Substitutions in conserved regions preceding and within the linker affect activity and flexibility of tRNase ZL, the long form of tRNase Z

The enzyme tRNase Z, a member of the metallo-β-lactamase family, endonucleolytically removes 3\u27 trailers from precursor tRNAs, preparing them for CCA addition and aminoacylation. The short form of tRNase Z, tRNase ZS, functions as a homodimer and is found in all prokaryotes and some eukaryotes. The long form, tRNase ZL, related to tRNase ZS through tandem duplication and found only in eukaryotes, possesses ~2,000-fold greater catalytic efficiency than tRNase ZS. tRNase ZL consists of related but diverged amino and carboxy domains connected by a flexible linker (also referred to as a flexible tether) and functions as a monomer. The amino domain retains the flexible arm responsible for substrate recognition and binding while the carboxy domain retains the active site. The linker region was explored by Ala-scanning through two conserved regions of D. melanogaster tRNase Z: NdomTprox, located at the carboxy end of the amino domain proximal to the linker, and Tflex, a flexible site in the linker. Periodic substitutions in a hydrophobic patch (F329 and L332) at the carboxy end of NdomTprox show 2,700 and 670-fold impairment relative to wild type, respectively, accompanied by reduced linker flexibility at N-T inside the Ndom- linker boundary. The Ala substitution for N378 in the Tflex region has 10-fold higher catalytic efficiency than wild type and locally decreased flexibility, while the Ala substitution at R382 reduces catalytic efficiency ~50-fold. These changes in pre-tRNA processing kinetics and protein flexibility are interpreted in light of a recent crystal structure for S. cerevisiae tRNase Z, suggesting transmission of local changes in hydrophobicity into the skeleton of the amino domain

The G1/S Specific Cyclin D2 Is a Regulator of HIV-1 Restriction in Non-proliferating Cells

Macrophages are a heterogeneous cell population strongly influenced by differentiation stimuli that become susceptible to HIV-1 infection after inactivation of the restriction factor SAMHD1 by cyclin-dependent kinases (CDK). Here, we have used primary human monocyte-derived macrophages differentiated through different stimuli to evaluate macrophage heterogeneity on cell activation and proliferation and susceptibility to HIV-1 infection. Stimulation of monocytes with GM-CSF induces a non-proliferating macrophage population highly restrictive to HIV-1 infection, characterized by the upregulation of the G1/S-specific cyclin D2, known to control early steps of cell cycle progression. Knockdown of cyclin D2, enhances HIV-1 replication in GM-CSF macrophages through inactivation of SAMHD1 restriction factor by phosphorylation. Co-immunoprecipitation experiments show that cyclin D2 forms a complex with CDK4 and p21, a factor known to restrict HIV-1 replication by affecting the function of the downstream cascade that leads to SAMHD1 deactivation. Thus, we demonstrate that cyclin D2 acts as regulator of cell cycle proteins affecting SAMHD1-mediated HIV-1 restriction in non-proliferating macrophage

Pharmacological modulation of SAMHD1 activity by CDK4/6 inhibitors improves anticancer therapy

