Search

© 2015, Public Library of Science. All rights reserved. This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is mad

OBJECTIVE: The authors examined the comorbidity of borderline personality disorder with other personality disorders in a series of consecutively admitted adolescents. For comparison, the comorbidity of borderline personality disorder with other personality disorders was also examined in a series of adults consecutively admitted to the same hospital during the same period. METHOD: A total of 138 adolescents and 117 adults were reliably assessed with the Personality Disorder Examination, a semistructured diagnostic interview for DSM-III-R personality disorders. Sixty-eight adolescents and 50 adults met the diagnostic criteria for borderline personality disorder. The co-occurrence of other personality disorders in the group of subjects with borderline personality disorder was statistically compared to that in the group without borderline personality disorder, for adolescents and adults separately. RESULTS: For the adults, Bonferroni-corrected chi-square analysis revealed significant diagnostic co-occurrence with borderline personality disorder for antisocial personality disorder only. For the adolescents, borderline personality disorder showed significant co-occurrence with schizotypal and passive-aggressive personality disorders. CONCLUSIONS: In the adults, borderline personality disorder was significantly comorbid only with another cluster B disorder. The adolescents, by comparison, displayed a broader pattern of comorbidity of borderline personality disorder, encompassing aspects of clusters A and C. These results suggest that the borderline personality disorder diagnosis may represent a more diffuse range of psychopathology in adolescents than in adults. [ABSTRACT FROM AUTHOR]

The flavoenzyme proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the oxidation of l-proline to Δ 1 -pyrroline-5-carboxylate. The enzyme is a target for chemical probe discovery because of its role in the metabolism of certain cancer cells. N -propargylglycine is the first and best characterized mechanism-based covalent inactivator of PRODH. This compound consists of a recognition module (glycine) that directs the inactivator to the active site and an alkyne warhead that reacts with the FAD after oxidative activation, leading to covalent modification of the FAD N5 atom. Here we report structural and kinetic data on analogs of N -propargylglycine with the goals of understanding the initial docking step of the inactivation mechanism and to test the allyl group as a warhead. The crystal structures of PRODH complexed with unreactive analogs in which N is replaced by S show how the recognition module mimics the substrate proline by forming ion pairs with conserved arginine and lysine residues. Further, the C atom adjacent to the alkyne warhead is optimally positioned for hydride transfer to the FAD, providing the structural basis for the first bond-breaking step of the inactivation mechanism. The structures also suggest new strategies for designing improved N -propargylglycine analogs. N -allylglycine, which consists of a glycine recognition module and allyl warhead, is shown to be a covalent inactivator; however, it is less efficient than N -propargylglycine in both enzyme inactivation and cellular assays. Crystal structures of the N -allylglycine-inactivated enzyme are consistent with covalent modification of the N5 by propanal.

Objective: Sutter Health launched system-wide general population standardized suicide screening with the Columbia-Suicide Severity Rating Scale (C-SSRS) screen (triage) version in 23 hospitals in 2019, replacing a one-question "danger to self" (DTS) assessment. This study analyzed the impact of C-SSRS implementation on screening rates, positive screenings, and documented psychiatric care within 90 days for all patients and a subgroup diagnosed with Major Depressive Disorder (MDD)., Methods: Adults seen at hospitals in the pre-period (July 1, 2017-June 30, 2019) and post-period (July 1, 2019-December 31, 2020) were identified using electronic health records. Outcomes were compared using chi-square statistics and interrupted time series (ITS) models., Results: Pre-period, 92.8% (740,984/798,653) of patients were screened by DTS versus 84.6% (504,015/595,915) by C-SSRS in the post-period. Positive screening rates were 1.5% pre-period and 2.2% post-period, and 9.2% pre-period versus 10.8% post-period for those with MDD. Among individuals with positive screenings, 64.0% (pre-period) had documented follow-up psychiatric care versus 52.5% post-period and 66.4% of those with moderate or high-risk. Among all patients seen there was an overall increase in documentation of psychiatric care within 90 days (0.87% pre- to 0.96% post-period). ITS models revealed a 9.6% decline in screening, 1.3% increase in positive screenings, and 12.9% decline in documented psychiatric care following C-SSRS implementation (all p  < 0.01)., Conclusions: Following implementation, there was meaningful increase in suicide risk identification, and an increase in the proportion of patients with documented psychiatric care. Observed relative declines in screening warrant future research examining opportunities and barriers to general population C-SSRS use., (© 2024 The Authors. Psychiatric Research and Clinical Practice published by Wiley Periodicals LLC on behalf of American Psychiatric Association.)

The tumor microenvironment is composed of a complex mixture of different cell types interacting under conditions of nutrient deprivation, but the metabolism therein is not fully understood due to difficulties in measuring metabolic fluxes and exchange of metabolites between different cell types in vivo . Genome-scale metabolic modeling enables estimation of such exchange fluxes as well as an opportunity to gain insight into the metabolic behavior of individual cell types. Here, we estimated the availability of nutrients and oxygen within the tumor microenvironment using concentration measurements from blood together with a metabolite diffusion model. In addition, we developed an approach to efficiently apply enzyme usage constraints in a comprehensive metabolic model of human cells. The combined modeling reproduced severe hypoxic conditions and the Warburg effect, and we found that limitations in enzymatic capacity contribute to cancer cells' preferential use of glutamine as a substrate to the citric acid cycle. Furthermore, we investigated the common hypothesis that some stromal cells are exploited by cancer cells to produce metabolites useful for the cancer cells. We identified over 200 potential metabolites that could support collaboration between cancer cells and cancer-associated fibroblasts, but when limiting to metabolites previously identified to participate in such collaboration, no growth advantage was observed. Our work highlights the importance of enzymatic capacity limitations for cell behaviors and exemplifies the utility of enzyme-constrained models for accurate prediction of metabolism in cells and tumor microenvironments., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)

Background: Despite significant progress in clinical management, colorectal cancer (CRC) remains the third most common cause of cancer-related deaths. A positive association between PYCR2 (pyrroline-5-carboxylate reductase-2), a terminal enzyme of proline metabolism, and CRC aggressiveness was recently reported. However, how PYCR2 promotes colon carcinogenesis remains ill understood., Methods: A comprehensive analysis was performed using publicly available cancer databases and CRC patient cohorts. Proteomics and biochemical evaluations were performed along with genetic manipulations and in vivo tumor growth assays to gain a mechanistic understanding., Results: PYCR2 expression was significantly upregulated in CRC and associated with poor patient survival, specifically among PYCR isoforms (PYCR1, 2, and 3). The genetic inhibition of PYCR2 inhibited the tumorigenic abilities of CRC cells and in vivo tumor growth. Coinciding with these observations was a significant decrease in cellular proline content. PYCR2 overexpression promoted the tumorigenic abilities of CRC cells. Proteomics (LC-MS/MS) analysis further demonstrated that PYCR2 loss of expression in CRC cells inhibits survival and cell cycle pathways. A subsequent biochemical analysis supported the causal role of PYCR2 in regulating CRC cell survival and the cell cycle, potentially by regulating the expression of MASTL, a cell-cycle-regulating protein upregulated in CRC. Further studies revealed that PYCR2 regulates Wnt/β-catenin-signaling in manners dependent on the expression of MASTL and the cancer stem cell niche., Conclusions: PYCR2 promotes MASTL/Wnt/β-catenin signaling that, in turn, promotes cancer stem cell populations and, thus, colon carcinogenesis. Taken together, our data highlight the significance of PYCR2 as a novel therapeutic target for effectively treating aggressive colon cancer.

