ZMYM3 regulates BRCA1 localization at damaged chromatin to promote DNA repair.

Chromatin connects DNA damage response factors to sites of damaged DNA to promote the signaling and repair of DNA lesions. The histone H2A variants H2AX, H2AZ, and macroH2A represent key chromatin constituents that facilitate DNA repair. Through proteomic screening of these variants, we identified ZMYM3 (zinc finger, myeloproliferative, and mental retardation-type 3) as a chromatin-interacting protein that promotes DNA repair by homologous recombination (HR). ZMYM3 is recruited to DNA double-strand breaks through bivalent interactions with both histone and DNA components of the nucleosome. We show that ZMYM3 links the HR factor BRCA1 to damaged chromatin through specific interactions with components of the BRCA1-A subcomplex, including ABRA1 and RAP80. By regulating ABRA1 recruitment to damaged chromatin, ZMYM3 facilitates the fine-tuning of BRCA1 interactions with DNA damage sites and chromatin. Consistent with a role in regulating BRCA1 function, ZMYM3 deficiency results in impaired HR repair and genome instability. Thus, our work identifies a critical chromatin-binding DNA damage response factor, ZMYM3, which modulates BRCA1 functions within chromatin to ensure the maintenance of genome integrity.

Rilonacept use in lupus pericarditis.

OBJECTIVES: Lupus pericarditis affects 22% of patients with systemic lupus erythematosus (SLE), is associated with worse outcomes, and often requires immunosuppression. Rilonacept is an interleukin-1 receptor antagonist approved for the treatment of recurrent idiopathic pericarditis, but its efficacy in lupus pericarditis is unknown. Here, we report the efficacy of rilonacept in a case series of patients with lupus pericarditis. METHODS: We describe a case series of 4 patients with refractory lupus pericarditis treated with rilonacept in the Johns Hopkins Lupus Center. All patients met the 2012 SLICC criteria for SLE. Refractory lupus pericarditis was defined as recurring or persistent typical pericardial pain symptoms despite standard-of-care treatment including at least one immunosuppressant. RESULTS: Four patients with refractory pericarditis were included. All patients were women, age ranged 26-44 years, 2 patients reported White, 1 Black, and 1 Hispanic ethnicity. Extra-pericardial SLE manifestations were heterogeneous among patients. Only 1 of 3 patient had elevated CRP (not measured in one). Two patients were previously treated with anakinra with initial response, but pericarditis redeveloped in both. Rilonacept led to complete resolution of pericardial symptoms in 3 patients, and partial resolution (40%) in 1, within 2 weeks. CONCLUSIONS: Rilonacept successfully treated lupus pericarditis in this case series. Rilonacept should be considered for the treatment of lupus pericarditis.

Diagnostic Schemas: Form and Function.

Diagnostic schemas are frameworks that depict organized clinical knowledge and serve as a bridge between problem representation and differential diagnosis generation. Schema-based problem solving is increasingly used among clinician educators and is widely featured in digital media. We examine the origins of schemas and their theoretical background, review existing literature on their applications in medicine, and explore their utility for learners and teachers.

Screen identifies bromodomain protein ZMYND8 in chromatin recognition of transcription-associated DNA damage that promotes homologous recombination.

How chromatin shapes pathways that promote genome-epigenome integrity in response to DNA damage is an issue of crucial importance. We report that human bromodomain (BRD)-containing proteins, the primary "readers" of acetylated chromatin, are vital for the DNA damage response (DDR). We discovered that more than one-third of all human BRD proteins change localization in response to DNA damage. We identified ZMYND8 (zinc finger and MYND [myeloid, Nervy, and DEAF-1] domain containing 8) as a novel DDR factor that recruits the nucleosome remodeling and histone deacetylation (NuRD) complex to damaged chromatin. Our data define a transcription-associated DDR pathway mediated by ZMYND8 and the NuRD complex that targets DNA damage, including when it occurs within transcriptionally active chromatin, to repress transcription and promote repair by homologous recombination. Thus, our data identify human BRD proteins as key chromatin modulators of the DDR and provide novel insights into how DNA damage within actively transcribed regions requires chromatin-binding proteins to orchestrate the appropriate response in concordance with the damage-associated chromatin context.