Funding: This research was funded by Instituto de Salud Carlos III, Fondo de Investigación Sanitaria (FIS) PI16/00103, PI17/00624 and CP14/00016 cofinanced by FEDER. EB is a research fellow from ISCIII-FIS (CP14/00016). EGV, MP, LG are research fellows from Generalitat de Catalunya AGAUR. RB is a research fellow from PERIS, Generalitat de Catalunya (PERIS SLT002/16/00059). IE is a research fellow from la Caixa Bank Foundation (LCF/BQ/IN18/11660017) cofunded by the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No. 713673.Sterile alpha motif and histidine-aspartic acid domain-containing protein 1 (SAMHD1) is a dNTP triphosphohydrolase involved in the regulation of the intracellular dNTP pool, linked to viral restriction, cancer development and autoimmune disorders. SAMHD1 function is regulated by phosphorylation through a mechanism controlled by cyclin-dependent kinases and tightly linked to cell cycle progression. Recently, SAMHD1 has been shown to decrease the efficacy of nucleotide analogs used as chemotherapeutic drugs. Here, we demonstrate that SAMHD1 can enhance or decrease the efficacy of various classes of anticancer drug, including nucleotide analogues, but also anti-folate drugs and CDK inhibitors. Importantly, we show that selective CDK4/6 inhibitors are pharmacological activators of SAMHD1 that act by inhibiting its inactivation by phosphorylation. Combinations of a CDK4/6 inhibitor with nucleoside or folate antimetabolites potently enhanced drug efficacy, resulting in highly synergic drug combinations (CI < 0.04). Mechanistic analyses reveal that cell cycle-controlled modulation of SAMHD1 function is the central process explaining changes in anticancer drug efficacy, therefore providing functional proof of the potential of CDK4/6 inhibitors as a new class of adjuvants to boost chemotherapeutic regimens. The evaluation of SAMHD1 expression in cancer tissues allowed for the identification of cancer types that would benefit from the pharmacological modulation of SAMHD1 function. In conclusion, these results indicate that the modulation of SAMHD1 function may represent a promising strategy for the improvement of current antimetabolite-based treatment

Sex and gender in infection and immunity: addressing the bottlenecks from basic science to public health and clinical applications

Although sex and gender are recognized as major determinants of health and immunity, their role israrely considered in clinical practice and public health. We identified six bottlenecks preventing theinclusion of sex and gender considerations from basic science to clinical practice, precision medicineand public health policies. (i) A terminology-related bottleneck, linked to the definitions of sex andgender themselves, and the lack of consensus on how to evaluate gender. (ii) A data-relatedbottleneck, due to gaps in sex-disaggregated data, data on trans/non-binary people and genderidentity. (iii) A translational bottleneck, limited by animal models and the underrepresentation ofgender minorities in biomedical studies. (iv) A statistical bottleneck, with inappropriate statisticalanalyses and results interpretation. (v) An ethical bottleneck posed by the underrepresentation ofpregnant people and gender minorities in clinical studies. (vi) A structural bottleneck, as systemicbias and discriminations affect not only academic research but also decision makers. We specifyguidelines for researchers, scientific journals, funding agencies and academic institutions to addressthese bottlenecks. Following such guidelines will support the development of more efficient andequitable care strategies for all

Sex and gender in infection and immunity: addressing the bottlenecks from basic science to public health and clinical applications

Although sex and gender are recognized as major determinants of health and immunity, their role is rarely considered in clinical practice and public health. We identified six bottlenecks preventing the inclusion of sex and gender considerations from basic science to clinical practice, precision medicine and public health policies. (i) A terminology-related bottleneck, linked to the definitions of sex and gender themselves, and the lack of consensus on how to evaluate gender. (ii) A data-related bottleneck, due to gaps in sex-disaggregated data, data on trans/non-binary people and gender identity. (iii) A translational bottleneck, limited by animal models and the underrepresentation of gender minorities in biomedical studies. (iv) A statistical bottleneck, with inappropriate statistical analyses and results interpretation. (v) An ethical bottleneck posed by the underrepresentation of pregnant people and gender minorities in clinical studies. (vi) A structural bottleneck, as systemic bias and discriminations affect not only academic research but also decision makers. We specify guidelines for researchers, scientific journals, funding agencies and academic institutions to address these bottlenecks. Following such guidelines will support the development of more efficient and equitable care strategies for all

Sex and gender in infection and immunity: addressing the bottlenecks from basic science to public health and clinical applications.

