Loss of CD4+ T cell-intrinsic arginase 1 accelerates Th1 response kinetics and reduces lung pathology during influenza infection.

Arginase 1 (Arg1), the enzyme catalyzing the conversion of arginine to ornithine, is a hallmark of IL-10-producing immunoregulatory M2 macrophages. However, its expression in T cells is disputed. Here, we demonstrate that induction of Arg1 expression is a key feature of lung CD4+ T cells during mouse in vivo influenza infection. Conditional ablation of Arg1 in CD4+ T cells accelerated both virus-specific T helper 1 (Th1) effector responses and its resolution, resulting in efficient viral clearance and reduced lung pathology. Using unbiased transcriptomics and metabolomics, we found that Arg1-deficiency was distinct from Arg2-deficiency and caused altered glutamine metabolism. Rebalancing this perturbed glutamine flux normalized the cellular Th1 response. CD4+ T cells from rare ARG1-deficient patients or CRISPR-Cas9-mediated ARG1-deletion in healthy donor cells phenocopied the murine cellular phenotype. Collectively, CD4+ T cell-intrinsic Arg1 functions as an unexpected rheostat regulating the kinetics of the mammalian Th1 lifecycle with implications for Th1-associated tissue pathologies.

Humanin selectively prevents the activation of pro-apoptotic protein BID by sequestering it into fibers.

Members of the B-cell lymphoma (BCL-2) protein family regulate mitochondrial outer membrane permeabilization (MOMP), a phenomenon in which mitochondria become porous and release death-propagating complexes during the early stages of apoptosis. Pro-apoptotic BCL-2 proteins oligomerize at the mitochondrial outer membrane during MOMP, inducing pore formation. Of current interest are endogenous factors that can inhibit pro-apoptotic BCL-2 mitochondrial outer membrane translocation and oligomerization. A mitochondrial-derived peptide, Humanin (HN), was reported being expressed from an alternate ORF in the mitochondrial genome and inhibiting apoptosis through interactions with the pro-apoptotic BCL-2 proteins. Specifically, it is known to complex with BAX and BID. We recently reported the fibrillation of HN and BAX into ß-sheets. Here, we detail the fibrillation between HN and BID. These fibers were characterized using several spectroscopic techniques, protease fragmentation with mass analysis, and EM. Enhanced fibrillation rates were detected with rising temperatures or pH values and the presence of a detergent. BID fibers are similar to those produced using BAX; however, the structures differ in final conformations of the BCL-2 proteins. BID fibers display both types of secondary structure in the fiber, whereas BAX was converted entirely to ß-sheets. The data show that two distinct segments of BID are incorporated into the fiber structure, whereas other portions of BID remain solvent-exposed and retain helical structure. Similar analyses show that anti-apoptotic BCL-xL does not form fibers with humanin. These results support a general mechanism of sequestration of pro-apoptotic BCL-2 proteins into fibers by HN to inhibit MOMP.

Mitochondrial General Control of Amino Acid Synthesis 5 Like 1 Regulates Glutaminolysis, Mammalian Target of Rapamycin Complex 1 Activity, and Murine Liver Regeneration.

BACKGROUND AND AIMS: The regenerative capacity of the liver plays a protective role against hepatotoxins and impaired regeneration exacerbates liver dysfunction in nonalcoholic fatty liver disease (NAFLD). Mitochondrial bioenergetic and -synthetic functions are important contributory factors in hepatic regeneration, and the control of mitochondrial protein acetylation is implicated in the mitochondrial susceptibility to liver stressors. Here, we evaluated the role of general control of amino acid synthesis 5 like 1 (GCN5L1), a mediator of mitochondrial metabolism and acetylation, in modulating murine liver regeneration (LR) in response to acute CCl4 -induced hepatotoxicity. APPROACH AND RESULTS: Initial metabolomic screening found that liver GCN5L1 knockout (LKO) mice have augmented glutaminolysis. Absence of GCN5L1 modified enzyme activity of liver-enriched glutaminase enzyme (glutaminase 2; GLS2), and GCN5L1 levels modulated GLS2 oligomerization and acetylation. This metabolic remodeling resulted in the elevation of α-ketoglutarate levels, which are known to activate mammalian target of rapamycin complex 1 (mTORC1). This signaling pathway was induced with increased phosphorylation of S6 kinase in LKO hepatocytes, and inhibition of glutaminolysis reversed aberrant mTORC1 signaling. At the same time, glutaminolysis, activity of GLS2, and activation of mTORC1 signaling were reversed by the genetic reintroduction of the mitochondrial isoform of GCN5L1 into LKO primary hepatocytes. Finally, LKO mice had a more robust regenerative capacity in response to CCl4 hepatoxicity, and this response was blunted by both the mTORC1 inhibitor, rapamycin, and by pharmacological blunting of glutaminolysis. CONCLUSIONS: These data point to a central role of glutaminolysis in modulating the regenerative capacity in the liver. Furthermore, inhibition of mitochondrial GCN5L1 to augment LR may be a useful strategy in disease states linked to hepatotoxicity.

