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Mycobacterium phage Adephagia is a cluster K phage that infects Mycobacterium smegmatis and some strains of Mycobacterium pathogens. Adephagia has a siphoviral virion morphology and is temperate. Its genome is 59,646 bp long and codes for one tRNA gene and 94 predicted protein-coding genes; most genes not associated with virion structure and assembly are functionally ill-defined. Here, we determined the Adephagia gene expression patterns in lytic and lysogenic growth and used structural predictions to assign additional gene functions. We characterized 66 nonstructural genes for their toxic phenotypes when expressed in M. smegmatis, and we show that 25 of these (38%) are either toxic or strongly inhibit growth, resulting in either reduced viability or small colony sizes. Some of these genes are predicted to be involved in DNA metabolism or regulation, but others are of unknown function. We also characterize the HicAB-like toxin-antitoxin (TA) system encoded by Adephagia (gp91 and gp90, respectively) and show that the gp90 antitoxin is lysogenically expressed, abrogates gp91 toxicity, and is required for normal lytic and lysogenic growth., Competing Interests: Conflicts of interest The author(s) declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

The vast majority of membrane phospholipids (PLs) include two asymmetrically positioned fatty acyls: oxidizable polyunsaturated fatty acids (PUFA) attached predominantly at the sn2 position, and non-oxidizable saturated/monounsaturated acids (SFA/MUFA) localized at the sn1 position. The peroxidation of PUFA-PLs, particularly sn2-arachidonoyl(AA)- and sn2-adrenoyl(AdA)-containing phosphatidylethanolamines (PE), has been associated with the execution of ferroptosis, a program of regulated cell death. There is a minor subpopulation (≈1-2 mol %) of doubly PUFA-acylated phospholipids (di-PUFA-PLs) whose role in ferroptosis remains enigmatic. Here we report that 15-lipoxygenase (15LOX) exhibits unexpectedly high pro-ferroptotic peroxidation activity towards di-PUFA-PEs. We revealed that peroxidation of several molecular species of di-PUFA-PEs occurred early in ferroptosis. Ferrostatin-1, a typical ferroptosis inhibitor, effectively prevented peroxidation of di-PUFA-PEs. Furthermore, co-incubation of cells with di-AA-PE and 15LOX produced PUFA-PE peroxidation and induced ferroptotic death. The decreased contents of di-PUFA-PEs in ACSL4 KO A375 cells was associated with lower levels of di-PUFA-PE peroxidation and enhanced resistance to ferroptosis. Thus, di-PUFA-PE species are newly identified phospholipid peroxidation substrates and regulators of ferroptosis, representing a promising therapeutic target for many diseases related to ferroptotic death., (© 2024 Wiley-VCH GmbH.)

Ferroptosis is a regulated form of cell death, the mechanism of which is still to be understood. 15-lipoxygenase (15LOX) complex with phosphatidylethanolamine (PE)-binding protein 1 (PEBP1) catalyzes the generation of pro-ferroptotic cell death signals, hydroperoxy-polyunsaturated PE. We focused on gaining new insights into the molecular basis of these pro-ferroptotic interactions using computational modeling and liquid chromatography-mass spectrometry experiments. Simulations of 15LOX-1/PEBP1 complex dynamics and interactions with lipids revealed that association with the membrane triggers a conformational change in the complex. This conformational change facilitates the access of stearoyl/arachidonoyl-PE (SAPE) substrates to the catalytic site. Furthermore, the binding of SAPE promotes tight interactions within the complex and induces further conformational changes that facilitate the oxidation reaction. The reaction yields two hydroperoxides as products, 15-HpETE-PE and 12-HpETE-PE, at a ratio of 5:1. A significant effect of PEBP1 is observed only on the predominant product. Moreover, combined experiments and simulations consistently demonstrate the significance of PEBP1 P112E mutation in generating ferroptotic cell death signals., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Approximately 70% of bacteriophage-encoded proteins are of unknown function. Elucidating these protein functions represents opportunities to discover new phage-host interactions and mechanisms by which the phages modulate host activities. Here, we describe a pipeline for prioritizing phage-encoded proteins for structural analysis and characterize the gp82 protein encoded by mycobacteriophage Phaedrus. Structural and solution studies of gp82 show it is a trimeric protein containing two domains. Co-precipitation studies with the host Mycobacterium smegmatis identified the ATPase MoxR as an interacting partner protein. Phaedrus gp82-MoxR interaction requires the presence of a loop sequence within gp82 that is highly exposed and disordered in the crystallographic structure. We show that Phaedrus gp82 overexpression in M. smegmatis retards the growth of M. smegmatis on solid medium, resulting in a small colony phenotype. Overexpression of gp82 containing a mutant disordered loop or the overexpression of MoxR both rescue this phenotype. Lastly, we show that recombinant gp82 reduces levels of MoxR-mediated ATPase activity in vitro that is required for its chaperone function, and that the disordered loop plays an important role in this phenotype. We conclude that Phaedrus gp82 binds to and reduces mycobacterial MoxR activity, leading to reduced function of host proteins that require MoxR chaperone activity for their normal activity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Programmed ferroptotic death eliminates cells in all major organs and tissues with imbalanced redox metabolism due to overwhelming iron-catalyzed lipid peroxidation under insufficient control by thiols (Glutathione (GSH)). Ferroptosis has been associated with the pathogenesis of major chronic degenerative diseases and acute injuries of the brain, cardiovascular system, liver, kidneys, and other organs, and its manipulation offers a promising new strategy for anticancer therapy. This explains the high interest in designing new small-molecule-specific inhibitors against ferroptosis. Given the role of 15-lipoxygenase (15LOX) association with phosphatidylethanolamine (PE)-binding protein 1 (PEBP1) in initiating ferroptosis-specific peroxidation of polyunsaturated PE, we propose a strategy of discovering antiferroptotic agents as inhibitors of the 15LOX/PEBP1 catalytic complex rather than 15LOX alone. Here we designed, synthesized, and tested a customized library of 26 compounds using biochemical, molecular, and cell biology models along with redox lipidomic and computational analyses. We selected two lead compounds, FerroLOXIN-1 and 2, which effectively suppressed ferroptosis in vitro and in vivo without affecting the biosynthesis of pro-/anti-inflammatory lipid mediators in vivo. The effectiveness of these lead compounds is not due to radical scavenging or iron-chelation but results from their specific mechanisms of interaction with the 15LOX-2/PEBP1 complex, which either alters the binding pose of the substrate [eicosatetraenoyl-PE (ETE-PE)] in a nonproductive way or blocks the predominant oxygen channel thus preventing the catalysis of ETE-PE peroxidation. Our successful strategy may be adapted to the design of additional chemical libraries to reveal new ferroptosis-targeting therapeutic modalities.