Although sex and gender are recognized as major determinants of health and immunity, their role is rarely considered in clinical practice and public health. We identified six bottlenecks preventing the inclusion of sex and gender considerations from basic science to clinical practice, precision medicine and public health policies. (i) A terminology-related bottleneck, linked to the definitions of sex and gender themselves, and the lack of consensus on how to evaluate gender. (ii) A data-related bottleneck, due to gaps in sex-disaggregated data, data on trans/non-binary people and gender identity. (iii) A translational bottleneck, limited by animal models and the underrepresentation of gender minorities in biomedical studies. (iv) A statistical bottleneck, with inappropriate statistical analyses and results interpretation. (v) An ethical bottleneck posed by the underrepresentation of pregnant people and gender minorities in clinical studies. (vi) A structural bottleneck, as systemic bias and discriminations affect not only academic research but also decision makers. We specify guidelines for researchers, scientific journals, funding agencies and academic institutions to address these bottlenecks. Following such guidelines will support the development of more efficient and equitable care strategies for all

Mutations in ELAC2 associated with hypertrophic cardiomyopathy impair mitochondrial tRNA 3'-end processing.

Mutations in either the mitochondrial or nuclear genomes are associated with a diverse group of human disorders characterized by impaired mitochondrial respiration. Within this group, an increasing number of mutations have been identified in nuclear genes involved in mitochondrial RNA metabolism, including ELAC2. The ELAC2 gene codes for the mitochondrial RNase Z, responsible for endonucleolytic cleavage of the 3' ends of mitochondrial pre-tRNAs. Here, we report the identification of 16 novel ELAC2 variants in individuals presenting with mitochondrial respiratory chain deficiency, hypertrophic cardiomyopathy (HCM), and lactic acidosis. We provide evidence for the pathogenicity of the novel missense variants by studying the RNase Z activity in an in vitro system. We also modeled the residues affected by a missense mutation in solved RNase Z structures, providing insight into enzyme structure and function. Finally, we show that primary fibroblasts from the affected individuals have elevated levels of unprocessed mitochondrial RNA precursors. Our study thus broadly confirms the correlation of ELAC2 variants with severe infantile-onset forms of HCM and mitochondrial respiratory chain dysfunction. One rare missense variant associated with the occurrence of prostate cancer (p.Arg781His) impairs the mitochondrial RNase Z activity of ELAC2, suggesting a functional link between tumorigenesis and mitochondrial RNA metabolism.Includes MRC, BBSRC, Wellcome Trust and NIHR

Molecular basis of innate immune activation pathways as regulators of susceptibility and clinical evolution of viral infections