Humanin induces conformational changes in the apoptosis regulator BAX and sequesters it into fibers, preventing mitochondrial outer-membrane permeabilization.

The mitochondrial, or intrinsic, apoptosis pathway is regulated mainly by members of the B-cell lymphoma 2 (BCL-2) protein family. BCL-2-associated X apoptosis regulator (BAX) plays a pivotal role in the initiation of mitochondria-mediated apoptosis as one of the factors causing mitochondrial outer-membrane permeabilization (MOMP). Of current interest are endogenous BAX ligands that inhibit its MOMP activity. Mitochondrial-derived peptides (MDPs) are a recently identified class of mitochondrial retrograde signaling molecules and are reported to be potent apoptosis inhibitors. Among them, humanin (HN) has been shown to suppress apoptosis by inhibiting BAX translocation to the mitochondrial outer membrane, but the molecular mechanism of this interaction is unknown. Here, using recombinant protein expression, along with light-scattering, CD, and fluorescence spectroscopy, we report that HN and BAX can form fibers together in vitro Results from negative stain EM experiments suggest that BAX undergoes secondary and tertiary structural rearrangements and incorporates into the fibers, and that its membrane-associating C-terminal helix is important for the fibrillation process. Additionally, HN mutations known to alter its anti-apoptotic activity affect fiber morphology. Our findings reveal for the first time a potential mechanism by which BAX can be sequestered by fibril formation, which can prevent it from initiating MOMP and committing the cell to apoptosis.

Bax expression is optimal at low oxygen tension and constant agitation.

Bax is a pro-apoptosis protein that translocates from the cytosol to the mitochondria membrane upon initiation of programed cell death. Bax subsequently disrupts the mitochondria membrane, resulting in the release of cytochrome C which activates the downstream caspases. The structure of inactive Bax has been solved, but despite intensive investigation, the mechanism by which it regulates apoptosis is not established. The low yield of Bax expression in E. coli hampers efforts to elucidate the mechanism. Thus, we undertook a systematic study aimed at improving the yield of Bax. Bacteria were grown in a computer-controlled fermenter and expression was induced by addition of Isopropyl ß-d-1-thiogalactopyranoside (IPTG). The Bax expression level decreased continuously when the dissolved oxygen level was kept at 30%, which is non-limiting for E. coli. Alternatively, when oxygen input was decreased with constant agitation and air flow (or kLa), Bax yield increased by a factor of three. To make sure the short chain fatty acids generated during micro-aerobic fermentation had no adverse effect, their concentrations were closely monitored with HPLC and their effect on cell growth and Bax expression were investigated additionally using shake flasks. Through proteomic analysis using Tandem Mass Tag (TMT) labeling, we identified degradation pathway within E. coli cells as a potential player behind the lower expression level.

A ubiquitin-binding protein, FAAP20, links RNF8-mediated ubiquitination to the Fanconi anemia DNA repair network.