Metabolic networks are interconnected and influence diverse cellular processes. The protein-metabolite interactions that mediate these networks are frequently low affinity and challenging to systematically discover. We developed mass spectrometry integrated with equilibrium dialysis for the discovery of allostery systematically (MIDAS) to identify such interactions. Analysis of 33 enzymes from human carbohydrate metabolism identified 830 protein-metabolite interactions, including known regulators, substrates, and products as well as previously unreported interactions. We functionally validated a subset of interactions, including the isoform-specific inhibition of lactate dehydrogenase by long-chain acyl-coenzyme A. Cell treatment with fatty acids caused a loss of pyruvate-lactate interconversion dependent on lactate dehydrogenase isoform expression. These protein-metabolite interactions may contribute to the dynamic, tissue-specific metabolic flexibility that enables growth and survival in an ever-changing nutrient environment.

Organisms must either synthesize or assimilate essential organic compounds to survive. The homocysteine synthase Met15 has been considered essential for inorganic sulfur assimilation in yeast since its discovery in the 1970s. As a result, MET15 has served as a genetic marker for hundreds of experiments that play a foundational role in eukaryote genetics and systems biology. Nevertheless, we demonstrate here through structural and evolutionary modeling, in vitro kinetic assays, and genetic complementation, that an alternative homocysteine synthase encoded by the previously uncharacterized gene YLL058W enables cells lacking Met15 to assimilate enough inorganic sulfur for survival and proliferation. These cells however fail to grow in patches or liquid cultures unless provided with exogenous methionine or other organosulfurs. We show that this growth failure, which has historically justified the status of MET15 as a classic auxotrophic marker, is largely explained by toxic accumulation of the gas hydrogen sulfide because of a metabolic bottleneck. When patched or cultured with a hydrogen sulfide chelator, and when propagated as colony grids, cells without Met15 assimilate inorganic sulfur and grow, and cells with Met15 achieve even higher yields. Thus, Met15 is not essential for inorganic sulfur assimilation in yeast. Instead, MET15 is the first example of a yeast gene whose loss conditionally prevents growth in a manner that depends on local gas exchange. Our results have broad implications for investigations of sulfur metabolism, including studies of stress response, methionine restriction, and aging. More generally, our findings illustrate how unappreciated experimental variables can obfuscate biological discovery., Competing Interests: Conflict of interest A.-R. C. is a member of the scientific advisory board for Flagship Labs 69, Inc (ProFound Therapeutics). T. I. is a cofounder of Data4Cure and has an equity interest. T. I. is on the Scientific Advisory Board of IDEAYA Biosciences, Inc, has an equity interest, and receives income. The terms of these arrangements have been reviewed and approved by the University of California San Diego in accordance with its conflict of interest policies. All other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Triosephosphate isomerase (TPI) deficiency (TPI Df) is an untreatable glycolytic enzymopathy that results in hemolytic anemia, progressive muscular impairment and irreversible brain damage. Although there is a 'common' mutation (TPIE105D), other pathogenic mutations have been described. We identified patients who were compound heterozygous for a newly described mutation, TPIQ181P, and the common TPIE105D mutation. Intriguingly, these patients lacked neuropathy or cognitive impairment. We then initiated biochemical and structural studies of TPIQ181P. Surprisingly, we found that purified TPIQ181P protein had markedly impaired catalytic properties whereas crystallographic studies demonstrated that the TPIQ181P mutation resulted in a highly disordered catalytic lid. We propose that genetic complementation occurs between the two alleles, one with little activity (TPIQ181P) and one with low stability (TPIE105D). Consistent with this, TPIQ181P/E105D fibroblasts exhibit a significant reduction in the TPI protein. These data suggest that impaired stability, and not catalytic activity, is a better predictor of TPI Df severity. Lastly, we tested two recently discovered chemical modulators of mutant TPI stability, itavastatin and resveratrol, and observed a significant increase in TPI in TPIQ181P/E105D patient cells., Competing Interests: Competing interests The authors declare the following potential conflicts of interest with respect to the research, authorship and/or publication of this article: The University of Pittsburgh has filed a provisional patent application on which M.J.P. and S.L.H are listed as inventors. No personal financial benefit has been realized, and there are no pending plans to commercialize that would create a competing financial interest., (© 2022. Published by The Company of Biologists Ltd.)