Background: During the COVID-19 pandemic, many Americans experienced new or worsened mental health conditions. Concurrently, much care switched from in-person to virtual care, highlighting the value of virtual care but also some of the underlying challenges., Methods: This paper explores one such challenge, the separation of mental health care from physical health care, and a potential solution, collaborative care. It is a team-based approach linking psychiatrists to primary care providers that can help break down the silos of care created through reimbursement models., Results: In this context of collaborative care, high quality virtual care further bridges the divide between physical and mental health care. Asynchronous virtual care for mental and behavioral health is an innovation that can create efficiencies while still supporting collaborative care., Discussion: The barriers and weaknesses of using virtual care exclusively for mental and behavioral health are discussed, as well as examples of policy changes which can improve mental health care through collaborative virtual care., Competing Interests: P.Y. reports employment and ownership in AsyncHealth Inc. All others have no conflict of interest., (© John Scott et al., 2023; Published by Mary Ann Liebert, Inc.)

Significance: Cells depend on well-functioning mitochondria for essential processes such as energy production, redox signaling, coordination of metabolic pathways, and cofactor biosynthesis. Mitochondrial dysfunction, metabolic decline, and protein stress have been implicated in the etiology of multiple late-onset diseases, including various ataxias, diabetes, sarcopenia, neuromuscular disorders, and neurodegenerative diseases such as parkinsonism, amyotrophic lateral sclerosis, and glaucoma. Recent Advances: New evidence supports that increased energy metabolism protects neuron function during aging. Key energy metabolic enzymes, however, are susceptible to oxidative damage making it imperative that the mitochondrial proteome is protected. More than 40 different enzymes have been identified as important factors for guarding mitochondrial health and maintaining a dynamic pool of mitochondria. Critical Issues: Understanding shared mechanisms of age-related disorders of neurodegenerative diseases such as glaucoma, Alzheimer's disease, and Parkinson's disease is important for developing new therapies. Functional mitochondrial shape and dynamics rely on complex interactions between mitochondrial proteases and membrane proteins. Identifying the sequence of molecular events that lead to mitochondrial dysfunction and metabolic stress is a major challenge. Future Directions: A critical need exists for new strategies that reduce mitochondrial protein stress and promote mitochondrial dynamics in age-related neurological disorders. Discovering how mitochondria-associated degradation is related to proteostatic mechanisms in mitochondrial compartments may reveal new opportunities for therapeutic interventions. Also, little is known about how protein and membrane contacts in the inner and outer mitochondrial membrane are regulated, even though they are pivotal for mitochondrial architecture. Future work will need to delineate the molecular details of these processes.

Implementation of suicide risk screening may improve prevention and facilitate mental health treatment. This study analyzed implementation of universal general population screening using the Columbia-Suicide Severity Rating Scale (C-SSRS) within hospitals. The study included adults seen at 23 hospitals from 7/1/2019-12/31/2020. We describe rates of screening, suicide risk, and documented subsequent psychiatric care (i.e., transfer/discharge to psychiatric acute care, or referral/consultation with system-affiliated behavioral health providers). Patients with suicide risk (including those with Major Depressive Disorder [MDD]) were compared to those without using Wilcoxon rank-sum -tests for continuous variables and χ2 tests for categorical variables. Results reported are statistically significant at p < 0.05 level. Among 595,915 patients, 84.5% were screened by C-SSRS with 2.2% of them screening positive (37.6% low risk [i.e., ideation only], and 62.4% moderate or high risk [i.e., with a plan, intent, or suicidal behaviors]). Of individuals with suicide risk, 52.5% had documentation of psychiatric care within 90 days. Individuals with suicide risk (vs. without) were male (48.1% vs 43.0%), Non-Hispanic White (55.0% vs 47.8%), younger (mean age 41.0 [SD: 17.7] vs. 49.8 [SD: 20.4]), housing insecure (12.5% vs 2.6%), with mental health diagnoses (80.3% vs 25.1%), including MDD (41.3% vs 6.7%). Universal screening identified 2.2% of screened adults with suicide risk; 62.4% expressed a plan, intent or suicidal behaviors, and 80.3% had mental health diagnoses. Documented subsequent psychiatric care likely underestimates true rates due to care fragmentation. These findings reinforce the need for screening, and research on whether screening leads to improved care and fewer suicides., (© Society of Behavioral Medicine 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Pyrroline-5-carboxylate reductase (PYCR) is a proline biosynthetic enzyme that catalyzes the NAD(P)H-dependent reduction of Δ 1 -pyrroline-5-carboxylate (P5C) to proline. Humans have three PYCR isoforms, with PYCR1 often upregulated in different types of cancers. Here, we studied the biochemical and structural properties of the Thr171Met variant of PYCR1, which is found in patients with malignant melanoma and lung adenocarcinoma. Although PYCR1 is strongly associated with cancer progression, characterization of a PYCR1 variant in cancer patients has not yet been reported. Thr171 is conserved in all three PYCR isozymes and is located near the P5C substrate binding site. We found that the amino acid replacement does not affect thermostability but has a profound effect on PYCR1 catalytic activity. The k cat of the PYCR1 variant T171M is 100- to 200-fold lower than wild-type PYCR1 when P5C is the variable substrate, and 10- to 25-fold lower when NAD(P)H is varied. A 1.84 Å resolution X-ray crystal structure of T171M reveals that the Met side chain invades the P5C substrate binding site, suggesting that the catalytic defect is due to steric clash preventing P5C from achieving the optimal pose for hydride transfer from NAD(P)H. These results suggest that any impact on PYCR1 function associated with T171M in cancer does not derive from increased catalytic activity., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Abstract: Adolescent pregnancy and the babies of teen mothers have been a focus of attention and concern for many years. The literature on the health of pregnant and parenting teens, however, is largely silent about the impact of stigma on them and their children. Stigma is a pervasive cultural attitude, which leads us to overlook potentially good outcomes for this vulnerable population and contributes to poor outcomes for these mothers and babies. Stigma is a cause of stress leading to well-known adverse effects on health and development for both mothers and babies. This article reviews manifestations of social stigma in our communities, in the offices of professionals, and in policies that embed bias in our social systems. The impact of inequity has been vividly revealed over the course of the global pandemic. We review findings related to interventions associated with better outcomes and offer suggestions for more humane care and policies., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

L-Thioproline (L-thiazolidine-4-carboxylate, L-T4C) is a cyclic sulfur-containing analog of L-proline found in multiple kingdoms of life. The oxidation of L-T4C leads to L-cysteine formation in bacteria, plants, mammals, and protozoa. The conversion of L-T4C to L-Cys in bacterial cell lysates has been attributed to proline dehydrogenase and L-Δ 1 -pyrroline-5-carboxylate (P5C) reductase (PYCR) enzymes but detailed kinetic studies have not been conducted. Here, we characterize the dehydrogenase activity of human PYCR isozymes 1 and 2 with L-T4C using NAD(P) + as the hydride acceptor. Both PYCRs exhibit significant L-T4C dehydrogenase activity; however, PYCR2 displays nearly tenfold higher catalytic efficiency (136 M -1  s -1 ) than PYCR1 (13.7 M -1  s -1 ). Interestingly, no activity was observed with either L-Pro or the analog DL-thiazolidine-2-carboxylate, indicating that the sulfur at the 4-position is critical for PYCRs to utilize L-T4C as a substrate. Inhibition kinetics show that L-Pro is a competitive inhibitor of PYCR1 [Formula: see text] with respect to L-T4C, consistent with these ligands occupying the same binding site. We also confirm by mass spectrometry that L-T4C oxidation by PYCRs leads to cysteine product formation. Our results suggest a new enzyme function for human PYCRs in the metabolism of L-T4C., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Thiazolidine carboxylates such as thiazolidine-4-carboxylate (T4C) and thiazolidine-2-carboxylate (T2C) are naturally occurring sulfur analogues of proline. These compounds have been observed to have both beneficial and toxic effects in cells. Given that proline dehydrogenase has been proposed to be a key enzyme in the oxidative metabolism of thioprolines, we characterized T4C and T2C as substrates of proline catabolic enzymes using proline utilization A (PutA), which is a bifunctional enzyme with proline dehydrogenase (PRODH) and l-glutamate-γ-semialdehyde dehydrogenase (GSALDH) activities. PutA is shown here to catalyze the FAD-dependent PRODH oxidation of both T4C and T2C with catalytic efficiencies significantly higher than with proline. Stopped-flow experiments also demonstrate that l-T4C and l-T2C reduce PutA-bound FAD at rates faster than proline. Unlike proline, however, oxidation of T4C and T2C does not generate a substrate for NAD + -dependent GSALDH. Instead, PutA/PRODH oxidation of T4C leads to cysteine formation, whereas oxidation of T2C generates an apparently stable Δ 4 -thiazoline-2-carboxylate species. Our results provide new insights into the metabolism of T2C and T4C.