The Clinical Usefulness of the Practice Resource for Driving after Stroke (PReDAS).

Occupational Therapists (OTs) have identified a critical need for organized, evidence-based resources to approach driving post-stroke. The Practice Resource for Driving After Stroke (PReDAS) is a resource to support the clinical reasoning and practice of health professionals for addressing driving in acute stroke care. The purpose of this pilot study is to evaluate the usefulness of the PReDAS to support clinician and patient decision-making about return to driving after stroke/Transient Ischemic Attack (TIA) in the acute care hospital setting. OTs, physicians, and patients diagnosed with stroke/TIA were surveyed regarding their experience with the PReDAS in acute care. Patient participants were also contacted for a follow-up questionnaire. OT, physician and patient stakeholders reported the PReDAS was useful to support decision-making for driving. The majority of patients recalled information provided in acute care and abstained from driving as advised. This study provides preliminary support for the clinical usefulness of the PReDAS.

A Scoping Review of Environmental Factors That Impact Driving with Arthritis: Considerations for Occupational Therapy.

Using the International Classification of Functioning, Disability, and Health, this scoping review mapped environmental barriers and facilitators that can influence driving with arthritis. A search of research databases located 2445 studies from which 19 were included. The predominant diagnosis researched was rheumatoid arthritis. The most common facilitators were vehicle adaptations (e.g., supplemental mirrors, seat cushions). Barriers included a lack of knowledge among clinicians to address behind-the-wheel concerns and nonuse/abandonment of such adaptions. Results highlight key clinical and research opportunities to support drivers with arthritis.

A Lysine-Targeted Affinity Label for Serine-ß-Lactamase Also Covalently Modifies New Delhi Metallo-ß-lactamase-1 (NDM-1).

The divergent sequences, protein structures, and catalytic mechanisms of serine- and metallo-ß-lactamases hamper the development of wide-spectrum ß-lactamase inhibitors that can block both types of enzymes. The O-aryloxycarbonyl hydroxamate inactivators of Enterobacter cloacae P99 class C serine-ß-lactamase are unusual covalent inhibitors in that they target both active-site Ser and Lys residues, resulting in a cross-link consisting of only two atoms. Many clinically relevant metallo-ß-lactamases have an analogous active-site Lys residue used to bind ß-lactam substrates, suggesting a common site to target with covalent inhibitors. Here, we demonstrate that an O-aryloxycarbonyl hydroxamate inactivator of serine-ß-lactamases can also serve as a classical affinity label for New Delhi metallo-ß-lactamase-1 (NDM-1). Rapid dilution assays, site-directed mutagenesis, and global kinetic fitting are used to map covalent modification at Lys211 and determine KI (140 µM) and kinact (0.045 min-1) values. Mass spectrometry of the intact protein and the use of ultraviolet photodissociation for extensive fragmentation confirm stoichiometric covalent labeling that occurs specifically at Lys211. A 2.0 Å resolution X-ray crystal structure of inactivated NDM-1 reveals that the covalent adduct is bound at the substrate-binding site but is not directly coordinated to the active-site zinc cluster. These results indicate that Lys-targeted affinity labels might be a successful strategy for developing compounds that can inactivate both serine- and metallo-ß-lactamases.

Impact of Diabetes on 30-Day Complications in Mastectomy and Implant-Based Breast Reconstruction.

