Implications of the structure of human uridine phosphorylase 1 on the development of novel inhibitors for improving the therapeutic window of fluoropyrimidine chemotherapy

<p>Abstract</p> <p>Background</p> <p>Uridine phosphorylase (UPP) is a key enzyme of pyrimidine salvage pathways, catalyzing the reversible phosphorolysis of ribosides of uracil to nucleobases and ribose 1-phosphate. It is also a critical enzyme in the activation of pyrimidine-based chemotherapeutic compounds such a 5-fluorouracil (5-FU) and its prodrug capecitabine. Additionally, an elevated level of this enzyme in certain tumours is believed to contribute to the selectivity of such drugs. However, the clinical effectiveness of these fluoropyrimidine antimetabolites is hampered by their toxicity to normal tissue. In response to this limitation, specific inhibitors of UPP, such as 5-benzylacyclouridine (BAU), have been developed and investigated for their ability to modulate the cytotoxic side effects of 5-FU and its derivatives, so as to increase the therapeutic index of these agents.</p> <p>Results</p> <p>In this report we present the high resolution structures of human uridine phosphorylase 1 (hUPP1) in ligand-free and BAU-inhibited conformations. The structures confirm the unexpected solution observation that the human enzyme is dimeric in contrast to the hexameric assembly present in microbial UPPs. They also reveal in detail the mechanism by which BAU engages the active site of the protein and subsequently disables the enzyme by locking the protein in a closed conformation. The observed inter-domain motion of the dimeric human enzyme is much greater than that seen in previous UPP structures and may result from the simpler oligomeric organization.</p> <p>Conclusion</p> <p>The structural details underlying hUPP1's active site and additional surfaces beyond these catalytic residues, which coordinate binding of BAU and other acyclouridine analogues, suggest avenues for future design of more potent inhibitors of this enzyme. Notably, the loop forming the back wall of the substrate binding pocket is conformationally different and substantially less flexible in hUPP1 than in previously studied microbial homologues. These distinctions can be utilized to discover novel inhibitory compounds specifically optimized for efficacy against the human enzyme as a step toward the development of more effective chemotherapeutic regimens that can selectively protect normal tissues with inherently lower UPP activity.</p

Characterization of the family of Mistic homologues

BACKGROUND: Mistic is a unique Bacillus subtilis protein with virtually no detectable homologues in GenBank, which appears to integrate into the bacterial membrane despite an overall hydrophilic composition. These unusual properties have been shown to be useful for high-yield recombinant expression of other membrane proteins through fusion to the C-terminus of Mistic. To better understand the structure and function of Mistic, we systematically searched for and characterized homologous proteins among closely related bacteria. RESULTS: Three homologues of Mistic were found with 62% to 93% residue identity, all only 84 residues in length, corresponding to the C-terminal residues of B. subtilis Mistic. In every case, the Mistic gene was found partially overlapping a downstream gene for a K(+ )channel protein. Residue variation amongst these sequences is restricted to loop regions of the protein's structure, suggesting that secondary structure elements and overall fold have been conserved. Additionally, all three homologues retain the functional ability to chaperone fusion partners to the membrane. CONCLUSION: The functional core of Mistic consists of 84 moderately conserved residues that are sufficient for membrane targeting and integration. Understanding the minimal structural and chemical complexity of Mistic will lead to insights into the mechanistic underpinnings of Mistic-chaperoned membrane integration, as well as how to optimize its use for the recombinant heterologous expression of other integral membrane proteins of interest

Assessing Sleep Habits in Italian Community-Dwelling Adolescents: Psychometric Properties of the School Sleep Habits Survey Scales

