1H, 15N, 13C backbone and Cß resonance assignments for UBQLN1 UBA and UBAA domains.

UBQLN1 functions in autophagy and proteasome-mediated protein degradation. It contains an N-terminal ubiquitin-like domain (UBL), a C-terminal ubiquitin-associated domain (UBA), and a flexible central region which functions as a chaperone to prevent protein aggregation. Here, we report the 1H, 15N, and 13C resonance assignments for the backbone (NH, N, C', Cα, and Hα) and sidechain Cß atoms of the UBQLN1 UBA and an N-terminally adjacent segment called the UBA-adjacent domain (UBAA). We find a subset of the resonances corresponding to the UBAA to have concentration-dependent chemical shifts, likely due to self-association. We also find the backbone amide nitrogen of T572 to be shifted upfield relative to the average value for a threonine amide nitrogen, a phenomenon likely caused by T572 Hγ1 engagement in a hydrogen bond with adjacent backbone carbonyl atoms. The assignments described in this manuscript can be used to study the protein dynamics of the UBQLN1 UBA and UBAA as well as the interaction of these domains with other proteins.

E6AP AZUL interaction with UBQLN1/2 in cells, condensates, and an AlphaFold-NMR integrated structure.

The E3 ligase E6AP/UBE3A has a dedicated binding site in the 26S proteasome provided by the RAZUL domain of substrate receptor hRpn10/S5a/PSMD4. Guided by RAZUL sequence similarity, we test and demonstrate here that the E6AP AZUL binds transiently to the UBA of proteasomal shuttle factor UBQLN1/2. Despite a weak binding affinity, E6AP AZUL is recruited to UBQLN2 biomolecular condensates in vitro and E6AP interacts with UBQLN1/2 in cellulo. Steady-state and transfer nuclear Overhauser effect (NOE) experiments indicate direct interaction of AZUL with UBQLN1 UBA. Intermolecular contacts identified by NOE spectroscopy (NOESY) data were combined with AlphaFold2-Multimer predictions to yield an AZUL:UBA model structure. We additionally identify an oligomerization domain directly adjacent to UBQLN1/2 UBA (UBA adjacent [UBAA]) that is α-helical and allosterically reconfigured by AZUL binding to UBA. These data lead to a model of E6AP recruitment to UBQLN1/2 by AZUL:UBA interaction and provide fundamental information on binding requirements for interactions in condensates and cells.

Pattern of corneal disorders in Ekiti: A tertiary eye center experience.

Purpose: The cornea is the most significant refractive medium in the eye. Pathologies affecting the cornea usually have a great impact on vision. The etiology of corneal disorder varies from one geographical location to another. The objective of this study was to determine the pattern of corneal disorders at Ekiti State University Teaching Hospital, Ado-Ekiti. Materials and Methods: A retrospective study of case records of patients with cornea disorders over a 5-year period was carried out. Demographic characteristics, presenting visual acuity, and risk factor for cornea disorders were retrieved. Data were entered into the Statistical Package for the Social Sciences version 20, and statistical significance was inferred at P < 0.05. Results: Corneal disorders accounted for 3.3% of the eye disorders seen during the period of study. The median age was 37 years. Males outnumbered females giving a ratio of 1.9:1 and the age range from 0.25 to 92 years. There were more females than males in the 11-20 years' age group. Students (84, 25.4%) and artisans (62, 18.8%) were the two leading occupational groups. Infectious cases constituted 27.2% of the cases. Visual acuity at presentation was <3/60 in 131 (39.7%) cases. Foreign body entry was the leading etiologic agent in 101 (30.6%) cases. Conclusion: Half of the patients were blind at presentation, and many of them presented after more than 1 week of the onset of symptoms. Corneal foreign body, trauma, and vernal keratoconjunctivitis were the leading known predisposing factors. There will be need to emphasize more on the role of protective eye devices among our people, especially those who engage in outdoor activities.