BACKGROUND: Diabetic patients are known to be at increased risk of postoperative complications after multiple types of surgery. However, conflicting evidence exists regarding the association between diabetes and wound complications in mastectomy and breast reconstruction. This study evaluates the impact of diabetes on surgical outcomes after mastectomy procedures and implant-based breast reconstruction. METHODS: The American College of Surgeons National Surgical Quality Improvement Program database review from 2010 to 2015 identified patients undergoing total, partial, or subcutaneous mastectomy, as well as immediate or delayed implant reconstruction. Primary outcomes included postoperative wound complications and implant failure. Preoperative variables and outcomes were compared between diabetic and nondiabetic patients. Multivariate regression was used to control for confounders. RESULTS: The following groups were identified: partial (n = 52,583), total (n = 41,540), and subcutaneous mastectomy (n = 3145), as well as immediate (n = 4663) and delayed (n = 4279) implant reconstruction. Diabetes was associated with higher rates of superficial incisional surgical site infection (SSI) in partial mastectomy (odds ratio [OR] = 8.66; P = 0.03). Diabetes was also associated with higher rates of deep incisional SSI (OR = 1.61; P = 0.01) in subcutaneous mastectomy and both superficial (OR = 1.56; P = 0.04) and deep incisional SSI (OR = 2.07; P = 0.04) in total mastectomy. Diabetes was not associated with any wound complications in immediate reconstruction but was associated with higher rates of superficial incisional SSI (OR = 17.46; P < 0.001) in the delayed reconstruction group. There was no association with implant failure in either group. CONCLUSIONS: Evaluation of the largest national cohort of mastectomy and implant reconstructive procedures suggests that diabetic patients are at significantly increased risk of 30-d postoperative infectious wound complications but present no difference in rates of early implant failure.

Diabetes is not associated with increased rates of free flap failure: Analysis of outcomes in 6030 patients from the ACS-NSQIP database.

BACKGROUND: Diabetes affects a significant proportion of the population in the United States. Microsurgical procedures are common in this patient population, and despite many conflicting reports in the literature, there are no large studies evaluating the direct association between diabetes and outcomes, specifically failure, following free flap reconstruction. In this study, we sought to determine the impact of diabetes on postoperative outcomes following free flap reconstruction using a national multi-institutional database. METHODS: We reviewed the American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) database to identify patients undergoing free flap reconstruction from 2010 to 2015. Preoperative variables and outcomes were compared between diabetic and nondiabetic patients. Univariate and multivariate analyses were performed to control for confounders. RESULTS: We identified 6030 eligible patients. No significant difference in flap failure rates was observed. However, diabetic patients presented significantly higher rates of wound complications, including deep incisional surgical site infection (SSI) (OR = 1.35; P = .01) and wound dehiscence (OR = 1.17; P = .03). Diabetic patients also presented a significantly longer hospital length of stay (LOS) (ß = .62; P < .001). CONCLUSIONS: Our study evaluated the largest national cohort of free flap procedures. These results suggest that diabetes is not associated with increased rates of flap failure. However, diabetic patients are at significantly higher risk of postoperative deep incisional SSI, wound dehiscence, and longer LOS. Our findings provide the most concrete evidence to date in support of free flap reconstruction in diabetic patients, but highlight the need for heightened clinical vigilance and wound care for optimal outcomes.

Predictors of Adverse Outcomes in the Management of Mandibular Fractures.

BACKGROUND: Mandibular fractures are the most common isolated facial fractures in the United States. Various risk factors have been associated with increased rates of postoperative complications. However, national outcome reports are limited and data are conflicting. Using a national multi-institutional database, the authors sought to analyze 30-day outcomes after mandibular fracture repair and determine risk factors for complications, readmission, and reoperation. METHODS: Retrospective review of the American College of Surgeons National Surgical Quality Improvement database was performed to identify patients undergoing surgical treatment of mandibular fractures between 2010 and 2015 using current procedural terminology codes. Primary outcomes included: wound complications, overall complications, as well as readmission and reoperation rates. Multivariate regression analysis was performed to adjust for confounders. RESULTS: A total of 953 patients were eligible for analysis. Mean patient age was 34.5 years, 84% were males, and 50% were active smokers. Wound complications, overall complications, 30-day reoperation, and readmission occurred in 4.0%, 7.9%, 2.2%, and 33% of patients, respectively. Age was a significant risk factor for 30-day readmission (odds ratio [OR] = 1.06, P = 0.01), reoperation (OR = 1.05, P = 0.01), and overall complications (OR = 1.03, P = 0.02) on multivariate analysis, and smoking was a significant risk factor for 30-day reoperation (OR = 4.86, P = 0.03). CONCLUSION: The analysis identified age as an independent risk factor for readmission, reoperation, and overall complications. Smokers were also nearly 5 times as likely to undergo additional surgery. This is particularly important given that over half of patients were active smokers, highlighting the importance of perioperative patient education and smoking cessation within this population.