The Fanconi anemia (FA) protein network is necessary for repair of DNA interstrand crosslinks (ICLs), but its control mechanism remains unclear. Here we show that the network is regulated by a ubiquitin signaling cascade initiated by RNF8 and its partner, UBC13, and mediated by FAAP20, a component of the FA core complex. FAAP20 preferentially binds the ubiquitin product of RNF8-UBC13, and this ubiquitin-binding activity and RNF8-UBC13 are both required for recruitment of FAAP20 to ICLs. Both RNF8 and FAAP20 are required for recruitment of FA core complex and FANCD2 to ICLs, whereas RNF168 can modulate efficiency of the recruitment. RNF8 and FAAP20 are needed for efficient FANCD2 monoubiquitination, a key step of the FA network; RNF8 and the FA core complex work in the same pathway to promote cellular resistance to ICLs. Thus, the RNF8-FAAP20 ubiquitin cascade is critical for recruiting FA core complex to ICLs and for normal function of the FA network.

MitoRACE: evaluating mitochondrial function in vivo and in single cells with subcellular resolution using multiphoton NADH autofluorescence.

KEY POINTS: We developed a novel metabolic imaging approach that provides direct measures of the rate of mitochondrial energy conversion with single-cell and subcellular resolution by evaluating NADH autofluorescence kinetics during the mitochondrial redox after cyanide experiment (mitoRACE). Measures of mitochondrial NADH flux by mitoRACE are sensitive to physiological and pharmacological perturbations in vivo. Metabolic imaging with mitoRACE provides a highly adaptable platform for evaluating mitochondrial function in vivo and in single cells with potential for broad applications in the study of energy metabolism. ABSTRACT: Mitochondria play a critical role in numerous cell types and diseases, and structure and function of mitochondria can vary greatly among cells or within different regions of the same cell. However, there are currently limited methodologies that provide direct assessments of mitochondrial function in vivo, and contemporary measures of mitochondrial energy conversion lack the spatial resolution necessary to address cellular and subcellular heterogeneity. Here, we describe a novel metabolic imaging approach that provides direct measures of mitochondrial energy conversion with single-cell and subcellular resolution by evaluating NADH autofluorescence kinetics during the mitochondrial redox after cyanide experiment (mitoRACE). MitoRACE measures the rate of NADH flux through the steady-state mitochondrial NADH pool by rapidly inhibiting mitochondrial energetic flux, resulting in an immediate, linear increase in NADH fluorescence proportional to the steady-state NADH flux rate, thereby providing a direct measure of mitochondrial NADH flux. The experiments presented here demonstrate the sensitivity of this technique to detect physiological and pharmacological changes in mitochondrial flux within tissues of living animals and reveal the unique capability of this technique to evaluate mitochondrial function with single-cell and subcellular resolution in different cell types in vivo and in cell culture. Furthermore, we highlight the potential applications of mitoRACE by showing that within single neurons, mitochondria in neurites have higher energetic flux rates than mitochondria in the cell body. Metabolic imaging with mitoRACE provides a highly adaptable platform for evaluating mitochondrial function in vivo and in single cells, with potential for broad applications in the study of energy metabolism.

Efficient Generation of ß-Globin-Expressing Erythroid Cells Using Stromal Cell-Derived Induced Pluripotent Stem Cells from Patients with Sickle Cell Disease.

Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells represent an ideal source for in vitro modeling of erythropoiesis and a potential alternative source for red blood cell transfusions. However, iPS cell-derived erythroid cells predominantly produce ε- and γ-globin without ß-globin production. We recently demonstrated that ES cell-derived sacs (ES sacs), known to express hemangioblast markers, allow for efficient erythroid cell generation with ß-globin production. In this study, we generated several iPS cell lines derived from bone marrow stromal cells (MSCs) and peripheral blood erythroid progenitors (EPs) from sickle cell disease patients, and evaluated hematopoietic stem/progenitor cell (HSPC) generation after iPS sac induction as well as subsequent erythroid differentiation. MSC-derived iPS sacs yielded greater amounts of immature hematopoietic progenitors (VEGFR2 + GPA-), definitive HSPCs (CD34 + CD45+), and megakaryoerythroid progenitors (GPA + CD41a+), as compared to EP-derived iPS sacs. Erythroid differentiation from MSC-derived iPS sacs resulted in greater amounts of erythroid cells (GPA+) and higher ß-globin (and ßS-globin) expression, comparable to ES sac-derived cells. These data demonstrate that human MSC-derived iPS sacs allow for more efficient erythroid cell generation with higher ß-globin production, likely due to heightened emergence of immature progenitors. Our findings should be important for iPS cell-derived erythroid cell generation. Stem Cells 2017;35:586-596.