Ferroptosis and necroptosis are two pro-inflammatory cell death programs contributing to major pathologies and their inhibition has gained attention to treat a wide range of disease states. Necroptosis relies on activation of RIP1 and RIP3 kinases. Ferroptosis is triggered by oxidation of polyunsaturated phosphatidylethanolamines (PUFA-PE) by complexes of 15-Lipoxygenase (15LOX) with phosphatidylethanolamine-binding protein 1 (PEBP1). The latter, also known as RAF kinase inhibitory protein, displays promiscuity towards multiple proteins. In this study we show that RIP3 K51A kinase inactive mice have increased ferroptotic burden and worse outcome after irradiation and brain trauma rescued by anti-ferroptotic compounds Liproxstatin-1 and Ferrostatin 16-86. Given structural homology between RAF and RIP3, we hypothesized that PEBP1 acts as a necroptosis-to-ferroptosis switch interacting with either RIP3 or 15LOX. Using genetic, biochemical, redox lipidomics and computational approaches, we uncovered that PEBP1 complexes with RIP3 and inhibits necroptosis. Elevated expression combined with higher affinity enables 15LOX to pilfer PEBP1 from RIP3, thereby promoting PUFA-PE oxidation and ferroptosis which sensitizes Rip3 K51A/K51A kinase-deficient mice to total body irradiation and brain trauma. This newly unearthed PEBP1/15LOX-driven mechanism, along with previously established switch between necroptosis and apoptosis, can serve multiple and diverse cell death regulatory functions across various human disease states., (Copyright © 2022. Published by Elsevier B.V.)

Hydroperoxy-eicosatetraenoyl-phosphatidylethanolamine (HpETE-PE) is a ferroptotic cell death signal. HpETE-PE is produced by the 15-Lipoxygenase (15LOX)/Phosphatidylethanolamine Binding Protein-1 (PEBP1) complex or via an Fe-catalyzed non-enzymatic radical reaction. Ferrostatin-1 (Fer-1), a common ferroptosis inhibitor, is a lipophilic radical scavenger but a poor 15LOX inhibitor arguing against 15LOX having a role in ferroptosis. In the current work, we demonstrate that Fer-1 does not affect 15LOX alone, however, it effectively inhibits HpETE-PE production by the 15LOX/PEBP1 complex. Computational molecular modeling shows that Fer-1 binds to the 15LOX/PEBP1 complex at three sites and could disrupt the catalytically required allosteric motions of the 15LOX/PEBP1 complex. Using nine ferroptosis cell/tissue models, we show that HpETE-PE is produced by the 15LOX/PEBP1 complex and resolve the long-existing Fer-1 anti-ferroptotic paradox., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Temporally harmonized elimination of damaged or unnecessary organelles and cells is a prerequisite of health. Under Type 2 inflammatory conditions, human airway epithelial cells (HAECs) generate proferroptotic hydroperoxy-arachidonoyl-phosphatidylethanolamines (HpETE-PEs) as proximate death signals. Production of 15-HpETE-PE depends on activation of 15-lipoxygenase-1 (15LO1) in complex with PE-binding protein-1 (PEBP1). We hypothesized that cellular membrane damage induced by these proferroptotic phospholipids triggers compensatory prosurvival pathways, and in particular autophagic pathways, to prevent cell elimination through programmed death. We discovered that PEBP1 is pivotal to driving dynamic interactions with both proferroptotic 15LO1 and the autophagic protein microtubule-associated light chain-3 (LC3). Further, the 15LO1-PEBP1-generated ferroptotic phospholipid, 15-HpETE-PE, promoted LC3-I lipidation to stimulate autophagy. This concurrent activation of autophagy protects cells from ferroptotic death and release of mitochondrial DNA. Similar findings are observed in Type 2 Hi asthma, where high levels of both 15LO1-PEBP1 and LC3-II are seen in HAECs, in association with low bronchoalveolar lavage fluid mitochondrial DNA and more severe disease. The concomitant activation of ferroptosis and autophagy by 15LO1-PEBP1 complexes and their hydroperoxy-phospholipids reveals a pathobiologic pathway relevant to asthma and amenable to therapeutic targeting., Competing Interests: The authors declare no competing interest.

Mutations in ganglioside-induced differentiation-associated protein 1 (GDAP1) alter mitochondrial morphology and result in several subtypes of the inherited peripheral neuropathy Charcot-Marie-Tooth disease; however, the mechanism by which GDAP1 functions has remained elusive. GDAP1 contains primary sequence homology to the GST superfamily; however, the question of whether GDAP1 is an active GST has not been clearly resolved. Here, we present biochemical evidence, suggesting that GDAP1 has lost the ability to bind glutathione without a loss of substrate binding activity. We have revealed that the α-loop, located within the H-site motif is the primary determinant for substrate binding. Using structural data of GDAP1, we have found that critical residues and configurations in the G-site which canonically interact with glutathione are altered in GDAP1, rendering it incapable of binding glutathione. Last, we have found that the overexpression of GDAP1 in HeLa cells results in a mitochondrial phenotype which is distinct from oxidative stress-induced mitochondrial fragmentation. This phenotype is dependent on the presence of the transmembrane domain, as well as a unique hydrophobic domain that is not found in canonical GSTs. Together, we data point toward a non-enzymatic role for GDAP1, such as a sensor or receptor., (© 2020 Federation of American Societies for Experimental Biology.)