Proline dehydrogenase (PRODH) is a flavoenzyme that catalyzes the first step of proline catabolism, the oxidation of l-proline to Δ 1 -pyrroline-5-carboxylate. PRODH has emerged as a cancer therapy target because of its involvement in the metabolic reprogramming of cancer cells. Here, we report the discovery of a new class of PRODH inactivator, which covalently and irreversibly modifies the FAD in a light-dependent manner. Two examples, 1,3-dithiolane-2-carboxylate and tetrahydrothiophene-2-carboxylate, have been characterized using X-ray crystallography (1.52-1.85 Å resolution), absorbance spectroscopy, and enzyme kinetics. The structures reveal that in the dark, these compounds function as classical reversible, proline analogue inhibitors. However, exposure of enzyme-inhibitor cocrystals to bright white light induces decarboxylation of the inhibitor and covalent attachment of the residual S-heterocycle to the FAD N5 atom, locking the cofactor into a reduced, inactive state. Spectroscopic measurements of the inactivation process in solution confirm the requirement for light and show that blue light is preferred. Enzyme activity assays show that the rate of inactivation is enhanced by light and that the inactivation is irreversible. We also demonstrate the photosensitivity of cancer cells to one of these compounds. A possible mechanism is proposed involving photoexcitation of the FAD, while the inhibitor is noncovalently bound in the active site, followed by electron transfer, decarboxylation, and radical combination steps. Our results could lead to the development of photopharmacological drugs targeting PRODH.

In many bacteria, the reactions of proline catabolism are catalyzed by the bifunctional enzyme known as proline utilization A (PutA). PutA catalyzes the two-step oxidation of l-proline to l-glutamate using distinct proline dehydrogenase (PRODH) and l-glutamate-γ-semialdehyde dehydrogenase (GSALDH) active sites, which are separated by over 40 Å and connected by a complex tunnel system. The tunnel system consists of a main tunnel that connects the two active sites and functions in substrate channeling, plus six ancillary tunnels whose functions are unknown. Here we used tunnel-blocking mutagenesis to probe the role of a dynamic ancillary tunnel (tunnel 2a) whose shape is modulated by ligand binding to the PRODH active site. The 1.90 Å resolution crystal structure of Geobacter sulfurreducens PutA variant A206W verified that the side chain of Trp206 cleanly blocks tunnel 2a without perturbing the surrounding structure. Steady-state kinetic measurements indicate the mutation impaired PRODH activity without affecting the GSALDH activity. Single-turnover experiments corroborated a severe impairment of PRODH activity with flavin reduction decreased by nearly 600-fold in A206W relative to wild-type. Substrate channeling is also significantly impacted as A206W exhibited a 3000-fold lower catalytic efficiency in coupled PRODH-GSALDH activity assays, which measure NADH formation as a function of proline. The structure suggests that Trp206 inhibits binding of the substrate l-proline by preventing the formation of a conserved glutamate-arginine ion pair and closure of the PRODH active site. Our data are consistent with tunnel 2a serving as an open space through which the glutamate of the ion pair travels during the opening and closing of the active site in response to binding l-proline. These results confirm the essentiality of the conserved ion pair in binding l-proline and support the hypothesis that the ion pair functions as a gate that controls access to the PRODH active site., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Aldehyde dehydrogenase 4A1 (ALDH4A1) catalyzes the final steps of both proline and hydroxyproline catabolism. It is a dual substrate enzyme that catalyzes the NAD + -dependent oxidations of L-glutamate-γ-semialdehyde to L-glutamate (proline metabolism), and 4-hydroxy-L-glutamate-γ-semialdehyde to 4-erythro-hydroxy-L-glutamate (hydroxyproline metabolism). Here we investigated the inhibition of mouse ALDH4A1 by the six stereoisomers of proline and 4-hydroxyproline using steady-state kinetics and X-ray crystallography. Trans-4-hydroxy-L-proline is the strongest of the inhibitors studied, characterized by a competitive inhibition constant of 0.7 mM, followed by L-proline (1.9 mM). The other compounds are very weak inhibitors (approximately 10 mM or greater). Insight into the selectivity for L-stereoisomers was obtained by solving crystal structures of ALDH4A1 complexed with trans-4-hydroxy-L-proline and trans-4-hydroxy-D-proline. The structures suggest that the 10-fold greater preference for the L-stereoisomer is due to a serine residue that hydrogen bonds to the amine group of trans-4-hydroxy-L-proline. In contrast, the amine group of the D-stereoisomer lacks a direct interaction with the enzyme due to a different orientation of the pyrrolidine ring. These results suggest that hydroxyproline catabolism is subject to substrate inhibition by trans-4-hydroxy-L-proline, analogous to the known inhibition of proline catabolism by L-proline. Also, drugs targeting the first enzyme of hydroxyproline catabolism, by elevating the level of trans-4-hydroxy-L-proline, may inadvertently impair proline catabolism by the inhibition of ALDH4A1., (© 2021 The Protein Society.)

Pyrroline-5-carboxylate reductase (PYCR in humans) catalyzes the final step of l-proline biosynthesis by catalyzing the reduction of L-Δ 1 -pyrroline-5-carboxylate (L-P5C) to l-proline using NAD(P)H as the hydride donor. In humans, three isoforms PYCR1, PYCR2, and PYCR3 are known. Recent genome-wide association and clinical studies have revealed that homozygous mutations in human PYCR2 lead to postnatal microcephaly and hypomyelination, including hypomyelinating leukodystrophy type 10. To uncover biochemical and structural insights into human PYCR2, we characterized the steady-state kinetics of the wild-type enzyme along with two protein variants, Arg119Cys and Arg251Cys, that were previously identified in patients with microcephaly and hypomyelination. Kinetic measurements with PYCR2 suggest a sequential binding mechanism with L-P5C binding before NAD(P)H and NAD(P) + releasing before L-Pro. Both disease-related variants are catalytically impaired. Depending on whether NADPH or NADH was used, the catalytic efficiency of the R119C protein variant was 40 or 366 times lower than that of the wild-type enzyme, while the catalytic efficiency of the R251C protein variant was 7 or 26 times lower than that of the wild-type enzyme. In addition, thermostability and circular dichroism measurements suggest that the R251C protein variant has a pronounced folding defect. These results are consistent with the involvement of Arg119Cys and Arg251Cys in disease pathology., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Proline utilization A (PutA) proteins are bifunctional proline catabolic enzymes that catalyze the 4-electron oxidation of l-proline to l-glutamate using spatially-separated proline dehydrogenase and l-glutamate-γ-semialdehyde dehydrogenase (GSALDH, a.k.a. ALDH4A1) active sites. The observation that l-proline inhibits both the GSALDH activity of PutA and monofunctional GSALDHs motivated us to study the inhibition of PutA by proline stereoisomers and analogs. Here we report five high-resolution crystal structures of PutA with the following ligands bound in the GSALDH active site: d-proline, trans-4-hydroxy-d-proline, cis-4-hydroxy-d-proline, l-proline, and trans-4-hydroxy-l-proline. Three of the structures are of ternary complexes of the enzyme with an inhibitor and either NAD + or NADH. To our knowledge, the NADH complex is the first for any GSALDH. The structures reveal a conserved mode of recognition of the inhibitor carboxylate, which results in the pyrrolidine rings of the d- and l-isomers having different orientations and different hydrogen bonding environments. Activity assays show that the compounds are weak inhibitors with millimolar inhibition constants. Curiously, although the inhibitors occupy the aldehyde binding site, kinetic measurements show the inhibition is uncompetitive. Uncompetitive inhibition may involve proline binding to a remote site or to the enzyme-NADH complex. Together, the structural and kinetic data expand our understanding of how proline-like molecules interact with GSALDH, reveal insight into the relationship between stereochemistry and inhibitor affinity, and demonstrate the pitfalls of inferring the mechanism of inhibition from crystal structures alone., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Pyrroline-5-carboxylate reductase 1 (PYCR1) catalyzes the biosynthetic half-reaction of the proline cycle by reducing Δ 1 -pyrroline-5-carboxylate (P5C) to proline through the oxidation of NAD(P)H. Many cancers alter their proline metabolism by up-regulating the proline cycle and proline biosynthesis, and knockdowns of PYCR1 lead to decreased cell proliferation. Thus, evidence is growing for PYCR1 as a potential cancer therapy target. Inhibitors of cancer targets are useful as chemical probes for studying cancer mechanisms and starting compounds for drug discovery; however, there is a notable lack of validated inhibitors for PYCR1. To fill this gap, we performed a small-scale focused screen of proline analogs using X-ray crystallography. Five inhibitors of human PYCR1 were discovered: l-tetrahydro-2-furoic acid, cyclopentanecarboxylate, l-thiazolidine-4-carboxylate, l-thiazolidine-2-carboxylate, and N -formyl l-proline (NFLP). The most potent inhibitor was NFLP, which had a competitive (with P5C) inhibition constant of 100 μm The structure of PYCR1 complexed with NFLP shows that inhibitor binding is accompanied by conformational changes in the active site, including the translation of an α-helix by 1 Å. These changes are unique to NFLP and enable additional hydrogen bonds with the enzyme. NFLP was also shown to phenocopy the PYCR1 knockdown in MCF10A H-RAS V12 breast cancer cells by inhibiting de novo proline biosynthesis and impairing spheroidal growth. In summary, we generated the first validated chemical probe of PYCR1 and demonstrated proof-of-concept for screening proline analogs to discover inhibitors of the proline cycle., Competing Interests: Conflict of interest—S.-M. F. has received funding from Bayer, Merck, and Black Belt Therapeutics and has consulted for Fund+., (© 2020 Christensen et al.)

