferase histidine protein methyltransferase 1 (Hpm1p) monomethylates H243 in the ribosomal protein Rpl3p and represents the only known histidine methyltransferase in Saccharomyces cerevisiae. Interestingly, the hpm1 deletion strain is highly pleiotropic, with many extraribosomal phenotypes including improved growth rates in alternative carbon sources. Here, we investigate how the loss of histidine methyltransferase Hpm1p results in diverse phenotypes, through use of targeted mass spectrometry (MS), growth assays, quantitative proteomics, and differential crosslinking MS. We confirmed the localization and stoichiometry of the H243 methylation site, found unreported sensitivities of Δhpm1 yeast to nonribosomal stressors, and identified differentially abundant proteins upon hpm1 knockout with clear links to the coordination of sugar metabolism. We adapted the emerging technique of quantitative large-scale stable isotope labeling of amino acids in cell culture crosslinking MS for yeast, which resulted in the identification of 1267 unique in vivo lysine-lysine crosslinks. By reproducibly monitoring over 350 of these in WT and Δhpm1, we detected changes to protein structure or protein-protein interactions in the ribosome, membrane proteins, chromatin, and mitochondria. Importantly, these occurred independently of changes in protein abundance and could explain a number of phenotypes of Δhpm1, not addressed by expression analysis. Further to this, some phenotypes were predicted solely from changes in protein structure or interactions and could be validated by orthogonal techniques. Taken together, these studies reveal a broad role for Hpm1p in yeast and illustrate how crosslinking MS will be an essential tool for understanding complex phenotypes.

Improved Interpretation of Protein Conformational Differences and Ligand Occupancy in Large-Scale Cross-Link Data.

Chemical cross-linking of proteins in complex samples, cells, or even tissues is emerging to provide unique structural information on proteins and complexes that exist within native or nativelike environments. The public database XLinkDB automatically maps cross-links to available structures based on sequence homology. Structures most likely to reflect protein conformations in the cross-linked sample are routinely identified by having cross-linked residues separated by Euclidean distances within the maximum span of the applied cross-linker. Solvent accessible surface distance (SASD), which considers the accessibility of the cross-linked residues and the path connecting them, is a better predictor of consistency than the Euclidean distance. However, SASDs of structures are not publicly available, and their calculation is computationally intensive. Here, we describe in XLinkDB version 4.0 the automatic calculation of SASDs using Jwalk for all cross-links mapped to structures, both with and without regard to ligands, and derive empirical maximum SASD spans for BDP-NHP and DSSO cross-linkers of 51 and 43 Å, respectively. We document ligands proximal to cross-links in structures and demonstrate how SASDs can be used to help infer sample protein conformations and ligand occupancy, highlighting cross-links sensitive to ADP binding in mitochondria isolated from HEK293 cells.