Mutations in the gene triosephosphate isomerase (TPI) lead to a severe multisystem condition that is characterized by hemolytic anemia, a weakened immune system, and significant neurologic symptoms such as seizures, distal neuropathy, and intellectual disability. No effective therapy is available. Here we report a compound heterozygous patient with a novel TPI pathogenic variant (NM_000365.5:c.569G>A:p.(Arg189Gln)) in combination with the common (NM_000365.5:c.315G>C:p.(Glu104Asp)) allele. We characterized the novel variant by mutating the homologous Arg in Drosophila using a genomic engineering system, demonstrating that missense mutations at this position cause a strong loss of function. Compound heterozygote animals were generated and exhibit motor behavioural deficits and markedly reduced protein levels. Furthermore, examinations of the TPI Arg189Gln /TPI Glu104Asp patient fibroblasts confirmed the reduction of TPI levels, suggesting that Arg189Gln may also affect the stability of the protein. The Arg189 residue participates in two salt bridges on the backside of the TPI enzyme dimer, and we reveal that a mutation at this position alters the coordination of the substrate-binding site and important catalytic residues. Collectively, these data reveal a new human pathogenic variant associated with TPI deficiency, identify the Arg189 salt bridge as critical for organizing the catalytic site of the TPI enzyme, and demonstrates that reduced TPI levels are associated with human TPI deficiency. These findings advance our understanding of the molecular pathogenesis of the disease, and suggest new therapeutic avenues for pre-clinical trials., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Accurate DNA replication is essential for genomic stability and cancer prevention. Homologous recombination is important for high-fidelity DNA damage tolerance during replication. How the homologous recombination machinery is recruited to replication intermediates is unknown. Here, we provide evidence that a Rad51 paralog-containing complex, the budding yeast Shu complex, directly recognizes and enables tolerance of predominantly lagging strand abasic sites. We show that the Shu complex becomes chromatin associated when cells accumulate abasic sites during S phase. We also demonstrate that purified recombinant Shu complex recognizes an abasic analog on a double-flap substrate, which prevents AP endonuclease activity and endonuclease-induced double-strand break formation. Shu complex DNA binding mutants are sensitive to methyl methanesulfonate, are not chromatin enriched, and exhibit increased mutation rates. We propose a role for the Shu complex in recognizing abasic sites at replication intermediates, where it recruits the homologous recombination machinery to mediate strand specific damage tolerance.

Accurate modeling of structural dynamics of proteins and their differentiation across different species can help us understand generic mechanisms of function shared by family members and the molecular basis of the specificity of individual members. We focused here on the family of lipoxygenases, enzymes that catalyze lipid oxidation, the mammalian and bacterial structures of which have been elucidated. We present a systematic method of approach for characterizing the sequence, structure, dynamics, and allosteric signaling properties of these enzymes using a combination of structure-based models and methods and bioinformatics tools applied to a data set of 88 structures. The analysis elucidates the signature dynamics of the lipoxygenase family and its differentiation among members, as well as key sites that enable its adaptation to specific substrate binding and allosteric activity.

sn2-15-Hydroperoxy-eicasotetraenoyl-phosphatidylethanolamines ( sn2-15-HpETE-PE) generated by mammalian 15-lipoxygenase/phosphatidylethanolamine binding protein-1 (15-LO/PEBP1) complex is a death signal in a recently identified type of programmed cell demise, ferroptosis. How the enzymatic complex selects sn2-ETE-PE as the substrate among 1 of ∼100 total oxidizable membrane PUFA phospholipids is a central, yet unresolved question. To unearth the highly selective and specific mechanisms of catalytic competence, we used a combination of redox lipidomics, mutational and computational structural analysis to show they stem from (i) reactivity toward readily accessible hexagonally organized membrane sn2-ETE-PEs, (ii) relative preponderance of sn2-ETE-PE species vs other sn2-ETE-PLs, and (iii) allosteric modification of the enzyme in the complex with PEBP1. This emphasizes the role of enzymatic vs random stochastic free radical reactions in ferroptotic death signaling.

Ferroptosis is a form of programmed cell death that is pathogenic to several acute and chronic diseases and executed via oxygenation of polyunsaturated phosphatidylethanolamines (PE) by 15-lipoxygenases (15-LO) that normally use free polyunsaturated fatty acids as substrates. Mechanisms of the altered 15-LO substrate specificity are enigmatic. We sought a common ferroptosis regulator for 15LO. We discovered that PEBP1, a scaffold protein inhibitor of protein kinase cascades, complexes with two 15LO isoforms, 15LO1 and 15LO2, and changes their substrate competence to generate hydroperoxy-PE. Inadequate reduction of hydroperoxy-PE due to insufficiency or dysfunction of a selenoperoxidase, GPX4, leads to ferroptosis. We demonstrated the importance of PEBP1-dependent regulatory mechanisms of ferroptotic death in airway epithelial cells in asthma, kidney epithelial cells in renal failure, and cortical and hippocampal neurons in brain trauma. As master regulators of ferroptotic cell death with profound implications for human disease, PEBP1/15LO complexes represent a new target for drug discovery., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Obtaining crystals for structure determination can be a difficult and time consuming proposition for any protein. Coiled-coil proteins and domains are found throughout nature, however, because of their physical properties and tendency to aggregate, they are traditionally viewed as being especially difficult to crystallize. Here, we utilize a variety of quick and simple techniques designed to identify a series of possible domain boundaries for a given coiled-coil protein, and then quickly characterize the behavior of these proteins in solution. With the addition of a strongly fluorescent tag (mRuby2), protein characterization is simple and straightforward. The target protein can be readily visualized under normal lighting and can be quantified with the use of an appropriate imager. The goal is to quickly identify candidates that can be removed from the crystallization pipeline because they are unlikely to succeed, affording more time for the best candidates and fewer funds expended on proteins that do not produce crystals. This process can be iterated to incorporate information gained from initial screening efforts, can be adapted for high-throughput expression and purification procedures, and is augmented by robotic screening for crystallization.