Engineered miniprotein host-small-molecule guest pairs could be utilized to design new processes within cells as well as investigate fundamental aspects of cell signaling mechanisms. However, the development of host-guest pairs capable of functioning in living systems has proven challenging. Moreover, few examples of host-guest pairs with stoichiometries other than 2:1 exist, significantly hindering the ability to study the influence of oligomerization state on signaling fidelity. Herein, we present an approach to identify host-guest systems for relatively small green fluorescent guests by incorporation into cyclic peptides. The optimal host-guest pair produced a 10-fold increase in green fluorescence signal upon binding. Biophysical characterization clearly demonstrated higher order supramolecular assembly, which could be visualized on the surface of living yeast cells using a turn-on fluorescence readout. This work further defines evolutionary design principles to afford host-guest pairs with stoichiometries other than 2:1 and enables the identification of spectrally orthogonal host-guest pairs., (© 2020 Wiley-VCH GmbH.)

Protein biochemistry protocols typically include disulfide bond reducing agents to guard against unwanted thiol oxidation and protein aggregation. Commonly used disulfide bond reducing agents include dithiothreitol, β-mercaptoethanol, glutathione, and the tris(alkyl)phosphine compounds tris(2-carboxyethyl)phosphine (TCEP) and tris(3-hydroxypropyl)phosphine (THPP). While studying the catalytic activity of the NAD(P)H-dependent enzyme Δ 1 -pyrroline-5-carboxylate reductase, we unexpectedly observed a rapid non-enzymatic chemical reaction between NAD + and the reducing agents TCEP and THPP. The product of the reaction exhibits a maximum ultraviolet absorbance peak at 334 nm and forms with an apparent association rate constant of 231-491 M -1 s -1 . The reaction is reversible, and nuclear magnetic resonance characterization ( 1 H, 13 C, and 31 P) of the product revealed a covalent adduct between the phosphorus of the tris(alkyl)phosphine reducing agent and the C4 atom of the nicotinamide ring of NAD + . We also report a 1.45 Å resolution crystal structure of short-chain dehydrogenase/reductase with the NADP + -TCEP reaction product bound in the cofactor binding site, which shows that the adduct can potentially inhibit enzymes. These findings serve to caution researchers when using TCEP or THPP in experimental protocols with NAD(P) + . Because NAD(P) + -dependent oxidoreductases are widespread in nature, our results may be broadly relevant.

Objectives: To determine the personnel costs and revenue generated by embedding a behavioral health nurse practitioner (BHNP) in primary care clinics to evaluate and manage adolescent behavioral health needs., Study Design: We estimated personnel costs and revenue from a quality improvement project undertaken at 4 clinic sites between August 1, 2016, and July 31, 2018, at a large multispecialty medical group in northern California., Methods: Costs were estimated by identifying the actual hours spent by the BHNP and for medical assistant (MA) support and using Bureau of Labor Statistics national data on wages and benefits. Revenue was estimated by analyzing Current Procedural Terminology (CPT) codes for BHNP visits from the Epic electronic health record and corresponding relative value units (RVUs), based on 135% of 2018 nationally unadjusted Medicare rates., Results: We estimate 2-year revenue of $144,449 and personnel costs (salary + benefits) of $90,431. The BHNP work totaled 1083 hours, and MA support totaled 312 hours. Using a nurse practitioner wage of $53.70/hour and an MA wage of $16.95/hour, total salary costs were $63,451; we then added benefits costs. Using the CPT codes assigned to the 768 encounters with 207 unique patients, we estimated generation of 1640 RVUs and total revenue of $144,449., Conclusions: This analysis found that personnel costs ($90,514) of a primary care-embedded BHNP are 63% of the potential revenue generated ($144,449). This analysis suggests that a primary care BHNP could be a cost-saving and patient-centered way to reduce the burden on primary care providers while meeting the growing needs of adolescents with behavioral health needs.

Proline accumulation and metabolism are associated with mechanisms of abiotic stress avoidance in plants. Proline accumulation generally improves osmotic stress tolerance whereas proline metabolism can have varying effects from ATP generation to the formation of reactive oxygen species. To further understand the roles of proline in stress protection, two peanut cultivars with contrasting tolerance to drought were examined by transcriptional and biochemical analyses during water stress. Plants exposed to polyethylene glycol had diminished relative water content and increased proline content; while, only the drought sensitive plants, cultivar Granoleico, showed lipid oxidative damage (measured as thiobarbituric acid reactive substances). The expression of proline biosynthesis genes (P5CS1, P5CS2a, P5CS2b, P5CR) was increased in both cultivars upon exposure to water stress. However, the relative expression of proline catabolism genes (ProDH1, ProDH2) was increased only in the sensitive cultivar during stress. Exogenous addition of proline and the proline analogue thiazolidine-4-carboxylic acid (T4C), both substrates of proline dehydrogenase, was also used to exacerbate and identify plant responses. Pretreatment of plants with T4C induced unique changes in the drought tolerant EC-98 cultivar such as higher mRNA levels of proline biosynthetic and catabolic ProDH genes, even in the absence of water stress. The increased levels of ProDH gene expression, potentially associated with higher T4C conversion to cysteine, may contribute to the tolerant phenotype., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the FAD-dependent 2-electron oxidation of l-proline to Δ 1 -pyrroline-5-carboxylate. PRODH has emerged as a possible cancer therapy target, and thus the inhibition of PRODH is of interest. Here we show that the proline analogue thiazolidine-2-carboxylate (T2C) is a mechanism-based inactivator of PRODH. Structures of the bifunctional proline catabolic enzyme proline utilization A (PutA) determined from crystals grown in the presence of T2C feature strong electron density for a 5-membered ring species resembling l-T2C covalently bound to the N5 of the FAD in the PRODH domain. The modified FAD exhibits a large butterfly bend angle, indicating that the FAD is locked into the 2-electron reduced state. Reduction of the FAD is consistent with the crystals lacking the distinctive yellow color of the oxidized enzyme and stopped-flow kinetic data showing that T2C is a substrate for the PRODH domain of PutA. A mechanism is proposed in which PRODH catalyzes the oxidation of T2C at the C atom adjacent to the S atom of the thiazolidine ring (C5). Then, the N5 atom of the reduced FAD attacks the C5 of the oxidized T2C species, resulting in the covalent adduct observed in the crystal structure. To our knowledge, this is the first report of T2C inactivating (or inhibiting) PRODH or any other flavoenzyme. These results may inform the design of new mechanism-based inactivators of PRODH for use as chemical probes to study the roles of proline metabolism in cancer.