RésuméObjectif: La cornée est le milieu de réfraction le plus important de l'œil. Les pathologies affectant la cornée ont généralement un grand impact sur la vision. L'étiologie du trouble cornéen varie d'un emplacement géographique à un autre. L'objectif de cette étude était de déterminer le schéma des troubles de la cornée à l'hôpital universitaire d'Ekiti State University, Ado Ekiti. Matériel et méthodes: Une étude rétrospective des dossiers des patients atteints de troubles de la cornée sur une période de 5 ans a été réalisée. Les caractéristiques démographiques, présentant l'acuité visuelle et le facteur de risque de troubles de la cornée ont été récupérées. Les données ont été entrées dans le progiciel statistique pour la version 20 des sciences sociales, et la signification statistique a été déduite à P <0,05. Résultats: Les troubles cornéens représentaient 3,3% des troubles oculaires observés pendant la période d'étude. L'âge médian était de 37 ans. Les hommes étaient plus nombreux que les femmes, ce qui.

Pattern of corneal disorders in Ekiti: a tertiary eye center experience

Purpose: The cornea is the most significant refractive medium in the eye. Pathologies affecting the cornea usually have a great impact on vision. The etiology of corneal disorder varies from one geographical location to another. The objective of this study was to determine the pattern of corneal disorders at Ekiti State University Teaching Hospital, Ado-Ekiti. Materials and Methods: A retrospective study of case records of patients with cornea disorders over a 5-year period was carried out. Demographic characteristics, presenting visual acuity, and risk factor for cornea disorders were retrieved. Data were entered into the Statistical Package for the Social Sciences version 20, and statistical significance was inferred at P < 0.05. Results: Corneal disorders accounted for 3.3% of the eye disorders seen during the period of study. The median age was 37 years. Males outnumbered females giving a ratio of 1.9:1 and the age range from 0.25 to 92 years. There were more females than males in the 11­20 years' age group. Students (84, 25.4%) and artisans (62, 18.8%) were the two leading occupational groups. Infectious cases constituted 27.2% of the cases. Visual acuity at presentation was <3/60 in 131 (39.7%) cases. Foreign body entry was the leading etiologic agent in 101 (30.6%) cases. Conclusion: Half of the patients were blind at presentation, and many of them presented after more than 1 week of the onset of symptoms. Corneal foreign body, trauma, and vernal keratoconjunctivitis were the leading known predisposing factors. There will be need to emphasize more on the role of protective eye devices among our people, especially those who engage in outdoor activities

Covalent Rpn13-Binding Inhibitors for the Treatment of Ovarian Cancer.

Substitution of the m,p-chloro groups of bis-benzylidinepiperidone RA190 for p-nitro, generating RA183, enhanced covalent drug binding to Cys88 of RPN13. Treatment of cancer cell lines with RA183 inhibited ubiquitin-mediated protein degradation, resulting in rapid accumulation of high-molecular-weight polyubiquitinated proteins, blockade of NFκB signaling, endoplasmic reticulum stress, an unfolded protein response, production of reactive oxygen species, and apoptotic cell death. High-grade ovarian cancer, triple-negative breast cancer, and multiple myeloma cell lines were particularly vulnerable to RA183. RA183 stabilized a tetraubiquitin-linked firefly luciferase reporter protein in cancer cell lines and mice, demonstrating in vitro and in vivo proteasomal inhibition, respectively. However, RA183 was rapidly cleared from plasma, likely reflecting its rapid degradation to the active compound RA9, as seen in human liver microsomes. Intraperitoneal administration of RA183 inhibited proteasome function and orthotopic tumor growth in mice bearing human ovarian cancer model ES2-luc ascites or syngeneic ID8-luc tumor.

Pharmacokinetics of dacarbazine (DTIC) in pregnancy.