Regulation of the actin-activated MgATPase activity of Acanthamoeba myosin II by phosphorylation of serine 639 in motor domain loop 2.

It had been proposed previously that only filamentous forms of Acanthamoeba myosin II have actin-activated MgATPase activity and that this activity is inhibited by phosphorylation of up to four serine residues in a repeating sequence in the C-terminal nonhelical tailpiece of the two heavy chains. We have reinvestigated these issues using recombinant WT and mutant myosins. Contrary to the earlier proposal, we show that two nonfilamentous forms of Acanthamoeba myosin II, heavy meromyosin and myosin subfragment 1, have actin-activated MgATPase that is down-regulated by phosphorylation. By mass spectroscopy, we identified five serines in the heavy chains that can be phosphorylated by a partially purified kinase preparation in vitro and also are phosphorylated in endogenous myosin isolated from the amoebae: four serines in the nonhelical tailpiece and Ser639 in loop 2 of the motor domain. S639A mutants of both subfragment 1 and full-length myosin had actin-activated MgATPase that was not inhibited by phosphorylation of the serines in the nonhelical tailpiece or their mutation to glutamic acid or aspartic acid. Conversely, S639D mutants of both subfragment 1 and full-length myosin were inactive, irrespective of the phosphorylation state of the serines in the nonhelical tailpiece. To our knowledge, this is the first example of regulation of the actin-activated MgATPase activity of any myosin by modification of surface loop 2.

Structural Relationships to Efficacy for Prazole-Derived Antivirals.

Here, an in vitro characterization of a family of prazole derivatives that covalently bind to the C73 site on Tsg101 and assay their ability to inhibit viral particle production is presented. Structurally, increased steric bulk on the 4-pyridyl of the prazole expands the prazole site on the UEV domain toward the ß-hairpin in the Ub-binding site and is coupled to increased inhibition of virus-like particle production in HIV-1. Increased bulk also increased toxicity, which is alleviated by increasing flexibility. Further, the formation of a novel secondary Tsg101 adduct for several of the tested compounds and the commercial drug lansoprazole. The secondary adduct involved the loss of the 4-pyridyl substituent to form an irreversible species, with implications for increasing the half-life of the active species or its specificity toward Tsg101 UEV. It is also determined that sulfide derivatives display effective viral inhibition, presumably through cellular sulfoxidation, allowing for delayed conversion within the cellular environment, and identify SARS-COV-2 as a target of prazole inhibition. These results open multiple avenues for the design of prazole derivatives for antiviral applications.

The mitochondrial thiolase ACAT1 regulates monocyte/macrophage type I interferon via epigenetic control.

Lipid-derived acetyl-CoA is shown to be the major carbon source for histone acetylation. However, there is no direct evidence demonstrating lipid metabolic pathway contribututions to this process. Mitochondrial acetyl-CoA acetyltransferase 1 (ACAT1) catalyzes the final step of ß-oxidation, the aerobic process catabolizing fatty acids (FA) into acetyl-CoA. To investigate this in the context of immunometabolism, we generated macrophage cell line lacking ACAT1. 13C-carbon tracing combined with mass spectrometry confirmed incorporation of FA-derived carbons into histone H3 and this incorporation was reduced in ACAT1 KO macrophage cells. RNA-seq identified a subset of genes downregulated in ACAT1 KO cells including STAT1/2 and interferon stimulated genes (ISGs). CHIP analysis demonstrated reduced acetyl-H3 binding to STAT1 promoter/enhancer regions. Increasing histone acetylation rescued STAT1/2 expression in ACAT1 KO cells. Concomitantly, ligand triggered IFNß release was blunted in ACAT1 KO cells and rescued by reconstitution of ACAT1. Furthermore, ACAT1 promotes FA-mediated histone acetylation in an acetylcarnitine shuttle-dependent manner. In patients with obesity, levels of ACAT1 and histone acetylation are abnormally elevated. Thus, our study identified a novel link between ACAT1 mediated FA metabolism and epigenetic modification on STAT1/2 that uncovers a regulatory role of lipid metabolism in innate immune signaling and opens novel avenues for interventions in human diseases such as obesity.