WhiB1 is a monomeric iron-sulfur cluster-containing transcription factor in the WhiB-like family that is widely distributed in actinobacteria including the notoriously persistent pathogen Mycobacterium tuberculosis (M. tuberculosis). WhiB1 plays multiple roles in regulating cell growth and responding to nitric oxide stress in M. tuberculosis, but its underlying mechanism is unclear. Here we report a 1.85 Å-resolution crystal structure of the [4Fe-4S] cluster-bound (holo-) WhiB1 in complex with the C-terminal domain of the σ70-family primary sigma factor σA of M. tuberculosis containing the conserved region 4 (σA4). Region 4 of the σ70-family primary sigma factors is commonly used by transcription factors for gene activation, and holo-WhiB1 has been proposed to activate gene expression via binding to σA4. The complex structure, however, unexpectedly reveals that the interaction between WhiB1 and σA4 is dominated by hydrophobic residues in the [4Fe-4S] cluster binding pocket, distinct from previously characterized canonical σ704-bound transcription activators. Furthermore, we show that holo-WhiB1 represses transcription by interaction with σA4in vitro and that WhiB1 must interact with σA4 to perform its essential role in supporting cell growth in vivo. Together, these results demonstrate that holo-WhiB1 regulates gene expression by a non-canonical mechanism relative to well-characterized σA4-dependent transcription activators., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Introduction: Approximately 25% of adolescents have behavioral disorders, yet few receive treatment. Primary care (PC) screening for depression and anxiety is recommended; however treatments, such as cognitive behavioral therapy (CBT), are rarely available in PC settings. Our aim was to determine whether the use of a CBT-based intervention (COPE for Teens) is associated with improved outcomes on measures of depression and anxiety, and to understand the patient experience., Methods: Health record data were examined, including questionnaires on depression (PHQ-A), anxiety (GAD-7), and experience with COPE. Differences between pre- and post-intervention scores were evaluated by paired t-tests. Questionnaire data were analyzed via thematic coding., Results: Thirty-seven patients (73% female; ages 12-18) completed pre- and post-intervention measures. Comparison showed decrease in PHQ-A scores by 2.1 (p = 0.0067) and GAD-7 scores by 2.3 (p = 0.0081). Questionnaire data demonstrate satisfaction with COPE., Discussion: Among these 37 adolescents, COPE provided effective PC-based behavioral treatment and a positive experience. Increased availability of COPE could improve care for adolescents., (Copyright © 2018 National Association of Pediatric Nurse Practitioners. Published by Elsevier Inc. All rights reserved.)

Heme oxygenase (HO) catalyzes heme degradation, a process crucial for regulating cellular levels of this vital, but cytotoxic, cofactor. Two HO isoforms, HO1 and HO2, exhibit similar catalytic mechanisms and efficiencies. They also share catalytic core structures, including the heme-binding site. Outside their catalytic cores are two regions unique to HO2: a 20-amino acid-long N-terminal extension and a C-terminal domain containing two heme regulatory motifs (HRMs) that bind heme independently of the core. Both HO isoforms contain a C-terminal hydrophobic membrane anchor; however, their sequences diverge. Here, using hydrogen-deuterium exchange MS, size-exclusion chromatography, and sedimentation velocity, we investigated how these divergent regions impact the dynamics and structure of the apo and heme-bound forms of HO1 and HO2. Our results reveal that heme binding to the catalytic cores of HO1 and HO2 causes similar dynamic and structural changes in regions (proximal, distal, and A6 helices) within and linked to the heme pocket. We observed that full-length HO2 is more dynamic than truncated forms lacking the membrane-anchoring region, despite sharing the same steady-state activity and heme-binding properties. In contrast, the membrane anchor of HO1 did not influence its dynamics. Furthermore, although residues within the HRM domain facilitated HO2 dimerization, neither the HRM region nor the N-terminal extension appeared to affect HO2 dynamics. In summary, our results highlight significant dynamic and structural differences between HO2 and HO1 and indicate that their dissimilar C-terminal regions play a major role in controlling the structural dynamics of these two proteins., (© 2019 Kochert et al.)

Approximately 49.5% of the adolescents report a mental health disorder; only about half of the children and adolescents with mental health disorders seek treatment from a mental health professional. Stigma and poor access to behavioral health providers are leading barriers to care. A large ambulatory health system implemented a BH navigation program to facilitate referrals from primary care physicians (PCPs), including pediatricians and family physicians, to BH providers. We studied PCP adoption of BH navigation services over a 4-year period, from July 2014 to June 2018. We retrieved operational data regarding service utilization, patient information from electronic health records and PCP information from administrative data, and surveyed PCPs for their appraisals of navigation services. Four thousand five hundred and fifty-five referrals were made for 3,912 patients from 290 PCPs (71% of PCPs in the health system). Depression (39%), anxiety (25%), and attention-deficit hyperactivity disorder (7%) were the most frequent reasons for referral. Referrals increased dramatically in the first half of the study period and decreased afterwards. Ninety-one percent of the PCPs agreed or strongly agreed that navigation enhanced their clinical care at 12-month survey. More than 90% of the PCPs rated the referral process, communication with navigation staff, and the overall experience as above average or excellent at 12 months. There was a decrease in these evaluation indicators after 2.5 years. The initial high referral volume reflects a need for BH navigation services. However, challenges remain to maintain positive PCP assessment in the face of such demand., (© Society of Behavioral Medicine 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Significance: Proline metabolism has complex roles in a variety of biological processes, including cell signaling, stress protection, and energy production. Proline also contributes to the pathogenesis of various disease-causing organisms. Understanding the mechanisms of how pathogens utilize proline is important for developing new strategies against infectious diseases. Recent Advances: The ability of pathogens to acquire amino acids is critical during infection. Besides protein biosynthesis, some amino acids, such as proline, serve as a carbon, nitrogen, or energy source in bacterial and protozoa pathogens. The role of proline during infection depends on the physiology of the host/pathogen interactions. Some pathogens rely on proline as a critical respiratory substrate, whereas others exploit proline for stress protection., Critical Issues: Disruption of proline metabolism and uptake has been shown to significantly attenuate virulence of certain pathogens, whereas in other pathogens the importance of proline during infection is not known. Inhibiting proline metabolism and transport may be a useful therapeutic strategy against some pathogens. Developing specific inhibitors to avoid off-target effects in the host, however, will be challenging. Also, potential treatments that target proline metabolism should consider the impact on intracellular levels of Δ 1 -pyrroline-5-carboxylate, a metabolite intermediate that can have opposing effects on pathogenesis., Future Directions: Further characterization of how proline metabolism is regulated during infection would provide new insights into the role of proline in pathogenesis. Biochemical and structural characterization of proline metabolic enzymes from different pathogens could lead to new tools for exploring proline metabolism during infection and possibly new therapeutic compounds.