Background. In the field of adolescent sleep research, different sleep surveys have been implemented; however, psychometric properties of these instruments have been investigated only minimally. Methods. In order to assess the psychometric properties of the Sleep–Wake Problems Behaviour Scale (SWP), the Sleepiness Scale (SLS), and the Morningness/Eveningness Questionnaire (ME), a moderately large sample of community-dwelling Italian adolescents (N = 778; 59.8% female; mean age = 15.77 years) was administered the Italian translation of the School Sleep Habits Survey. Results. Internal consistency estimates values were satisfactory for all measures; dimensionality analyses suggested a unidimensional structure for SWP, SLS and ME, respectively. Goodness-of-fit statistics for the one-factor model of the SLS, SWP, and ME scale items were adequate for all measures. Non-redundant taxometric analysis results consistently suggested a dimensional latent structure for the SLS, SWP, and ME, respectively. Conclusion. Our findings supported the use of the SLS, SWP, and ME total scores as measures of sleepiness, sleep-wake problem, and morningness/eveningness, at least among Italian community-dwelling adolescents, and encourage practitioners to rely on the conventional percentiles in order to interpret the SLS, SWP, and ME total scores

Active Site Conformational Dynamics in Human Uridine Phosphorylase 1

Uridine phosphorylase (UPP) is a central enzyme in the pyrimidine salvage pathway, catalyzing the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate. Human UPP activity has been a focus of cancer research due to its role in activating fluoropyrimidine nucleoside chemotherapeutic agents such as 5-fluorouracil (5-FU) and capecitabine. Additionally, specific molecular inhibitors of this enzyme have been found to raise endogenous uridine concentrations, which can produce a cytoprotective effect on normal tissues exposed to these drugs. Here we report the structure of hUPP1 bound to 5-FU at 2.3 Å resolution. Analysis of this structure reveals new insights as to the conformational motions the enzyme undergoes in the course of substrate binding and catalysis. The dimeric enzyme is capable of a large hinge motion between its two domains, facilitating ligand exchange and explaining observed cooperativity between the two active sites in binding phosphate-bearing substrates. Further, a loop toward the back end of the uracil binding pocket is shown to flexibly adjust to the varying chemistry of different compounds through an “induced-fit” association mechanism that was not observed in earlier hUPP1 structures. The details surrounding these dynamic aspects of hUPP1 structure and function provide unexplored avenues to develop novel inhibitors of this protein with improved specificity and increased affinity. Given the recent emergence of new roles for uridine as a neuron protective compound in ischemia and degenerative diseases, such as Alzheimer's and Parkinson's, inhibitors of hUPP1 with greater efficacy, which are able to boost cellular uridine levels without adverse side-effects, may have a wide range of therapeutic applications

Prolonged depression of knee extensor torque complexity following eccentric exercise

Neuromuscular fatigue reduces the temporal structure, or complexity, of muscle torque output. Exercise-induced muscle damage reduces muscle torque output for considerably longer than high-intensity fatiguing contractions. We hypothesised that muscle damaging eccentric exercise would lead to a persistent decrease in torque complexity, whereas fatiguing exercise would not. Ten healthy participants performed five isometric contractions (6 s contraction, 4 s rest) at 50% maximal voluntary contraction (MVC) before, immediately after, 10, 30 and 60 minutes, and 24 hours after eccentric (muscle damaging) and isometric (fatiguing) exercise. These contractions were also repeated 48 hours and one week after eccentric exercise. Torque and surface EMG signals were sampled throughout each test. Complexity and fractal scaling were quantified using approximate entropy (ApEn) and the detrended fluctuation analysis ? exponent (DFA ?). Global, central and peripheral perturbations were quantified using MVCs with femoral nerve stimulation. Complexity decreased following both eccentric (ApEn, mean (SD), from 0.39 (0.10) to 0.20 (0.12), P < 0.001) and isometric exercise (from 0.41 (0.13) to 0.09 (0.04); P < 0.001). After eccentric exercise ApEn and DFA ? required 24 hours to recover to baseline levels, but only 10 minutes following isometric exercise. MVC torque remained reduced (from 233.6 (74.2) to 187.5 (64.7) N.m) 48 hours after eccentric exercise, with such changes only evident up to 60 minutes following isometric exercise (MVC torque, from 246.1 (77.2) to 217.9 (71.8) N.m). The prolonged depression in maximal muscle torque output is therefore accompanied by a prolonged reduction in torque complexity