Bimaxillary Orthognathic Surgery Is Associated With an Increased Risk of Early Complications.

INTRODUCTION: Orthognathic surgery plays an important role in restoring aesthetic facial contour, correcting dental malocclusion, and the surgical treatment of obstructive sleep apnea. However, the rate of complications following bimaxillary as compared with single-jaw orthognathic surgery remains unclear. The authors therefore sought to evaluate complication rates following bimaxillary as compared with single-jaw orthognathic surgery MATERIALS AND METHODS:: The American College of Surgeons National Surgical Quality Improvement Program database was used to identify comparison groups. Preoperative characteristics and postoperative outcomes were compared between groups. The listed procedures have different operating times and characteristics with longer time expected in the bimaxillary osteotomies group. Regression analyses were performed to control for potential confounders. RESULTS: The 3 groups of interest included patients who underwent mandibular osteotomies (n = 126), LeFort I osteotomy (n = 194), and bimaxillary osteotomies (n = 190). These procedures have different operating times, with a longer time expected with bimaxillary osteotomies. Patients undergoing bimaxillary osteotomies had significantly higher rates of early wound complications, overall complications, longer mean operative time, and mean hospital length of stay. Performing bimaxillary osteotomies in the outpatient setting was an independent risk factor for wound complications (OR = 12.58; 95% CI: 1.66-95.20; P = 0.01), while an ASA class of 3 or more was an independent risk factor for overall complications (OR = 3.61; 95% CI: 1.02-12.75; P = 0.04) and longer hospital length of stay (ß = 4.96; 95% CI: 2.64 - 7.29; P < 0.001). CONCLUSIONS: Surgery in the outpatient setting as well as patient American Society of Anesthesiology physical status class 3 or higher were independent factors for postoperative adverse events in patients undergoing bimaxillary surgery. Our findings highlight the importance of addressing modifiable risk factors preoperatively and the need for closer postoperative monitoring in this patient population for optimal outcomes.

Combined Primary Cleft Lip and Palate Repair: Is It Safe?

BACKGROUND: Single-stage primary cleft lip and palate (PCLP) repair is controversial in the United States, and most patients are treated with a staged approach. In this study, early postoperative complications of the single-stage approach as compared to primary cleft lip (PCL) or primary cleft palate (PCP) alone were evaluated. This study represents the largest cohort of patients undergoing combined cleft lip and palate repair. METHODS: The American College of Surgeons National Surgical Quality Improvement Program-Pediatric database was used to identify patients undergoing single-stage PCLP, PCL, or PCP repairs. Preoperative factors and postoperative outcomes were compared between the 3 groups, as well as within the PCLP group between patients with and without complications. Univariate and multivariate analyses were performed. RESULTS: A TOTAL OF:: 181 patients were included in the single-stage PCLP group, 1007 in the PCP group and 783 in the PCL group. There was no difference in the rates of early complications between the 3 groups. Within the PCLP group, cardiac risk factors (ß = 35.19; 95% confidence interval [CI] 7.88-75.21; P = 0.04) and complications (ß = 77.31; 95% CI 35.82-118.79; P < 0.001) were significant risk factors for longer operative time. CONCLUSION: Analysis of a national database showed that single-stage PCLP repair is not associated with increased risk of early postoperative complications as compared to primary lip or palate repair alone. In-depth long-term analyses of craniofacial morphology, fistulae rate, speech, and dental outcomes are essential for a comprehensive assessment of the effects of combined cleft lip and palate repair.

Obesity and Lower Extremity Reconstruction: Evaluating Body Mass Index as an Independent Risk Factor for Early Complications.