PURPOSE: The purpose of this report is to describe, for the first time, the pharmacokinetics of dacarbazine (DTIC) and its metabolites [5-[3-methyl-triazen-1-yl]-imidazole-4-carboxamide (MTIC), 5-[3-hydroxymethyl-3-methyl-triazen-1-yl]-imidazole-4-carboxamide (HMMTIC) and 5-aminoimidazole-4-carboxamide (AIC)] during pregnancy (n = 2) and postpartum (n = 1). METHODS: Non-compartmental DTIC, MTIC, HMMTIC, and AIC pharmacokinetics (PK) were estimated in one case at 29 week gestation and 18 day postpartum and a second case at 32 week gestation, in women receiving DTIC in combination with doxorubicin, bleomycin, and vinblastine for treatment of Hodgkin's lymphoma. Drug concentrations were measured by HPLC. RESULTS: In the subject who completed both pregnancy and postpartum study days, DTIC area under the concentration-time curve (AUC) was 27% higher and metabolite AUCs were lower by 27% for HMMTIC, 38% for MTIC, and 83% of AIC during pregnancy compared to postpartum. At 7 and 9 year follow-up, both subjects were in remission of their Hodgkin's lymphoma. CONCLUSIONS: Based on these two case reports, pregnancy appears to decrease the metabolism of the pro-drug dacarbazine, likely through inhibition of CYP1A2 activity. Lower concentrations of active metabolites and decreased efficacy may result, although both these subjects experienced long-term remission of their Hodgkin's lymphoma.

A Phase I Clinical Trial of the Poly(ADP-ribose) Polymerase Inhibitor Veliparib and Weekly Topotecan in Patients with Solid Tumors.

Purpose: To determine the dose limiting toxicities (DLT), maximum tolerated dose (MTD), and recommended phase II dose (RP2D) of veliparib in combination with weekly topotecan in patients with solid tumors. Correlative studies were included to assess the impact of topotecan and veliparib on poly(ADP-ribose) levels in peripheral blood mononuclear cells, serum pharmacokinetics of both agents, and potential association of germline repair gene mutations with outcome.Experimental Design: Eligible patients had metastatic nonhematologic malignancies with measurable disease. Using a 3 + 3 design, patients were treated with veliparib orally twice daily on days 1-3, 8-10, and 15-17 and topotecan intravenously on days 2, 9, and 16 every 28 days. Tumor responses were assessed by RECIST.Results: Of 58 patients enrolled, 51 were evaluable for the primary endpoint. The MTD and RP2D was veliparib 300 mg twice daily on days 1-3, 8-10, and 15-17 along with topotecan 3 mg/m2 on days 2, 9, and 16 of a 28-day cycle. DLTs were grade 4 neutropenia lasting >5 days. The median number of cycles was 2 (1-26). The objective response rate was 10%, with 1 complete and 4 partial responses. Twenty-two patients (42%) had stable disease ranging from 4 to 26 cycles. Patients with germline BRCA1, BRCA2, or RAD51D mutations remained on study longer than those without homologous recombination repair (HRR) gene mutations (median 4 vs. 2 cycles).Conclusions: Weekly topotecan in combination with veliparib has a manageable safety profile and appears to warrant further investigation. Clin Cancer Res; 24(4); 744-52. ©2017 AACR.

Small molecule inhibitors of mesotrypsin from a structure-based docking screen.

PRSS3/mesotrypsin is an atypical isoform of trypsin, the upregulation of which has been implicated in promoting tumor progression. To date there are no mesotrypsin-selective pharmacological inhibitors which could serve as tools for deciphering the pathological role of this enzyme, and could potentially form the basis for novel therapeutic strategies targeting mesotrypsin. A virtual screen of the Natural Product Database (NPD) and Food and Drug Administration (FDA) approved Drug Database was conducted by high-throughput molecular docking utilizing crystal structures of mesotrypsin. Twelve high-scoring compounds were selected for testing based on lowest free energy docking scores, interaction with key mesotrypsin active site residues, and commercial availability. Diminazene (CID22956468), along with two similar compounds presenting the bis-benzamidine substructure, was validated as a competitive inhibitor of mesotrypsin and other human trypsin isoforms. Diminazene is the most potent small molecule inhibitor of mesotrypsin reported to date with an inhibitory constant (Ki) of 3.6±0.3 µM. Diminazene was subsequently co-crystalized with mesotrypsin and the crystal structure was solved and refined to 1.25 Å resolution. This high resolution crystal structure can now offer a foundation for structure-guided efforts to develop novel and potentially more selective mesotrypsin inhibitors based on similar molecular substructures.