Isoquercetin for thromboinflammation in sickle cell disease: a randomized double-blind placebo-controlled trial.

ABSTRACT: Data from a small trial in patients with cancer suggest that isoquercetin (IQ) treatment lowered thrombosis biomarkers and prevented clinical thrombosis, but, to our knowledge, no studies of IQ have been conducted to target thromboinflammation in adults with sickle cell disease (SCD). We conducted a randomized, double-blind, placebo-controlled trial in adults with steady-state SCD (hemoglobin SS [HbSS], HbSß0thal, HbSß+thal, or HbSC). The primary outcome was the change in plasma soluble P-selectin (sP-selectin) after treatment compared with baseline, analyzed in the intention-to-treat population. Between November 2019 and July 2022, 46 patients (aged 40 ± 11 years, 56% female, 75% under hydroxyurea treatment) were randomized to receive IQ (n = 23) or placebo (n = 23). IQ was well tolerated and all the adverse events (AEs; n = 21) or serious AEs (n = 14) recorded were not attributable to the study drug. The mean posttreatment change for sP-selectin showed no significant difference between the treatment groups (IQ, 0.10 ± 6.53 vs placebo, 0.74 ± 4.54; P = .64). In patients treated with IQ, whole-blood coagulation (P = .03) and collagen-induced platelet aggregation (P = .03) were significantly reduced from the baseline. Inducible mononuclear cell tissue factor gene expression and plasma protein disulfide isomerase reductase activity were also significantly inhibited (P = .003 and P = .02, respectively). Short-term fixed-dose IQ in patients with SCD was safe with no off-target bleeding and was associated with changes from the baseline in the appropriate direction for several biomarkers of thromboinflammation. The trial was registered at www.clinicaltrials.gov as #NCT04514510.

Macrodomain Mac1 of SARS-CoV-2 Nonstructural Protein 3 Hydrolyzes Diverse ADP-ribosylated Substrates.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for a global pandemic that resulted in more than 6-million deaths worldwide. The virus encodes several non-structural proteins (Nsps) that contain elements capable of disrupting cellular processes. Among these Nsp proteins, Nsp3 contains macrodomains, e.g., Mac1, Mac2, Mac3, with potential effects on host cells. Mac1 has been shown to increase SARS-CoV-2 virulence and disrupt ADP-ribosylation pathways in mammalian cells. ADP-ribosylation results from the transfer of the ADP-ribose moiety of NAD + to various acceptors, e.g., proteins, DNA, RNA, contributing on a cell's biological processes. ADP-ribosylation is the mechanism of action of bacterial toxins, e.g., Pseudomonas toxins, diphtheria toxin that disrupt protein biosynthetic and signaling pathways. On the other hand, some viral macrodomains cleavage ADP-ribose-acceptor bond, generating free ADP-ribose. By this reaction, the macrodomain-containing proteins interfere ADP-ribose homeostasis in host cells. Here, we examined potential hydrolytic activities of SARS-CoV-2 Mac1, 2, and 3 on substrates containing ADP-ribose. Mac1 cleaved α-NAD + , but not ß-NAD + , consistent with stereospecificity at the C-1" bond. In contrast to ARH1 and ARH3, Mac1 did not require Mg 2+ for optimal activity. Mac1 also hydrolyzed O -acetyl-ADP-ribose and ADP-ribose-1"-phosphat, but not Mac2 and Mac3. However, Mac1 did not cleave α-ADP-ribose-(arginine) and ADP-ribose-(serine)-histone H3 peptide, suggesting that Mac1 hydrolyzes ADP-ribose attached to O- and N-linked functional groups, with specificity at the catalytic site in the ADP-ribose moiety. We conclude that SARS-CoV-2 Mac1 may exert anti-viral activity by reversing host-mediated ADP-ribosylation. New insights on Nsp3 activities may shed light on potential SARS-CoV-2 therapeutic targets. IMPORTANCE: SARS-CoV-2, the virus responsible for COVID-19, encodes 3 macrodomain-containing proteins, e.g., Mac1, Mac2, Mac3, within non-structural proteins 3 (Nsp3). Mac1 was shown previously to hydrolyze ADP-ribose-phosphate. Inactivation of Mac1 reduced viral proliferation. Here we report that Mac1, but not Mac2 and Mac3, has multiple activities, i.e., Mac1 hydrolyzed. α-NAD + and O -acetyl-ADP-ribose. However, Mac1 did not hydrolyze ß-NAD + , ADP-ribose-serine on a histone 3 peptide (aa1-21), and ADP-ribose-arginine, exhibiting substrate selectivity. These data suggest that Mac1 may have multi-function as a α-NAD + consumer for viral replication and a disruptor of host-mediated ADP-ribosylation pathways. Understanding Mac1's mechanisms of action is important to provide possible therapeutic targets for COVID-19.