El sistema immune innat és el primer en respondre davant patògens que amenacen l’hoste. La resposta immune innata es caracteritza per induir una activació immune innata, iniciar la senyalització d’IFNs i activar vies inflamatòries que generen un estat antiviral capaç de limitar la propagació de patògens i induir la mort cel·lular de cèl·lules afectades. La identificació de factors de restricció del VIH, com SAMHD1, ha proporcionat coneixements que han relacionat el metabolisme dels nucleòtids, l’activació immune innata i la patogènesi del virus. Per tant, aquesta tesi avalua la relació entre factors virals i de l’hoste que poden afectar la regulació del sistema immune innat i activació de vies d’interferó on potencialment poden controlar la replicació viral i progressió de malalties associades. Gràcies a l’estudi in-vitro de la infecció del VIH, hem identificat que el virus indueix un mecanisme antiviral innat associat a la producció d’interferó de tipus I i activació de gens estimulats per interferó (ISGs). D’altra banda, el VIH indueix una aturada en el cicle cel·lular a la fase G2/M, mediada per p21, que causa un augment en els nivells de mort cel·lular en macròfags derivats de monòcits (MDMs). D’altra banda, la proteïna accessòria Vpx de VIH-2/SIV, té com a objectiu disminuir l’expressió de SAMHD1 i TASOR. Al mateix temps, Vpx inicia una cascada de senyalització d’activació immune innata caracteritzada per la inducció d’IFNs i expressió d’ISGs que poden ser claus en el disseny d’estratègies dirigides a la reactivació latent del VIH. El descobriment que mutacions en SAMHD1 i TREX1 estan associades a la malaltia autoimmune del síndrome d’Aicardi-Goutières (AGS) i el fet que ambdós gens tinguin un paper en la replicació del VIH, suggereix que la avaluació de sensors d’àcid nucleic en base a AGS pot potencialment identificar factors de l’hoste que regulen la replicació viral. De fet, un cribratge de gens d’AGS en macròfags primaris ha identificat ADAR1 com un regulador negatiu de la via de senyalització RLR-MAVS que contribueix a l’activació immune innata i la producció d’IFNs. Les cèl·lules on s’ha disminuït l’expressió d’ADAR1 mostren un augment de l’expressió de sensors d’ARN com MDA5 i RIG-I, un augment en la fosforilació del factor de transcripció IRF7 i un augment en la producció d’IFNs de tipus I. L’avaluació in-vitro d’ADAR1, ha mostrat que ADAR1 té un efecte proviral davant el VIH o antiviral davant de VHC i VPH, suggerint que el rol d’ADAR1 depèn de la combinació específica entre virus i hoste. Finalment, les variacions genètiques d’ADAR1 in-vivo s’associen a mal pronòstic clínic i afecten a la resposta del tractament i a la progressió de la malaltia. En resum, aquesta tesi demostra que la disfunció en vies de detecció d’àcids nucleics intracel·lulars, que desencadenen a una forta activació immune innata i les signatures IFN, són prometedores pel disseny de noves estratègies terapèutiques que tenen com a objectiu modular la replicació viral, alterar la progressió de la malaltia i afectar el resultat final de la infecció.El sistema inmune innato es el primero en responder ante patógenos que amenazan el huésped. La respuesta inmune innata se caracteriza por inducir una activación inmune innata, iniciar la señalización de IFNs y activar vías inflamatorias que generan un estado antiviral capaz de limitar la propagación del patógeno e inducir la muerte celular de células afectadas. La identificación de factores de restricción del VIH, como SAMHD1, ha proporcionado conocimientos que han relacionado el metabolismo de los nucleótidos, la activación inmune innata y la patogénesis del virus. Por lo tanto, esta tesis quiere evaluar la relación entre factores virales y del huésped que pueden afectar a la regulación del sistema inmune innato y activación de vías de interferón donde potencialmente pueden controlar la replicación viral y progresión de enfermedades asociadas. Gracias al estudio in-vitro de la infección del VIH, hemos identificado que el virus induce un mecanismo antiviral innato asociado a la producción de interferón de tipo I y activación de genes estimulados por interferón (ISGs). Por otra parte, el VIH induce una parada en el ciclo celular en la fase G2 / M, mediada por p21, que causa un aumento en los niveles de muerte celular en macrófagos derivados de monocitos (MDMS). Por otra parte, la proteína accesoria Vpx de VIH-2/SIV, tiene como objetivo disminuir la expresión de SAMHD1 y TASOR. Al mismo tiempo, VPX inicia una cascada de señalización de activación inmune innata caracterizada por la inducción de IFNs y expresión de ISGs que pueden ser claves en el diseño de estrategias dirigidas a la reactivación latente del VIH. El descubrimiento que mutaciones en SAMHD1 y TREX1 están asociadas a la enfermedad autoinmune del