Shroom-mediated remodeling of the actomyosin cytoskeleton is a critical driver of cellular shape and tissue morphology that underlies the development of many tissues including the neural tube, eye, intestines, and vasculature. Shroom uses a conserved SD2 domain to direct the subcellular localization of Rho-associated kinase (Rock), which in turn drives changes in the cytoskeleton and cellular morphology through its ability to phosphorylate and activate non-muscle myosin II. Here, we present the structure of the human Shroom-Rock binding module, revealing an unexpected stoichiometry for Shroom in which two Shroom SD2 domains bind independent surfaces on Rock. Mutation of interfacial residues impaired Shroom-Rock binding in vitro and resulted in altered remodeling of the cytoskeleton and loss of Shroom-mediated changes in cellular morphology. Additionally, we provide the first direct evidence that Shroom can function as a Rock activator. These data provide molecular insight into the Shroom-Rock interface and demonstrate that Shroom directly participates in regulating cytoskeletal dynamics, adding to its known role in Rock localization., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The five-subunit yeast Paf1 complex (Paf1C) regulates all stages of transcription and is critical for the monoubiquitylation of histone H2B (H2Bub), a modification that broadly influences chromatin structure and eukaryotic transcription. Here, we show that the histone modification domain (HMD) of Paf1C subunit Rtf1 directly interacts with the ubiquitin conjugase Rad6 and stimulates H2Bub independently of transcription. We present the crystal structure of the Rtf1 HMD and use site-specific, in vivo crosslinking to identify a conserved Rad6 interaction surface. Utilizing ChIP-exo analysis, we define the localization patterns of the H2Bub machinery at high resolution and demonstrate the importance of Paf1C in targeting the Rtf1 HMD, and thereby H2Bub, to its appropriate genomic locations. Finally, we observe HMD-dependent stimulation of H2Bub in a transcription-free, reconstituted in vitro system. Taken together, our results argue for an active role for Paf1C in promoting H2Bub and ensuring its proper localization in vivo., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Triosephosphate isomerase (TPI) deficiency is a poorly understood disease characterized by hemolytic anemia, cardiomyopathy, neurologic dysfunction, and early death. TPI deficiency is one of a group of diseases known as glycolytic enzymopathies, but is unique for its severe patient neuropathology and early mortality. The disease is caused by missense mutations and dysfunction in the glycolytic enzyme, TPI. Previous studies have detailed structural and catalytic changes elicited by disease-associated TPI substitutions, and samples of patient erythrocytes have yielded insight into patient hemolytic anemia; however, the neuropathophysiology of this disease remains a mystery. This study combines structural, biochemical, and genetic approaches to demonstrate that perturbations of the TPI dimer interface are sufficient to elicit TPI deficiency neuropathogenesis. The present study demonstrates that neurologic dysfunction resulting from TPI deficiency is characterized by synaptic vesicle dysfunction, and can be attenuated with catalytically inactive TPI. Collectively, our findings are the first to identify, to our knowledge, a functional synaptic defect in TPI deficiency derived from molecular changes in the TPI dimer interface.

The conserved budding yeast Rad51 paralogues, including Rad55, Rad57, Csm2 and Psy3 are indispensable for homologous recombination (HR)-mediated chromosome damage repair. Rad55 and Rad57 are associated in a heterodimer, while Csm2 and Psy3 form the Shu complex with Shu1 and Shu2. Here we show that Rad55 bridges an interaction between Csm2 with Rad51 and Rad52 and, using a fully reconstituted system, demonstrate that the Shu complex synergizes with Rad55-Rad57 and Rad52 to promote nucleation of Rad51 on single-stranded DNA pre-occupied by replication protein A (RPA). The csm2-F46A allele is unable to interact with Rad55, ablating the ability of the Shu complex to enhance Rad51 presynaptic filament assembly in vitro and impairing HR in vivo. Our results reveal that Rad55-Rad57, the Shu complex and Rad52 act as a functional ensemble to promote Rad51-filament assembly, which has important implications for understanding the role of the human RAD51 paralogues in Fanconi anaemia and cancer predisposition.

Triosephosphate isomerase (TPI) is a glycolytic enzyme which homodimerizes for full catalytic activity. Mutations of the TPI gene elicit a disease known as TPI Deficiency, a glycolytic enzymopathy noted for its unique severity of neurological symptoms. Evidence suggests that TPI Deficiency pathogenesis may be due to conformational changes of the protein, likely affecting dimerization and protein stability. In this report, we genetically and physically characterize a human disease-associated TPI mutation caused by an I170V substitution. Human TPI(I170V) elicits behavioral abnormalities in Drosophila. An examination of hTPI(I170V) enzyme kinetics revealed this substitution reduced catalytic turnover, while assessments of thermal stability demonstrated an increase in enzyme stability. The crystal structure of the homodimeric I170V mutant reveals changes in the geometry of critical residues within the catalytic pocket. Collectively these data reveal new observations of the structural and kinetic determinants of TPI Deficiency pathology, providing new insights into disease pathogenesis., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Shroom3 is an actin-associated regulator of cell morphology that is required for neural tube closure, formation of the lens placode, and gut morphogenesis in mice and has been linked to chronic kidney disease and directional heart looping in humans. Numerous studies have shown that Shroom3 likely regulates these developmental processes by directly binding to Rho-kinase and facilitating the assembly of apically positioned contractile actomyosin networks. We have characterized the molecular basis for the neural tube defects caused by an ENU-induced mutation that results in an arginine-to-cysteine amino acid substitution at position 1838 of mouse Shroom3. We show that this substitution has no effect on Shroom3 expression or localization but ablates Rock binding and renders Shroom3 non-functional for the ability to regulate cell morphology. Our results indicate that Rock is the major downstream effector of Shroom3 in the process of neural tube morphogenesis. Based on sequence conservation and biochemical analysis, we predict that the Shroom-Rock interaction is highly conserved across animal evolution and represents a signaling module that is utilized in a variety of biological processes., (© 2014. Published by The Company of Biologists Ltd.)