Structural and immunological differences in Plasmodium falciparum sexual stage transmission-blocking vaccines comprised of Pfs25-EPA nanoparticles.

Development of a malaria vaccine that blocks transmission of different parasite stages to humans and mosquitoes is considered critical for elimination efforts. A vaccine using Pfs25, a protein on the surface of zygotes and ookinetes, is under investigation as a transmission-blocking vaccine (TBV) that would interrupt parasite passage from mosquitoes to humans. The most extensively studied Pfs25 TBVs use Pichia pastoris-produced recombinant forms of Pfs25, chemically conjugated to a recombinant carrier protein, ExoProtein A (EPA). The recombinant form of Pfs25 first used in humans was identified as Pfs25H, which contained a total of 14 heterologous amino acid residues located at the amino- and carboxyl-termini including a His6 affinity tag. A second recombinant Pfs25, identified as Pfs25M, was produced to remove the heterologous amino acid residues and conjugated to EPA (Pfs25M-EPA). Here, monomeric Pfs25M was characterized biochemically and biophysically for identity, purity, and integrity including protein structure to assess its comparability with Pfs25H. Although the biological activities of Pfs25H and Pfs25M, whether generated by monomeric forms or conjugated nanoparticles, appeared similar, fine-mapping studies with two transmission-blocking monoclonal antibodies detected structural and immunological differences. In addition, evaluation of antisera generated against conjugated Pfs25H or Pfs25M nanoparticles in nonhuman primates identified polyclonal IgG that recognized these structural differences.

Structural insights into the catalytic mechanism of Escherichia coli selenophosphate synthetase.

Selenophosphate synthetase (SPS) catalyzes the synthesis of selenophosphate, the selenium donor for the biosynthesis of selenocysteine and 2-selenouridine residues in seleno-tRNA. Selenocysteine, known as the 21st amino acid, is then incorporated into proteins during translation to form selenoproteins which serve a variety of cellular processes. SPS activity is dependent on both Mg(2+) and K(+) and uses ATP, selenide, and water to catalyze the formation of AMP, orthophosphate, and selenophosphate. In this reaction, the gamma phosphate of ATP is transferred to the selenide to form selenophosphate, while ADP is hydrolyzed to form orthophosphate and AMP. Most of what is known about the function of SPS has derived from studies investigating Escherichia coli SPS (EcSPS) as a model system. Here we report the crystal structure of the C17S mutant of SPS from E. coli (EcSPS(C17S)) in apo form (without ATP bound). EcSPS(C17S) crystallizes as a homodimer, which was further characterized by analytical ultracentrifugation experiments. The glycine-rich N-terminal region (residues 1 through 47) was found in the open conformation and was mostly ordered in both structures, with a magnesium cofactor bound at the active site of each monomer involving conserved aspartate residues. Mutating these conserved residues (D51, D68, D91, and D227) along with N87, also found at the active site, to alanine completely abolished AMP production in our activity assays, highlighting their essential role for catalysis in EcSPS. Based on the structural and biochemical analysis of EcSPS reported here and using information obtained from similar studies done with SPS orthologs from Aquifex aeolicus and humans, we propose a catalytic mechanism for EcSPS-mediated selenophosphate synthesis.