BACKGROUND: The prevalence of obesity in the United States continues to grow and is estimated to affect over a quarter of the working-age population. Some studies have identified obesity as a risk factor for flap failure and complications in free flap-based breast reconstruction, but its clinical significance is less clear in nonbreast reconstruction. The role of obesity as a risk factor for failure and complications following lower extremity reconstruction has not been well described, and the limited existing literature demonstrates conflicting results. METHODS: The American College of Surgeons National Surgical Quality Improvement Program database was reviewed to identify patients undergoing local- or free-flap reconstruction of the lower extremity between 2010 and 2015. Preoperative variables and outcomes were compared between obese (body mass index ≥ 30) and nonobese patients. Chi-square analysis and Fisher's exact test were used for categorical variables and t-tests for continuous variables. Multivariate regression was performed to control for confounders. RESULTS: Univariate analysis of medical and surgical outcomes revealed that obese patients undergoing local flaps of the lower extremity required a significantly longer operative time (187.7 ± 123.2 vs. 166.2 ± 111.7 minutes; p = 0.003) and had significantly higher rates of superficial surgical site infection (SSI; 7.2% vs. 4.5%; p = 0.04). On univariate analysis, there were no significant differences in any postoperative outcomes between obese and nonobese patients undergoing microvascular free flaps of the lower extremity.On multivariate regression analysis, obesity was not an independent risk factor for superficial SSI (odds ratio = 1.01, p = 0.98) or increased operative time (ß = 16.01, p = 0.14) for local flaps of the lower extremity. CONCLUSION: Evaluation of a large, multicenter, validated and risk-adjusted nationwide cohort demonstrated that obesity is not an independent risk factor for early complications following lower extremity reconstruction, suggesting that these procedures may be performed safely in the obese patient population.

Tracking the Catalytic Cycle of Adenylate Kinase by Ultraviolet Photodissociation Mass Spectrometry.

The complex interplay of dynamic protein plasticity and specific side-chain interactions with substrate molecules that allows enzymes to catalyze reactions has yet to be fully unraveled. Top-down ultraviolet photodissociation (UVPD) mass spectrometry is used to track snapshots of conformational fluctuations in the phosphotransferase adenylate kinase (AK) throughout its active reaction cycle by characterization of complexes containing AK and each of four different adenosine phosphate ligands. Variations in efficiencies of UVPD backbone cleavages were consistently observed for three α-helices and the adenosine binding regions for AK complexes representing different steps of the catalytic cycle, implying that these stretches of the protein sample various structural microstates as the enzyme undergoes global open-to-closed transitions. Focusing on the conformational impact of recruiting or releasing the Mg2+ cofactor highlights two loop regions for which fragmentation increases upon UVPD, signaling an increase in loop flexibility as the metal cation disrupts the loop interactions with the substrate ligands. Additionally, the observation of holo ions and variations in UVPD backbone cleavage efficiency at R138 implicate this conserved active site residue in stabilizing the donor phosphoryl group during catalysis. This study showcases the utility of UVPD-MS to provide insight into conformational fluctuations of single residues for active enzymes.

Expanding the Scope of Cross-Link Identifications by Incorporating Collisional Activated Dissociation and Ultraviolet Photodissociation Methods.

With the advent of new cross-linking chemistries, analytical technologies, and search algorithms, cross-linking has become an increasingly popular strategy for evaluating tertiary and quaternary structures of proteins. Collisional activated dissociation remains the primary MS/MS method for identifications of peptide cross-links in high throughput workflows. Ultraviolet photodissociation (UVPD) at 193 nm has emerged as an alternative ion activation method well-suited for characterization of peptides and has been found in some cases to identify different peptides or provide distinctive sequence information than obtained by collisional activation methods. Complementary high energy collision dissociation (HCD) and UVPD were used in the present study to characterize protein cross-linking for bovine serum albumin, hemoglobin, and E. coli ribosome. Cross-links identified by HCD and UVPD using bis(sulfosuccinimidyl)suberate (BS3), a homobifunctional amine-to-amine cross-linker, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM), a heterofunctional amine-to-carboxylic acid cross-linker, were evaluated in the present study. While more unique BS3 cross-links were identified by HCD, UVPD, and HCD provided a complementary panel of DMTMM cross-links which extended the degree of structural insight obtained for the proteins.

Comparison of outcomes with surgical cut-down versus percutaneous transfemoral transcatheter aortic valve replacement: TAVR transfemoral access comparisons between surgical cut-down and percutaneous approach.