Phage-encoded serine integrases are large serine recombinases that mediate integrative and excisive site-specific recombination of temperate phage genomes. They are well suited for use in heterologous systems and for synthetic genetic circuits as the attP and attB attachment sites are small (<50 bp), there are no host factor or DNA supercoiling requirements, and they are strongly directional, doing only excisive recombination in the presence of a recombination directionality factor. Combining different recombinases that function independently and without cross-talk to construct complex synthetic circuits is desirable, and several different serine integrases are available. However, we show here that these functions are not reliably predictable, and we describe a pair of serine integrases encoded by mycobacteriophages Bxz2 and Peaches with unusual and unpredictable specificities. The integrases share only 59% amino acid sequence identity and the attP sites have fewer than 50% shared bases, but they use the same attB site and there is non-reciprocal cross-talk between the two systems. The DNA binding specificities do not result from differences in specific DNA contacts but from the constraints imposed by the configuration of the component half-sites within each of the attachment site DNAs., (© 2013.)

The recombination directionality factor, Xis, is a DNA bending protein that determines the outcome of integrase-mediated site-specific recombination by redesign of higher-order protein-DNA architectures. Although the attachment site DNA of mycobacteriophage Pukovnik is likely to contain four sites for Xis binding, Xis crystals contain five subunits in the asymmetric unit, four of which align into a Xis filament and a fifth that is generated by an unusual domain swap. Extensive intersubunit contacts stabilize a bent filament-like arrangement with Xis monomers aligned head to tail. The structure implies a DNA bend of ~120°, which is in agreement with DNA bending measured in vitro. Formation of attR-containing intasomes requires only Int and Xis, distinguishing Pukovnik from lambda. Therefore, we conclude that, in Pukovnik, Xis-induced DNA bending is sufficient to promote intramolecular Int-mediated bridges during intasome formation., (© 2013.)

Rho-associated coiled coil containing protein kinase (Rho-kinase or Rock) is a well-defined determinant of actin organization and dynamics in most animal cells characterized to date. One of the primary effectors of Rock is non-muscle myosin II. Activation of Rock results in increased contractility of myosin II and subsequent changes in actin architecture and cell morphology. The regulation of Rock is thought to occur via autoinhibition of the kinase domain via intramolecular interactions between the N-terminus and the C-terminus of the kinase. This autoinhibited state can be relieved via proteolytic cleavage, binding of lipids to a Pleckstrin Homology domain near the C-terminus, or binding of GTP-bound RhoA to the central coiled-coil region of Rock. Recent work has identified the Shroom family of proteins as an additional regulator of Rock either at the level of cellular distribution or catalytic activity or both. The Shroom-Rock complex is conserved in most animals and is essential for the formation of the neural tube, eye, and gut in vertebrates. To address the mechanism by which Shroom and Rock interact, we have solved the structure of the coiled-coil region of Rock that binds to Shroom proteins. Consistent with other observations, the Shroom binding domain is a parallel coiled-coil dimer. Using biochemical approaches, we have identified a large patch of residues that contribute to Shrm binding. Their orientation suggests that there may be two independent Shrm binding sites on opposing faces of the coiled-coil region of Rock. Finally, we show that the binding surface is essential for Rock colocalization with Shroom and for Shroom-mediated changes in cell morphology.

Polymerase associated factor 1 complex (Paf1C) broadly influences gene expression by regulating chromatin structure and the recruitment of RNA-processing factors during transcription elongation. The Plus3 domain of the Rtf1 subunit mediates Paf1C recruitment to genes by binding a repeating domain within the elongation factor Spt5 (suppressor of Ty). Here we provide a molecular description of this interaction by reporting the structure of human Rtf1 Plus3 in complex with a phosphorylated Spt5 repeat. We find that Spt5 binding is mediated by an extended surface containing phosphothreonine recognition and hydrophobic interfaces that interact with residues outside the Spt5 motif. Changes within these interfaces diminish binding of Spt5 in vitro and chromatin localization of Rtf1 in vivo. The structure reveals the basis for recognition of the repeat motif of Spt5, a key player in the recruitment of gene regulatory factors to RNA polymerase II.

Triosephosphate isomerase (TPI) is a glycolytic enzyme that converts dihydroxyacetone phosphate (DHAP) into glyceraldehyde 3-phosphate (GAP). Glycolytic enzyme dysfunction leads to metabolic diseases collectively known as glycolytic enzymopathies. Of these enzymopathies, TPI deficiency is unique in the severity of neurological symptoms. The Drosophila sugarkill mutant closely models TPI deficiency and encodes a protein prematurely degraded by the proteasome. This led us to question whether enzyme catalytic activity was crucial to the pathogenesis of TPI sugarkill neurological phenotypes. To study TPI deficiency in vivo we developed a genomic engineering system for the TPI locus that enables the efficient generation of novel TPI genetic variants. Using this system we demonstrate that TPI sugarkill can be genetically complemented by TPI encoding a catalytically inactive enzyme. Furthermore, our results demonstrate a non-metabolic function for TPI, the loss of which contributes significantly to the neurological dysfunction in this animal model.

The Saccharomyces cerevisiae Shu complex, consisting of Shu1, Shu2, Csm2 and Psy3, promotes error-free homologous recombination (HR) by an unknown mechanism. Recent structural analysis of two Shu proteins, Csm2 and Psy3, has revealed that these proteins are Rad51 paralogues and mediate DNA binding of this complex. We show in vitro that the Csm2-Psy3 heterodimer preferentially binds synthetic forked DNA or 3'-DNA overhang substrates resembling structures used during HR in vivo. We find that Csm2 interacts with Rad51 and the Rad51 paralogues, the Rad55-Rad57 heterodimer and that the Shu complex functions in the same epistasis group as Rad55-Rad57. Importantly, Csm2's interaction with Rad51 is dependent on Rad55, whereas Csm2's interaction with Rad55 occurs independently of Rad51. Consistent with the Shu complex containing Rad51 paralogues, the methyl methanesulphonate sensitivity of Csm2 is exacerbated at colder temperatures. Furthermore, Csm2 and Psy3 are needed for efficient recruitment of Rad55 to DNA repair foci after DNA damage. Finally, we observe that the Shu complex preferentially promotes Rad51-dependent homologous recombination over Rad51-independent repair. Our data suggest a model in which Csm2-Psy3 recruit the Shu complex to HR substrates, where it interacts with Rad51 through Rad55-Rad57 to stimulate Rad51 filament assembly and stability, promoting error-free repair.