An Acrobatic Substrate Metamorphosis Reveals a Requirement for Substrate Conformational Dynamics in Trypsin Proteolysis.

The molecular basis of enzyme catalytic power and specificity derives from dynamic interactions between enzyme and substrate during catalysis. Although considerable effort has been devoted to understanding how conformational dynamics within enzymes affect catalysis, the role of conformational dynamics within protein substrates has not been addressed. Here, we examine the importance of substrate dynamics in the cleavage of Kunitz-bovine pancreatic trypsin inhibitor protease inhibitors by mesotrypsin, finding that the varied conformational dynamics of structurally similar substrates can profoundly impact the rate of catalysis. A 1.4-Å crystal structure of a mesotrypsin-product complex formed with a rapidly cleaved substrate reveals a dramatic conformational change in the substrate upon proteolysis. By using long all-atom molecular dynamics simulations of acyl-enzyme intermediates with proteolysis rates spanning 3 orders of magnitude, we identify global and local dynamic features of substrates on the nanosecond-microsecond time scale that correlate with enzymatic rates and explain differential susceptibility to proteolysis. By integrating multiple enhanced sampling methods for molecular dynamics, we model a viable conformational pathway between substrate-like and product-like states, linking substrate dynamics on the nanosecond-microsecond time scale with large collective substrate motions on the much slower time scale of catalysis. Our findings implicate substrate flexibility as a critical determinant of catalysis.

Combinatorial protein engineering of proteolytically resistant mesotrypsin inhibitors as candidates for cancer therapy.

Engineered protein therapeutics offer advantages, including strong target affinity, selectivity and low toxicity, but like natural proteins can be susceptible to proteolytic degradation, thereby limiting their effectiveness. A compelling therapeutic target is mesotrypsin, a protease up-regulated with tumour progression, associated with poor prognosis, and implicated in tumour growth and progression of many cancers. However, with its unique capability for cleavage and inactivation of proteinaceous inhibitors, mesotrypsin presents a formidable challenge to the development of biological inhibitors. We used a powerful yeast display platform for directed evolution, employing a novel multi-modal library screening strategy, to engineer the human amyloid precursor protein Kunitz protease inhibitor domain (APPI) simultaneously for increased proteolytic stability, stronger binding affinity and improved selectivity for mesotrypsin inhibition. We identified a triple mutant APPIM17G/I18F/F34V, with a mesotrypsin inhibition constant (Ki) of 89 pM, as the strongest mesotrypsin inhibitor yet reported; this variant displays 1459-fold improved affinity, up to 350 000-fold greater specificity and 83-fold improved proteolytic stability compared with wild-type APPI. We demonstrated that APPIM17G/I18F/F34V acts as a functional inhibitor in cell-based models of mesotrypsin-dependent prostate cancer cellular invasiveness. Additionally, by solving the crystal structure of the APPIM17G/I18F/F34V-mesotrypsin complex, we obtained new insights into the structural and mechanistic basis for improved binding and proteolytic resistance. Our study identifies a promising mesotrypsin inhibitor as a starting point for development of anticancer protein therapeutics and establishes proof-of-principle for a novel library screening approach that will be widely applicable for simultaneously evolving proteolytic stability in tandem with desired functionality for diverse protein scaffolds.

Mesotrypsin Has Evolved Four Unique Residues to Cleave Trypsin Inhibitors as Substrates.