OBJECTIVES: The objective is to compare the short-term (30 days) and late (12 months) vascular adverse events in patients undergoing transfemoral (TF)-transcatheter aortic valve replacement (TAVR) by surgical cut-down (SC) vs. percutaneous (PC) approaches. BACKGROUND: Programs continue to utilize both approaches in TF-TAVR. There are limited data comparing outcomes by SC vs. PC approaches and long-term effects of endovascular intervention facilitated hemostasis on late vascular adverse events. METHODS: A total of 146 men and women aged 79.7 ± 10.0 years with severe aortic stenosis deemed extreme or high risk for surgery underwent TAVR via TF access. 61 had SC and 85 had PC approaches. Valve Academic Research Consortium (VARC-2) outcomes were assessed at an average of 12.1 months after TAVR. RESULTS: Hospital length of stay (LOS) post-TAVR was shorter for the PC group compared to the SC group (5.1 ± 3.9 vs. 8.2 ± 6.6 days; P < 0.001). More patients were discharged directly to home in the PC than the SC group (85.9% vs. 68.9%, P < 0.05). At 30 days, there were 13/61 (21.3%) and 16/85 (18.8%; P < 0.05) of any vascular events, and 2/61 (3.3%) and 2/85 (2.4%; P = 0.73) major vascular events in the SC and PC groups, respectively. There was no difference in all-cause mortality between the SC (14/61; 23%) and PC groups [17/85 (20%); P = 0.34]. There was no difference in any [4/33 (12%) vs. 3/43 (7%); P = 0.84] or major vascular adverse events [1/33 (3%) vs. 1/43 (2%); P = 0.79] in subjects that underwent adjunctive endovascular intervention compared to those who did not, respectively. There were no statistically significant univariate or multivariate predictors of any vascular event at 12 months when comparing SC to PC groups. CONCLUSION: For TF TAVR, the PC approach, when compared to the SC approach, is associated with a shorter hospital LOS and higher rate of direct discharge to home with similar risk of vascular complications, late vascular adverse events, and all-cause mortality at 12 months.

Single-Stage Primary Cleft Lip and Palate Repair: A Review of the Literature.

BACKGROUND: Single-stage cleft lip and palate repair is a debated surgical approach. While some studies have described favorable outcomes, concerns include the effect on craniomaxillofacial growth and increased risk of complications. To this date, there has not been a comprehensive appraisal of available data following combined cleft lip and palate repair. METHODS: An extensive literature review was performed to identify all relevant articles. The level of evidence of these articles was graded according to the Oxford Centre for Evidence-Based Medicine Levels of Evidence Scale. RESULTS: A total of 22 relevant articles were identified, all of which were retrospective in nature. Patient age at the time of surgery ranged from 1 month to 10 years, the longest duration of follow-up was 18 years, and the largest study included 106 patients. Review of the literature shows that overall surgical outcomes following combined cleft lip and palate repair are encouraging. An increased rate of postoperative fistulas with associated speech abnormalities in some studies is noteworthy. Importantly, there is no evidence to suggest an impact on craniomaxillofacial growth, and psychosocial outcomes and parental satisfaction seem to be improved with single-stage surgery as compared with the staged approach. CONCLUSIONS: Our review shows overall favorable outcomes associated with combined cleft lip and palate repair. The limited follow-up time or nature of evaluated outcomes in some studies may underrepresent the true rate of adverse events, and highlights the need for additional long-term studies with standardized follow-up. To our knowledge, our review is the first to evaluate existing data regarding outcomes following combined cleft lip and palate repair.

Structural Characterization of Native Proteins and Protein Complexes by Electron Ionization Dissociation-Mass Spectrometry.

Mass spectrometry (MS) has played an increasingly important role in the identification and structural and functional characterization of proteins. In particular, the use of tandem mass spectrometry has afforded one of the most versatile methods to acquire structural information for proteins and protein complexes. The unique nature of electron capture dissociation (ECD) for cleaving protein backbone bonds while preserving noncovalent interactions has made it especially suitable for the study of native protein structures. However, the intra- and intermolecular interactions stabilized by hydrogen bonds and salt bridges can hinder the separation of fragments even with preactivation, which has become particularly problematic for the study of large macromolecular proteins and protein complexes. Here, we describe the capabilities of another activation method, 30 eV electron ionization dissociation (EID), for the top-down MS characterization of native protein-ligand and protein-protein complexes. Rich structural information that cannot be delivered by ECD can be generated by EID. EID allowed for the comparison of the gas-phase and the solution-phase structural stability and unfolding process of human carbonic anhydrase I (HCA-I). In addition, the EID fragmentation patterns reflect the structural similarities and differences among apo-, Zn-, and Cu,Zn-superoxide dismutase (SOD1) dimers. In particular, the structural changes due to Cu-binding and a point mutation (G41D) were revealed by EID-MS. The performance of EID was also compared to that of 193 nm ultraviolet photodissociation (UVPD), which allowed us to explore their qualitative similarities and differences as potential valuable tools for the MS study of native proteins and protein complexes.