Reactive oxygen species (ROS) play essential roles in cell signaling, survival, and homeostasis. Aberrant ROS lead to disease and contribute to the aging process. Numerous enzymes and vigilant antioxidant pathways are required to regulate ROS for normal cellular health. Mitochondria are a major source of ROS, and mechanisms to prevent elevated ROS during oxidative phosphorylation require super oxide dismutase (SOD) activity. SOD2, also known as MnSOD, is targeted to mitochondria and is instrumental in regulating ROS by conversion of superoxides to hydrogen peroxide, which is further broken down into H(2)O and oxygen. Here, we describe the identification of a novel mutation within the mitochondrial SOD2 enzyme in Drosophila that results in adults with an extremely shortened life span, sensitivity to hyperoxia, and neuropathology. Additional studies demonstrate that this novel mutant, SOD2(bewildered), exhibits abnormal brain morphology, suggesting a critical role for this protein in neurodevelopment. We investigated the basis of this neurodevelopmental defect and discovered an increase in aberrant axonal that could underlie the aberrant neurodevelopment and brain morphology defects. This novel allele, SOD2(bewildered), provides a unique opportunity to study the effects of increased mitochondrial ROS on neural development, axonal targeting, and neural cell degeneration in vivo.

Shroom (Shrm) proteins are essential regulators of cell shape and tissue morpho-logy during animal development that function by interacting directly with the coiled-coil region of Rho kinase (Rock). The Shrm-Rock interaction is sufficient to direct Rock subcellular localization and the subsequent assembly of contractile actomyosin networks in defined subcellular locales. However, it is unclear how the Shrm-Rock interaction is regulated at the molecular level. To begin investigating this issue, we present the structure of Shrm domain 2 (SD2), which mediates the interaction with Rock and is required for Shrm function. SD2 is a unique three-segmented dimer with internal symmetry, and we identify conserved residues on the surface and within the dimerization interface that are required for the Rock-Shrm interaction and Shrm activity in vivo. We further show that these residues are critical in both vertebrate and invertebrate Shroom proteins, indicating that the Shrm-Rock signaling module has been functionally and molecularly conserved. The structure and biochemical analysis of Shrm SD2 indicate that it is distinct from other Rock activators such as RhoA and establishes a new paradigm for the Rock-mediated assembly of contractile actomyosin networks.

The conserved Paf1 complex localizes to the coding regions of genes and facilitates multiple processes during transcription elongation, including the regulation of histone modifications. However, the mechanisms that govern Paf1 complex recruitment to active genes are undefined. Here we describe a previously unrecognized domain within the Cdc73 subunit of the Paf1 complex, the Cdc73 C-domain, and demonstrate its importance for Paf1 complex occupancy on transcribed chromatin. Deletion of the C-domain causes phenotypes associated with elongation defects without an apparent loss of complex integrity. Simultaneous mutation of the C-domain and another subunit of the Paf1 complex, Rtf1, causes enhanced mutant phenotypes and loss of histone H3 lysine 36 trimethylation. The crystal structure of the C-domain reveals unexpected similarity to the Ras family of small GTPases. Instead of a deep nucleotide-binding pocket, the C-domain contains a large but comparatively flat surface of highly conserved residues, devoid of ligand. Deletion of the C-domain results in reduced chromatin association for multiple Paf1 complex subunits. We conclude that the Cdc73 C-domain probably constitutes a protein interaction surface that functions with Rtf1 in coupling the Paf1 complex to the RNA polymerase II elongation machinery.

yFACT (heterodimers of Saccharomyces cerevisiae Spt16-Pob3 combined with Nhp6) binds to and alters the properties of nucleosomes. The essential function of yFACT is not disrupted by deletion of the N-terminal domain (NTD) of Spt16 or by mutation of the middle domain of Pob3, but either alteration makes yeast cells sensitive to DNA replication stress. We have determined the structure of the Spt16 NTD and find evidence for a conserved potential peptide-binding site. Pob3-M also contains a putative binding site, and we show that these two sites perform an overlapping essential function. We find that yFACT can bind the N-terminal tails of some histones and that this interaction is important for yFACT-nucleosome binding. However, neither the Spt16 NTD nor a key residue in the putative Pob3-M-binding site was required for interactions with histone N termini or for yFACT-mediated nucleosome reorganization in vitro. Instead, both potential binding sites interact functionally with the C-terminal docking domain of the histone H2A. yFACT therefore appears to make multiple contacts with different sites within nucleosomes, and these interactions are partially redundant with one another. The docking domain of H2A is identified as an important participant in maintaining stability during yFACT-mediated nucleosome reorganization, suggesting new models for the mechanism of this activity.

During HIV-1 entry, the highly conserved gp41 N-trimer pocket region becomes transiently exposed and vulnerable to inhibition. Using mirror-image phage display and structure-assisted design, we have discovered protease-resistant D-amino acid peptides (D-peptides) that bind the N-trimer pocket with high affinity and potently inhibit viral entry. We also report high-resolution crystal structures of two of these D-peptides in complex with a pocket mimic that suggest sources of their high potency. A trimeric version of one of these peptides is the most potent pocket-specific entry inhibitor yet reported by three orders of magnitude (IC(50) = 250 pM). These results are the first demonstration that D-peptides can form specific and high-affinity interactions with natural protein targets and strengthen their promise as therapeutic agents. The D-peptides described here address limitations associated with current L-peptide entry inhibitors and are promising leads for the prevention and treatment of HIV/AIDS.