Heterokonts, Alveolata protists, green algae from Charophyta and Chlorophyta divisions, and all Embryophyta plants possess an aldehyde dehydrogenase (ALDH) gene named ALDH12. Here, we provide a biochemical characterization of two ALDH12 family members from the lower plant Physcomitrella patens and higher plant Zea mays. We show that ALDH12 encodes an NAD + -dependent glutamate γ-semialdehyde dehydrogenase (GSALDH), which irreversibly converts glutamate γ-semialdehyde (GSAL), a mitochondrial intermediate of the proline and arginine catabolism, to glutamate. Sedimentation equilibrium and small-angle X-ray scattering analyses reveal that in solution both plant GSALDHs exist as equilibrium between a domain-swapped dimer and the dimer-of-dimers tetramer. Plant GSALDHs share very low-sequence identity with bacterial, fungal, and animal GSALDHs (classified as ALDH4), which are the closest related ALDH superfamily members. Nevertheless, the crystal structure of ZmALDH12 at 2.2-Å resolution  shows that nearly all key residues involved in the recognition of GSAL are identical to those in ALDH4, indicating a close functional relationship with ALDH4. Phylogenetic analysis suggests that the transition from ALDH4 to ALDH12 occurred during the evolution of the endosymbiotic plant ancestor, prior to the evolution of green algae and land plants. Finally, ALDH12 expression in maize and moss is downregulated in response to salt and drought stresses, possibly to maintain proline levels. Taken together, these results provide molecular insight into the biological roles of the plant ALDH12 family., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

A key factor for flavoenzyme activity is the reduction potential of the bound flavin. The reduction potentials of protein-bound flavins span approximately a 500-mV range consistent with flavoenzymes having critical roles in metabolism and a variety of biological processes. Redox potentials of flavoenzymes have traditionally been determined using an electrode-based system with either direct or indirect electrochemical coupling between the protein and the working electrode. An electrode independent method, however, is also now commonly used and involves calculating the unknown flavin reduction potential of the protein from the known reduction potential of a reference or indicator dye. Here, the "classic" potentiometric method and the xanthine/xanthine oxidase methods are described. Both methods rely on equilibrium between protein-bound flavin and redox dyes. The potentiometric method measures the equilibrated redox potential of the protein-dye mixture whereas the xanthine/xanthine oxidase technique relies on slow continuous enzymatic reduction to maintain a constant equilibrium between the protein and the dyes. Because electrochemical equipment is not required, the xanthine/xanthine oxidase method is more accessible and convenient for researchers seeking to determine reduction potentials of flavoproteins or other biological redox centers such as hemes. The xanthine/xanthine oxidase method has been used to determine flavin reduction potentials from +132 to -417mV, demonstrating it is suitable for characterizing the redox properties of most flavoproteins., (© 2019 Elsevier Inc. All rights reserved.)

Interest in how proline contributes to cancer biology is expanding because of the emerging role of a novel proline metabolic cycle in cancer cell survival, proliferation, and metastasis. Proline biosynthesis and degradation involve the shared intermediate Δ 1 -pyrroline-5-carboxylate (P5C), which forms l-glutamate-γ-semialdehyde (GSAL) in a reversible non-enzymatic reaction. Proline is synthesized from glutamate or ornithine through GSAL/P5C, which is reduced to proline by P5C reductase (PYCR) in a NAD(P)H-dependent reaction. The degradation of proline occurs in the mitochondrion and involves two oxidative steps catalyzed by proline dehydrogenase (PRODH) and GSAL dehydrogenase (GSALDH). PRODH is a flavin-dependent enzyme that couples proline oxidation with reduction of membrane-bound quinone, while GSALDH catalyzes the NAD + -dependent oxidation of GSAL to glutamate. PRODH and PYCR form a metabolic relationship known as the proline-P5C cycle, a novel pathway that impacts cellular growth and death pathways. The proline-P5C cycle has been implicated in supporting ATP production, protein and nucleotide synthesis, anaplerosis, and redox homeostasis in cancer cells. This Perspective details the structures and reaction mechanisms of PRODH and PYCR and the role of the proline-P5C cycle in cancer metabolism. A major challenge in the field is to discover inhibitors that specifically target PRODH and PYCR isoforms for use as tools for studying proline metabolism and the functions of the proline-P5C cycle in cancer. These molecular probes could also serve as lead compounds in cancer drug discovery targeting the proline-P5C cycle.

Homooligomerization of proline utilization A (PutA) bifunctional flavoenzymes is intimately tied to catalytic function and substrate channeling. PutA from Bradyrhizobium japonicum (BjPutA) is unique among PutAs in that it forms a tetramer in solution. Curiously, a dimeric BjPutA hot spot mutant was previously shown to display wild-type catalytic activity despite lacking the tetrameric structure. These observations raised the question of what is the active oligomeric state of BjPutA. Herein, we investigate the factors that contribute to tetramerization of BjPutA in vitro. Negative-stain electron microscopy indicates that BjPutA is primarily dimeric at nanomolar concentrations, suggesting concentration-dependent tetramerization. Further, sedimentation-velocity analysis of BjPutA at high (micromolar) concentration reveals that although the binding of active-site ligands does not alter oligomeric state, reduction of the flavin adenine dinucleotide cofactor results in dimeric protein. Size-exclusion chromatography coupled with multiangle light scattering and small-angle x-ray scattering analysis also reveals that reduced BjPutA is dimeric. Taken together, these results suggest that the BjPutA oligomeric state is dependent upon both enzyme concentration and the redox state of the flavin cofactor. This is the first report, to our knowledge, of redox-linked oligomerization in the PutA family., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Proline utilization A (PutA) is a bifunctional flavoenzyme that catalyzes the two-step oxidation of l-proline to l-glutamate using spatially separated proline dehydrogenase (PRODH) and l-glutamate-γ-semialdehyde dehydrogenase (GSALDH) active sites. Substrate inhibition of the coupled PRODH-GSALDH reaction by proline is a common kinetic feature of PutAs, yet the structural basis for this phenomenon remains unknown. To understand the mechanism of substrate inhibition, we determined the 2.15 Å resolution crystal structure of Bradyrhizobium japonicum PutA complexed with proline. Proline was discovered in five locations remote from the PRODH active site. Most notably, strong electron density indicated that proline bound tightly to the GSAL binding site of the GSALDH active site. The pose and interactions of proline bound in this site are remarkably similar to those of the natural aldehyde substrate, GSAL, implying that proline inhibits the GSALDH reaction of PutA. Kinetic measurements show that proline is a competitive inhibitor of the PutA GSALDH reaction. Together, the structural and kinetic data show that substrate inhibition of the PutA coupled reaction is due to proline binding in the GSAL site., Competing Interests: The authors declare no conflict of interest.

Escherichia coli proline utilization A (EcPutA) is the archetype of trifunctional PutA flavoproteins, which function both as regulators of the proline utilization operon and bifunctional enzymes that catalyze the four-electron oxidation of proline to glutamate. EcPutA shifts from a self-regulating transcriptional repressor to a bifunctional enzyme in a process known as functional switching. The flavin redox state dictates the function of EcPutA. Upon proline oxidation, the flavin becomes reduced, triggering a conformational change that causes EcPutA to dissociate from the put regulon and bind to the cellular membrane. Major structure/function domains of EcPutA have been characterized, including the DNA-binding domain, proline dehydrogenase (PRODH) and l-glutamate-γ-semialdehyde dehydrogenase catalytic domains, and an aldehyde dehydrogenase superfamily fold domain. Still lacking is an understanding of the membrane-binding domain, which is essential for EcPutA catalytic turnover and functional switching. Here, we provide evidence for a conserved C-terminal motif (CCM) in EcPutA having a critical role in membrane binding. Deletion of the CCM or replacement of hydrophobic residues with negatively charged residues within the CCM impairs EcPutA functional and physical membrane association. Furthermore, cell-based transcription assays and limited proteolysis indicate that the CCM is essential for functional switching. Using fluorescence resonance energy transfer involving dansyl-labeled liposomes, residues in the α-domain are also implicated in membrane binding. Taken together, these experiments suggest that the CCM and α-domain converge to form a membrane-binding interface near the PRODH domain. The discovery of the membrane-binding region will assist efforts to define flavin redox signaling pathways responsible for EcPutA functional switching.