Human mesotrypsin is highly homologous to other mammalian trypsins, and yet it is functionally unique in possessing resistance to inhibition by canonical serine protease inhibitors and in cleaving these inhibitors as preferred substrates. Arg-193 and Ser-39 have been identified as contributors to the inhibitor resistance and cleavage capability of mesotrypsin, but it is not known whether these residues fully account for the unusual properties of mesotrypsin. Here, we use human cationic trypsin as a template for engineering a gain of catalytic function, assessing mutants containing mesotrypsin-like mutations for resistance to inhibition by bovine pancreatic trypsin inhibitor (BPTI) and amyloid precursor protein Kunitz protease inhibitor (APPI), and for the ability to hydrolyze these inhibitors as substrates. We find that Arg-193 and Ser-39 are sufficient to confer mesotrypsin-like resistance to inhibition; however, compared with mesotrypsin, the trypsin-Y39S/G193R double mutant remains 10-fold slower at hydrolyzing BPTI and 2.5-fold slower at hydrolyzing APPI. We identify two additional residues in mesotrypsin, Lys-74 and Asp-97, which in concert with Arg-193 and Ser-39 confer the full catalytic capability of mesotrypsin for proteolysis of BPTI and APPI. Novel crystal structures of trypsin mutants in complex with BPTI suggest that these four residues function cooperatively to favor conformational dynamics that assist in dissociation of cleaved inhibitors. Our results reveal that efficient inhibitor cleavage is a complex capability to which at least four spatially separated residues of mesotrypsin contribute. These findings suggest that inhibitor cleavage represents a functional adaptation of mesotrypsin that may have evolved in response to positive selection pressure.

Prevalence of harmful/traditional medication use in traumatic eye injury.

AIM: Ocular trauma of varying aetiologies do occur frequently, however when different traditional/harmful substances are applied before presentation in the hospital, prognosis in terms of visual outcome following treatment may be worse than expected. This study is aimed at determining the prevalence of harmful/traditional eye medication practices among patients with traumatic eye injury in a tertiary institution. STUDY DESIGN/SETTING: It is a retrospective study of patients seen at the eye clinic of the Ekiti State University Teaching Hospital (a state government owned hospital), Ado Ekiti, from January to December 2009. METHODS: A review of case notes (medical records) of patients with history of ocular trauma both open and closed globe injury and who presented to the eye clinic of the Ekiti State University Teaching Hospital, Ado Ekiti from January to December 2009 was carried out. Demographic data of the patients, nature of the ocular trauma, substances applied to the eyes, visual acuity at presentation and after treatment were extracted. Frequencies and percentages were used in analysing the data. RESULT: A total of 1420 new patients attended the eye clinic during the study period (January to December 2009). Forty eight (3.4%) applied various substances into their eyes after sustaining ocular injury. Substances applied include Kerosene 25%, cassava water 20.8%, breast milk 12.5%, personal urine 10.8%, and cow urine 8.3%. Nearly half of the patients 23 (47.9%) presented with low vision and after treatment there was no visual improvement in almost all of them, 22 (45.8%). The period before presentation ranges between 1hr -2 weeks post injury. However, the number of monocular blindness reduced from 8 (16.7%) to 5 (10.4%) after treatment. CONCLUSION: Kerosene and Cassava water were the common substances applied to the injured eye. The use of these harmful and traditional eye medications on injured eyes can reduce further the visual prognosis despite ophthalmic intervention.

Utilization of Eye Care Services Among Staff of a Tertiary Hospital.

PURPOSE: To determine the level of utilization of eye care services and to identify the barriers to uptake of eye care services among the staff in a hospital. DESIGN: A cross-sectional study conducted at a university teaching hospital. METHODS: A total of 250 staff members were selected using a proportionate sampling among the segment of study population. Data were collected using semistructured questionnaires, including demographic data, awareness about eye clinic and the services rendered, facilities utilized by staff in receiving eye treatment, and reasons for not utilizing the hospital eye care services. Data analysis was done using SPSS version 15. RESULTS: The majority (66%) of the staff were younger than 40 years. Around 229 staff members (91.6%) were aware of the clinic, whereas 222 (88.8%) were aware of at least 1 of the various services rendered. They received treatment from chemists (30.7%), private hospitals (26.3%), and optical shops (16.1%). The hospital eye clinic (11.8%) was the least chosen place to receive eye treatment. The reasons for nonutilization of eye care services were lack of finance (42.1%), poor staff attitude (23.7%), fear of damage to the eye (15.3%), high cost of treatment (9.7%), and ignorance of its existence (9.2%). Visual impairment was seen in 14 of the staff (5.6%), whereas blindness was seen in 1 (0.4%). CONCLUSIONS: The level of utilization of eye care services in the hospital by the staff is poor and very low compared with other facilities, although the majority had previous history of eye complaints.