An important issue for chromatin remodeling complexes is how their bromodomains recognize particular acetylated lysine residues in histones. The Rsc4 subunit of the yeast remodeler RSC contains an essential tandem bromodomain (TBD) that binds acetylated K14 of histone H3 (H3K14ac). We report a series of crystal structures that reveal a compact TBD that binds H3K14ac in the second bromodomain and, remarkably, binds acetylated K25 of Rsc4 itself in the first bromodomain. Endogenous Rsc4 is acetylated only at K25, and Gcn5 is identified as necessary and sufficient for Rsc4 K25 acetylation in vivo and in vitro. Rsc4 K25 acetylation inhibits binding to H3K14ac, and mutation of Rsc4 K25 results in altered growth rates. These data suggest an autoregulatory mechanism in which Gcn5 performs both the activating (H3K14ac) and inhibitory (Rsc4 K25ac) modifications, perhaps to provide temporal regulation. Additional regulatory mechanisms are indicated as H3S10 phosphorylation inhibits Rsc4 binding to H3K14ac peptides.

We report the crystal structure of the middle domain of the Pob3 subunit (Pob3-M) of S. cerevisiae FACT (yFACT, facilitates chromatin transcription), which unexpectedly adopts an unusual double pleckstrin homology (PH) architecture. A mutation within a conserved surface cluster in this domain causes a defect in DNA replication that is suppressed by mutation of replication protein A (RPA). The nucleosome reorganizer yFACT therefore interacts in a physiologically important way with the central single-strand DNA (ssDNA) binding factor RPA to promote a step in DNA replication. Purified yFACT and RPA display a weak direct physical interaction, although the genetic suppression is not explained by simple changes in affinity between the purified proteins. Further genetic analysis suggests that coordinated function by yFACT and RPA is important during nucleosome deposition. These results support the model that the FACT family has an essential role in constructing nucleosomes during DNA replication, and suggest that RPA contributes to this process.

We report the crystal structure of the catalytic domain of human ADAR2, an RNA editing enzyme, at 1.7 angstrom resolution. The structure reveals a zinc ion in the active site and suggests how the substrate adenosine is recognized. Unexpectedly, inositol hexakisphosphate (IP6) is buried within the enzyme core, contributing to the protein fold. Although there are no reports that adenosine deaminases that act on RNA (ADARs) require a cofactor, we show that IP6 is required for activity. Amino acids that coordinate IP6 in the crystal structure are conserved in some adenosine deaminases that act on transfer RNA (tRNA) (ADATs), related enzymes that edit tRNA. Indeed, IP6 is also essential for in vivo and in vitro deamination of adenosine 37 of tRNAala by ADAT1.

E1 enzymes activate ubiquitin or related proteins and pass them to E2 enzymes. A recent structure and associated biochemical studies (Huang et al., 2005) show how an E1 binds its cognate E2 and indicates that large, conformational changes will be an integral component of the E1 reaction cycle.

The attachment of the small protein ubiquitin to other proteins, a process known as ubiquitylation, is a widespread form of post-translational modification that regulates numerous cellular functions in eukaryotes. Ubiquitylation is performed by complexes of E2 and E3 enzymes that are assembled and select substrates via a series of protein-protein interactions. Recent structure determinations of the ubiquitylation machinery have revealed some of the various protein-protein interfaces involved.

A new structure reveals how substrates are recognized for conjugation to the ubiquitin-like protein SUMO, a posttranslational modification that functions in activation of transcription factors, nuclear import, and cell division.

While the signaling properties of ubiquitin depend on the topology of polyubiquitin chains, little is known concerning the molecular basis of specificity in chain assembly and recognition. UEV/Ubc complexes have been implicated in the assembly of Lys63-linked polyubiquitin chains that act as a novel signal in postreplicative DNA repair and I kappa B alpha kinase activation. The crystal structure of the Mms2/Ubc13 heterodimer shows the active site of Ubc13 at the intersection of two channels that are potential binding sites for the two substrate ubiquitins. Mutations that destabilize the heterodimer interface confer a marked UV sensitivity, providing direct evidence that the intact heterodimer is necessary for DNA repair. Selective mutations in the channels suggest a molecular model for specificity in the assembly of Lys63-linked polyubiquitin signals.

The yeast Tup1 and Ssn6 proteins form a transcriptional repression complex that represses transcription of a broad array of genes. It has been shown that the N-terminal domain of the Tup1 protein interacts with a region of the Ssn6 protein that consists of 10 tandem copies of a tetratricopeptide motif. In this work, we use a surface plasmon resonance assay to measure the affinity of the N-terminal domain of Tup1 for a minimal 3-TPR domain of Saccharomyces cerevisiae Ssn6 that is sufficient for binding to Tup1. This domain of Ssn6 binds with comparable affinity to S. cerevisiae and Candida albicans Tup1, but with 100-fold lower affinity to Tup1 protein containing a point mutation that gives rise to a defect in repression in vivo. Results from studies using analytical ultracentrifugation, CD spectroscopy, limited proteolysis, and (1)H NMR show that this domain of Tup1 is primarily alpha-helical and forms a stable tetramer that is highly nonglobular in shape. X-ray diffraction recorded from poorly ordered crystals of the Tup1 tetramerization domain contains fiber diffraction typical of a coiled coil. Our results are used to propose a model for the structure of the N-terminal domain of Tup1 and its interaction with the Ssn6 protein.