Proline has important roles in multiple biological processes such as cellular bioenergetics, cell growth, oxidative and osmotic stress response, protein folding and stability, and redox signaling. The proline catabolic pathway, which forms glutamate, enables organisms to utilize proline as a carbon, nitrogen, and energy source. FAD-dependent proline dehydrogenase (PRODH) and NAD + -dependent glutamate semialdehyde dehydrogenase (GSALDH) convert proline to glutamate in two sequential oxidative steps. Depletion of PRODH and GSALDH in humans leads to hyperprolinemia, which is associated with mental disorders such as schizophrenia. Also, some pathogens require proline catabolism for virulence. A unique aspect of proline catabolism is the multifunctional proline utilization A (PutA) enzyme found in Gram-negative bacteria. PutA is a large (>1000 residues) bifunctional enzyme that combines PRODH and GSALDH activities into one polypeptide chain. In addition, some PutAs function as a DNA-binding transcriptional repressor of proline utilization genes. This review describes several attributes of PutA that make it a remarkable flavoenzyme: (1) diversity of oligomeric state and quaternary structure; (2) substrate channeling and enzyme hysteresis; (3) DNA-binding activity and transcriptional repressor function; and (4) flavin redox dependent changes in subcellular location and function in response to proline (functional switching)., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Pipecolate, an intermediate of the lysine catabolic pathway, is oxidized to Δ 1 -piperideine-6-carboxylate (P6C) by the flavoenzyme l-pipecolate oxidase (PIPOX). P6C spontaneously hydrolyzes to generate α-aminoadipate semialdehyde, which is then converted into α-aminoadipate acid by α-aminoadipatesemialdehyde dehydrogenase. l-pipecolate was previously reported to protect mammalian cells against oxidative stress. Here, we examined whether PIPOX is involved in the mechanism of pipecolate stress protection. Knockdown of PIPOX by small interference RNA abolished pipecolate protection against hydrogen peroxide-induced cell death in HEK293 cells suggesting a critical role for PIPOX. Subcellular fractionation analysis showed that PIPOX is localized in the mitochondria of HEK293 cells consistent with its role in lysine catabolism. Signaling pathways potentially involved in pipecolate protection were explored by treating cells with small molecule inhibitors. Inhibition of both mTORC1 and mTORC2 kinase complexes or inhibition of Akt kinase alone blocked pipecolate protection suggesting the involvement of these signaling pathways. Phosphorylation of the Akt downstream target, forkhead transcription factor O3 (FoxO3), was also significantly increased in cells treated with pipecolate, further implicating Akt in the protective mechanism and revealing FoxO3 inhibition as a potentially key step. The results presented here demonstrate that pipecolate metabolism can influence cell signaling during oxidative stress to promote cell survival and suggest that the mechanism of pipecolate protection parallels that of proline, which is also metabolized in the mitochondria. J. Cell. Biochem. 118: 1678-1688, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Proline utilization A from Escherichia coli (EcPutA) is a multifunctional flavoenzyme that oxidizes proline to glutamate through proline dehydrogenase (PRODH) and Δ 1 -pyrroline-5-carboxylate dehydrogenase (P5CDH) activities, while also switching roles as a DNA-bound transcriptional repressor and a membrane-bound catabolic enzyme. This phenomenon, termed functional switching, occurs through a redox-mediated mechanism in which flavin reduction triggers a conformational change that increases EcPutA membrane binding affinity. Structural studies have shown that reduction of the FAD cofactor causes the ribityl moiety to undergo a crankshaft motion, indicating that the orientation of the ribityl chain is a key element of PutA functional switching. Here, we test the role of a conserved histidine that bridges from the FAD pyrophosphate to the backbone amide of a conserved leucine residue in the PRODH active site. An EcPutA mutant (H487A) was characterized by steady-state and rapid-reaction kinetics, and cell-based reporter gene experiments. The catalytic activity of H487A is severely diminished (>50-fold) with membrane vesicles as the electron acceptor, and H487A exhibits impaired lipid binding and in vivo transcriptional repressor activity. Rapid-reaction kinetic experiments demonstrate that H487A is 3-fold slower than wild-type EcPutA in a conformational change step following reduction of the FAD cofactor. Furthermore, the reduction potential (E m ) of H487A is ∼40 mV more positive than that of wild-type EcPutA, and H487A has an attenuated ability to catalyze the reverse PRODH chemical step of reoxidation by P5C. In this process, significant red semiquinone forms in contrast to the same reaction with wild-type EcPutA, in which facile two-electron reoxidation occurs without the formation of a measurable amount of semiquinone. These results indicate that His487 is critically important for the proline/P5C chemical step, conformational change kinetics, and functional switching in EcPutA.

The bifunctional flavoenzyme proline utilization A (PutA) catalyzes the two-step oxidation of proline to glutamate using separate proline dehydrogenase (PRODH) and l-glutamate-γ-semialdehyde dehydrogenase active sites. Because PutAs catalyze sequential reactions, they are good systems for studying how metabolic enzymes communicate via substrate channeling. Although mechanistically similar, PutAs vary widely in domain architecture, oligomeric state, and quaternary structure, and these variations represent different structural solutions to the problem of sequestering a reactive metabolite. Here, we studied PutA from Corynebacterium freiburgense (CfPutA), which belongs to the uncharacterized 3B class of PutAs. A 2.7 Å resolution crystal structure showed the canonical arrangement of PRODH, l-glutamate-γ-semialdehyde dehydrogenase, and C-terminal domains, including an extended interdomain tunnel associated with substrate channeling. The structure unexpectedly revealed a novel open conformation of the PRODH active site, which is interpreted to represent the non-activated conformation, an elusive form of PutA that exhibits suboptimal channeling. Nevertheless, CfPutA exhibited normal substrate-channeling activity, indicating that it isomerizes into the active state under assay conditions. Sedimentation-velocity experiments provided insight into the isomerization process, showing that CfPutA dimerizes in the presence of a proline analog and NAD + These results are consistent with the morpheein model of enzyme hysteresis, in which substrate binding induces conformational changes that promote assembly of a high-activity oligomer. Finally, we used domain deletion analysis to investigate the function of the C-terminal domain. Although this domain contains neither catalytic residues nor substrate sites, its removal impaired both catalytic activities, suggesting that it may be essential for active-site integrity., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Pyrroline-5-carboxylate reductase (PYCR) is the final enzyme in proline biosynthesis, catalyzing the NAD(P)H-dependent reduction of Δ 1 -pyrroline-5-carboxylate (P5C) to proline. Mutations in the PYCR1 gene alter mitochondrial function and cause the connective tissue disorder cutis laxa. Furthermore, PYCR1 is overexpressed in multiple cancers, and the PYCR1 knock-out suppresses tumorigenic growth, suggesting that PYCR1 is a potential cancer target. However, inhibitor development has been stymied by limited mechanistic details for the enzyme, particularly in light of a previous crystallographic study that placed the cofactor-binding site in the C-terminal domain rather than the anticipated Rossmann fold of the N-terminal domain. To fill this gap, we report crystallographic, sedimentation-velocity, and kinetics data for human PYCR1. Structures of binary complexes of PYCR1 with NADPH or proline determined at 1.9 Å resolution provide insight into cofactor and substrate recognition. We see NADPH bound to the Rossmann fold, over 25 Å from the previously proposed site. The 1.85 Å resolution structure of a ternary complex containing NADPH and a P5C/proline analog provides a model of the Michaelis complex formed during hydride transfer. Sedimentation velocity shows that PYCR1 forms a concentration-dependent decamer in solution, consistent with the pentamer-of-dimers assembly seen crystallographically. Kinetic and mutational analysis confirmed several features seen in the crystal structure, including the importance of a hydrogen bond between Thr-238 and the substrate as well as limited cofactor discrimination., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Proline utilization A (PutA) is a bifunctional flavoenzyme with proline dehydrogenase (PRODH) and Δ 1 -pyrroline-5-carboxylate (P5C) dehydrogenase (P5CDH) domains that catalyses the two-step oxidation of proline to glutamate. Trifunctional PutAs also have an N-terminal ribbon-helix-helix (RHH) DNA-binding domain and moonlight as autogenous transcriptional repressors of the put regulon. A unique property of trifunctional PutA is the ability to switch functions from DNA-bound repressor to membrane-associated enzyme in response to cellular nutritional needs and proline availability. In the present study, we attempt to construct a trifunctional PutA by fusing the RHH domain of Escherichia coli PutA (EcRHH) to the bifunctional Rhodobacter capsulatus PutA (RcPutA) in order to explore the modular design of functional switching in trifunctional PutAs. The EcRHH-RcPutA chimaera retains the catalytic properties of RcPutA while acquiring the oligomeric state, quaternary structure and DNA-binding properties of EcPutA. Furthermore, the EcRHH-RcPutA chimaera exhibits proline-induced lipid association, which is a fundamental characteristic of functional switching. Unexpectedly, RcPutA lipid binding is also activated by proline, which shows for the first time that bifunctional PutAs exhibit a limited form of functional switching. Altogether, these results suggest that the C-terminal domain (CTD), which is conserved by trifunctional PutAs and certain bifunctional PutAs, is essential for functional switching in trifunctional PutAs., (© 2016 The Author(s).)