Impact of G12 Mutations on the Structure of K-Ras Probed by Ultraviolet Photodissociation Mass Spectrometry.

Single-residue mutations at Gly12 (G12X) in the GTP-ase protein K-Ras can lead to activation of different downstream signaling pathways, depending on the identity of the mutation, through a poorly defined mechanism. Herein, native mass spectrometry combined with top-down ultraviolet photodissociation (UVPD) was employed to investigate the structural changes occurring from G12X mutations of K-Ras. Complexes between K-Ras or the G12X mutants and guanosine 5'-diphosphate (GDP) or GDPnP (a stable GTP analogue) were transferred to the gas phase by nano-electrospray ionization and characterized using UVPD. Variations in the efficiencies of backbone cleavages were observed upon substitution of GDPnP for GDP as well as for the G12X mutants relative to wild-type K-Ras. An increase in the fragmentation efficiency in the segment containing the first 50 residues was observed for the K-Ras/GDPnP complexes relative to the K-Ras/GDP complexes, whereas a decrease in fragmentation efficiency occurred in the segment containing the last 100 residues. Within these general regions, the specific residues at which changes in fragmentation efficiency occurred correspond to the phosphate and guanine binding regions, respectively, and are indicative of a change in the binding motif upon replacement of the ligand (GDP versus GDPnP). Notably, unique changes in UVPD were observed for each G12X mutant with the cysteine and serine mutations exhibiting similar UVPD changes whereas the valine mutation was significantly different. These findings suggest a mechanism that links the identity of the G12X substitution to different downstream effects through long-range conformational or dynamic effects as detected by variations in UVPD fragmentation.

A New Covalent Inhibitor of Class C ß-Lactamases Reveals Extended Active Site Specificity.

O-Aryloxycarbonyl hydroxamates have previously been shown to efficiently inactivate class C ß-lactamases by cross-linking serine and lysine residues in the active site. A new analogue of these inhibitors, D-(R)-O-(phenoxycarbonyl)-N-[(4-amino-4-carboxy-1-butyl)oxycarbonyl]hydroxylamine, designed to inactivate certain low-molecular mass dd-peptidases, has now been synthesized. Although the new molecule was found to be only a poor inactivator of the latter enzymes, it proved, unexpectedly, to be a very effective inactivator (ki = 3.5 × 10(4) M(-1) s(-1)) of class C ß-lactamases, more so than the original lead compound, O-phenoxycarbonyl-N-(benzyloxycarbonyl)hydroxylamine. Furthermore, the mechanism of inactivation is different. Mass spectrometry demonstrated that ß-lactamase inactivation by the new molecule involved formation of an O-alkoxycarbonylhydroxamate with the nucleophilic active site serine residue. This acyl-enzyme did not cyclize to cross-link the active site as did that from the lead compound. Model building suggested that the rapid enzyme acylation by the new molecule may occur because of favorable interaction between the polar terminus of its side chain and elements of the Ω loop that abuts the active site, Arg 204 in particular. This interaction should be considered in the design of new covalent ß-lactamase inhibitors. The initially formed acyl-enzyme partitions (ratio of ∼ 1) between hydrolysis, which regenerates the active enzyme, and formation of an inert second acyl-enzyme. Structural modeling suggests that the latter intermediate arises from conformational movement of the acyl group away from the reaction center, probably enforced by the inflexibility of the acyl group. The new molecule is thus a mechanism-based inhibitor in which an inert complex is formed by noncovalent rearrangement. Phosphyl analogues of the new molecule were efficient inactivators of neither dd-peptidases nor ß-lactamases.