ARH Family of ADP-Ribose-Acceptor Hydrolases.

The ARH family of ADP-ribose-acceptor hydrolases consists of three 39-kDa members (ARH1-3), with similarities in amino acid sequence. ARH1 was identified based on its ability to cleave ADP-ribosyl-arginine synthesized by cholera toxin. Mammalian ADP-ribosyltransferases (ARTCs) mimicked the toxin reaction, with ARTC1 catalyzing the synthesis of ADP-ribosyl-arginine. ADP-ribosylation of arginine was stereospecific, with ß-NAD+ as substrate and, α-anomeric ADP-ribose-arginine the reaction product. ARH1 hydrolyzed α-ADP-ribose-arginine, in addition to α-NAD+ and O-acetyl-ADP-ribose. Thus, ADP-ribose attached to oxygen-containing or nitrogen-containing functional groups was a substrate. Arh1 heterozygous and knockout (KO) mice developed tumors. Arh1-KO mice showed decreased cardiac contractility and developed myocardial fibrosis. In addition to Arh1-KO mice showed increased ADP-ribosylation of tripartite motif-containing protein 72 (TRIM72), a membrane-repair protein. ARH3 cleaved ADP-ribose from ends of the poly(ADP-ribose) (PAR) chain and released the terminal ADP-ribose attached to (serine)protein. ARH3 also hydrolyzed α-NAD+ and O-acetyl-ADP-ribose. Incubation of Arh3-KO cells with H2O2 resulted in activation of poly-ADP-ribose polymerase (PARP)-1, followed by increased nuclear PAR, increased cytoplasmic PAR, leading to release of Apoptosis Inducing Factor (AIF) from mitochondria. AIF, following nuclear translocation, stimulated endonucleases, resulting in cell death by Parthanatos. Human ARH3-deficiency is autosomal recessive, rare, and characterized by neurodegeneration and early death. Arh3-KO mice developed increased brain infarction following ischemia-reperfusion injury, which was reduced by PARP inhibitors. Similarly, PARP inhibitors improved survival of Arh3-KO cells treated with H2O2. ARH2 protein did not show activity in the in vitro assays described above for ARH1 and ARH3. ARH2 has a restricted tissue distribution, with primary involvement of cardiac and skeletal muscle. Overall, the ARH family has unique functions in biological processes and different enzymatic activities.

The PPR domain of mitochondrial RNA polymerase is an exoribonuclease required for mtDNA replication in Drosophila melanogaster.

Mitochondrial DNA (mtDNA) replication and transcription are of paramount importance to cellular energy metabolism. Mitochondrial RNA polymerase is thought to be the primase for mtDNA replication. However, it is unclear how this enzyme, which normally transcribes long polycistronic RNAs, can produce short RNA oligonucleotides to initiate mtDNA replication. We show that the PPR domain of Drosophila mitochondrial RNA polymerase (PolrMT) has 3'-to-5' exoribonuclease activity, which is indispensable for PolrMT to synthesize short RNA oligonucleotides and prime DNA replication in vitro. An exoribonuclease-deficient mutant, PolrMTE423P, partially restores mitochondrial transcription but fails to support mtDNA replication when expressed in PolrMT-mutant flies, indicating that the exoribonuclease activity is necessary for mtDNA replication. In addition, overexpression of PolrMTE423P in adult flies leads to severe neuromuscular defects and a marked increase in mtDNA transcript errors, suggesting that exoribonuclease activity may contribute to the proofreading of mtDNA transcription.

Interactions of the acidic domain and SRF interacting motifs with the NKX3.1 homeodomain.