Aldehyde dehydrogenases (ALDHs) catalyze the NAD(P) + -dependent oxidation of aldehydes to carboxylic acids and are important for metabolism and detoxification. Although the ALDH superfamily fold is well established, some ALDHs contain an uncharacterized domain of unknown function (DUF) near the C terminus of the polypeptide chain. Herein, we report the first structure of a protein containing the ALDH superfamily DUF. Proline utilization A from Sinorhizobium meliloti (SmPutA) is a 1233-residue bifunctional enzyme that contains the DUF in addition to proline dehydrogenase and l-glutamate-γ-semialdehyde dehydrogenase catalytic modules. Structures of SmPutA with a proline analog bound to the proline dehydrogenase site and NAD + bound to the ALDH site were determined in two space groups at 1.7-1.9 Å resolution. The DUF consists of a Rossmann dinucleotide-binding fold fused to a three-stranded β-flap. The Rossmann domain resembles the classic ALDH superfamily NAD + -binding domain, whereas the flap is strikingly similar to the ALDH superfamily dimerization domain. Paradoxically, neither structural element performs its implied function. Electron density maps show that NAD + does not bind to the DUF Rossmann fold, and small-angle X-ray scattering reveals a novel dimer that has never been seen in the ALDH superfamily. The structure suggests that the DUF is an adapter domain that stabilizes the aldehyde substrate binding loop and seals the substrate-channeling tunnel via tertiary structural interactions that mimic the quaternary structural interactions found in non-DUF PutAs. Kinetic data for SmPutA indicate a substrate-channeling mechanism, in agreement with previous studies of other PutAs., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) hydrolyzes dUTP to dUMP and pyrophosphate to maintain the cellular thymine-uracil ratio. dUTPase is also a target for cancer chemotherapy. However, the mechanism defining its substrate affinity remains unclear. Sequence comparisons of various dUTPases revealed that Arabidopsis thaliana dUTPase has a unique tryptophan at position 93, which potentially contributes to its degree of substrate affinity. To better understand the roles of tryptophan 93, A. thaliana dUTPase was studied., Results: Enzyme assays showed that A. thaliana dUTPase belongs to a high-affinity group of isozymes, which also includes the enzymes from Escherichia coli and Mycobacterium tuberculosis. Enzymes from Homo sapiens and Saccharomyces cerevisiae are grouped as low-affinity dUTPases. The structure of the homo-trimeric A. thaliana dUTPase showed three active sites, each with a different set of ligand interactions between the amino acids and water molecules. On an α-helix, tryptophan 93 appears to keep serine 89 in place via a water molecule and to specifically direct the ligand. Upon being oriented in the active site, the C-terminal residues close the active site to promote the reaction., Conclusions: In the high-affinity group, the prefixed direction of the serine residues was oriented by a positively charged residue located four amino acids away, while low-affinity enzymes possess small hydrophobic residues at the corresponding sites.

Objective: Binge-eating disorder (BED) is associated with elevated rates of mood and substance use disorders, but the significance of such comorbidity is ambiguous. We compared personality disorder and eating disorder psychopathology in four subgroups of BED patients: those with mood disorders, those with substance use disorders, those with both, and those with neither., Method: Subjects were 347 patients who met DSM-IV research criteria for BED. Semistructured interviews evaluated lifetime DSM-IV axis I disorders, DSM-IV personality disorder features, and eating disorder psychopathology., Results: Among these patients, 129 had co-occurring mood disorder, 34 had substance use disorder, 60 had both, and 124 had neither. Groups differed on personality disorder features, with those having mood disorder and both mood and substance use disorders showing the highest frequencies. Although groups did not differ in body mass index or binge eating frequency, they did differ on eating disorder psychopathology-with the groups having mood disorder and both comorbidities demonstrating higher eating, weight, and shape concerns. No differences were observed between groups with respect to ages of onset for specific eating behaviors, but some differences were observed for ages of disorder onset., Conclusion: Mood and substance use disorders co-occur frequently among patients with BED. Compared with a previous work, the additional comparison group (those with both mood and substance use disorders) and the control group (those with neither) afforded better discrimination regarding the significance of these comorbidities. Our findings suggest approaches to subtyping BED based on psychiatric comorbidity, and may also have implications for treatment., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The amino acid proline has a unique biological role in stress adaptation. Proline metabolism is manipulated under stress by multiple and complex regulatory pathways and can profoundly influence cell death and survival in microorganisms, plants, and animals. Though the effects of proline are mediated by diverse signaling pathways, a common theme appears to be the generation of reactive oxygen species (ROS) due to proline oxidation being coupled to the respiratory electron transport chain. Considerable research has been devoted to understand how plants exploit proline metabolism in response to abiotic and biotic stress. Here, we review potential mechanisms by which proline metabolism influences plant senescence, namely in the petal and leaf. Recent studies of petal senescence suggest proline content is manipulated to meet energy demands of senescing cells. In the flower and leaf, proline metabolism may influence ROS signaling pathways that delay senescence progression. Future studies focusing on the mechanisms by which proline metabolic shifts occur during senescence may lead to novel methods to rescue crops under stress and to preserve post-harvest agricultural products.

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.

The Atlantic Forest, which has a vast epiphytic richness, is a priority area for preservation, listed as one of the five most important world hotspots. Vascular epiphyte richness, composition and community structure were studied in two fragments, one of the ombrophilous (29º43'42"S and 50º22'00"W) and the other of the seasonal (29º40'54"S and 51º06'56"W) forest, both belonging to the Atlantic Forest biome in the Sinos River basin, Rio Grande do Sul, Brazil. In each fragment, 40 trees, divided into four ecological zones, were analyzed. In each zone, the occurrence of the species was recorded, and the importance value of each species was calculated according to the frequency of phorophytes and intervals, and cover scores. The Shannon index was calculated for the two communities. In the fragment of the ombrophilous forest (F1), 30 epiphytic species were recorded, and in the seasonal forest (F2), 25. The highest importance value was found for Microgramma squamulosa (Kaulf.) de la Sota in both fragments. The diversity indexes for F1 (H'=2.72) and F2 (H'=2.55) were similar and reflected the subtropical location of the areas. The decrease in mean richness in both fragments in zone 3 (internal crown) to zone 4 (external crown) may be associated with time and space availability for epiphyte occupation and microclimate variations. Exclusive species were found in the areas, which suggest that a greater number of preserved fragments may result in a greater number of preserved epiphytic species in the Sinos River basin.