NKX3.1 is a prostate tumor suppressor belonging to the NK-2 family of homeodomain (HD) transcription factors. NK-2 family members often possess a stretch of 10-15 residues enriched in acidic amino acids, the acidic domain (AD), in the flexible, disordered region N-terminal to the HD. Interactions between the N-terminal region of NKX3.1 and its homeodomain affect protein stability and DNA binding. CD spectroscopy measuring the thermal unfolding of NKX3.1 constructs showed a 2 degrees C intramolecular stabilization of the HD by the N-terminal region containing the acidic domain (residues 85-96). CD of mixtures of various N-terminal peptides with a construct containing just the HD showed that the acidic domain and the following region, the SRF interacting (SI) motif (residues 99-105), was necessary for this stabilization. Phosphorylation of the acidic domain is known to slow proteasomal degradation of NKX3.1 in prostate cells, and NMR spectroscopy was used to measure and map the interaction of the HD with phosphorylated and nonphosphorylated forms of the AD peptide. The interaction with the phosphorylated AD peptide was considerably stronger (K(d) = 0.5 +/- 0.2 mM), resulting in large chemical shift perturbations for residues Ser150 and Arg175 in the HD, as well as a 2 degrees C increase in the HD thermal stability compared to that of the nonphosphorylated form. NKX3.1 constructs with AD phosphorylation site threonine residues (89 and 93) mutated to glutamate were 4 degrees C more stable than HD alone. Using polymer theory, effective concentrations for interactions between domains connected by flexible linkers are predicted to be in the millimolar range, and thus, the weak intramolecular interactions observed here could conceivably modulate or compete with stronger, intermolecular interactions with the NKX3.1 HD.

The ARH and Macrodomain Families of α-ADP-ribose-acceptor Hydrolases Catalyze α-NAD+ Hydrolysis.

ADP-ribosyltransferases transfer ADP-ribose from ß-NAD+ to acceptors; ADP-ribosylated acceptors are cleaved by ADP-ribosyl-acceptor hydrolases (ARHs) and proteins containing ADP-ribose-binding modules termed macrodomains. On the basis of the ADP-ribosyl-arginine hydrolase 1 (ARH1) stereospecific hydrolysis of α-ADP-ribosyl-arginine and the hypothesis that α-NAD+ is generated as a side product of ß-NAD+/ NADH metabolism, we proposed that α-NAD+ was a substrate of ARHs and macrodomain proteins. Here, we report that ARH1, ARH3, and macrodomain proteins (i.e., MacroD1, MacroD2, C6orf130 (TARG1), Af1521, hydrolyzed α-NAD+ but not ß-NAD+. ARH3 had the highest α-NADase specific activity. The ARH and macrodomain protein families, in stereospecific reactions, cleave ADP-ribose linkages to N- or O- containing functional groups; anomerization of α- to ß-forms (e.g., α-ADP-ribosyl-arginine to ß-ADP-ribose- (arginine) protein) may explain partial hydrolysis of ADP-ribosylated acceptors with an increase in content of ADP-ribosylated substrates. Af1521 and ARH3 crystal structures with bound ADP-ribose revealed similar ADP-ribose-binding pockets with the catalytic residues of the ARH and macrodomain protein families in the N-terminal helix and loop. Although the biological roles of the ARHs and macrodomain proteins differ, they share enzymatic and structural properties that may regulate metabolites such as α-NAD+.

Sirt1 carboxyl-domain is an ATP-repressible domain that is transferrable to other proteins.

Sirt1 is an NAD+-dependent protein deacetylase that regulates many physiological functions, including stress resistance, adipogenesis, cell senescence and energy production. Sirt1 can be activated by energy deprivation, but the mechanism is poorly understood. Here, we report that Sirt1 is negatively regulated by ATP, which binds to the C-terminal domain (CTD) of Sirt1. ATP suppresses Sirt1 activity by impairing the CTD's ability to bind to the deacetylase domain as well as its ability to function as the substrate recruitment site. ATP, but not NAD+, causes a conformational shift to a less compact structure. Mutations that prevent ATP binding increase Sirt1's ability to promote stress resistance and inhibit adipogenesis under high-ATP conditions. Interestingly, the CTD can be attached to other proteins, thereby converting them into energy-regulated proteins. These discoveries provide insight into how extreme energy deprivation can impact Sirt1 activity and underscore the complex nature of Sirt1 structure and regulation.