síndrome de Aicardi-Goutières (AGS) y el hecho de que ambos tengan un papel en la replicación del VIH, sugiere que la evaluación de sensores de ácido nucleico en base a AGS puede potencialmente identificar factores del huésped que regulan la replicación viral. De hecho, un cribado de genes de AGS en macrófagos primarios ha identificado ADAR1 como un regulador negativo de la vía de señalización RLR-MAVS que contribuye a la activación inmune innata y la producción de IFNs. Las células donde se ha disminuido la expresión de ADAR1 muestran un aumento de la expresión de sensores de ARN como MDA5 y RIG-I, un aumento en la fosforilación del factor de transcripción IRF7 y un aumento en la producción de IFNs de tipo I. La evaluación in-vitro de ADAR1, ha mostrado que ADAR1 tiene un efecto proviral ante el VIH o antiviral ante VHC y VPH, sugiriendo que el rol de ADAR1 depende de la combinación específica entre virus y huésped. Finalmente, las variaciones genéticas de ADAR1 in-vivo se asocian a mal pronóstico clínico y afectan a la respuesta del tratamiento y la progresión de la enfermedad. En resumen, esta tesis demuestra que la disfunción en vías de detección de ácidos nucleicos intracelulares, que desencadenan una fuerte activación inmune innata y patrón de IFNs, son prometedoras para el diseño de nuevas estrategias terapéuticas que tienen como objetivo modular la replicación viral, alterar la progresión de la enfermedad y afectar el resultado final de la infección.The innate immune system is the first to respond against a pathogen invasion that threatens the host. An innate immune response is characterized by inducing innate immune activation, IFN signaling and inflammatory pathways that generate an antiviral state able to limit pathogen spread and induce cell death of damaged cells. The identification of HIV host restriction factors, such as SAMHD1, has provided insights linking nucleotide metabolism, innate immune activation and viral pathogenesis. Hence, this thesis evaluates the relationship between viral and host factors that strongly affect innate immune modulation and IFN pathways that have the potential to alter viral outcome and progression of associated diseases. Through the study of HIV infection, we have identified that HIV induces an innate antiviral mechanism associated to IFN-I production and interferon stimulated gene activation. Moreover, HIV induces a cell cycle arrest at G2/M mediated by p21 leading to elevated levels of cell death in monocyte derived macrophages. On the other hand, accessory protein Vpx from HIV-2/SIV, which targets the downregulation of SAMHD1 and TASOR, also triggers innate immune activation signatures characterized by induction of IFNs and expression of ISGs which may hold key in designing strategies targeting reactivation of latent HIV. The discovery that mutations in SAMHD1 and TREX1 are associated to autoimmune disease Aicardi-Goutières syndrome (AGS) and the fact that both genes have a role in HIV replication, suggests that evaluation of nucleic acid sensors in AGS may have potential for identifying host factors regulating viral replication. Indeed, a screening of AGS genes in primary macrophages has identified ADAR1 as a negative regulator of RIG-I like receptor RLR-MAVS signaling pathway contributing to innate immune activation and IFN production. ADAR1 downregulated cells show increase expression of RNA sensors, MDA5 and RIG-I, enhanced phosphorylation of transcriptional factor IRF7 and increase type I IFN production. ADAR1 in-vitro evaluation have shown a proviral role in HIV, but an antiviral role in HCV and HPV, suggesting ADAR1 role depends on the specific virus host combination. Finally, ADAR1 in-vivo genetic variations are associated to poor clinical outcomes and affect treatment response and disease progression. In summary, this thesis demonstrates that dysfunction of intracellular nucleic acid sensing pathways that trigger strong innate immune activation and IFN signatures hold promising in the design of novel therapeutic strategies aiming to modulate viral replication and disease progression and infection outcomes.Universitat Autònoma de Barcelona. Programa de Doctorat en Immunologia Avançad

Substitutions in conserved regions preceding and within the linker affect activity and flexibility of tRNase ZL, the long form of tRNase Z.

The enzyme tRNase Z, a member of the metallo-β-lactamase family, endonucleolytically removes 3' trailers from precursor tRNAs, preparing them for CCA addition and aminoacylation. The short form of tRNase Z, tRNase ZS, functions as a homodimer and is found in all prokaryotes and some eukaryotes. The long form, tRNase ZL, related to tRNase ZS through tandem duplication and found only in eukaryotes, possesses ~2,000-fold greater catalytic efficiency than tRNase ZS. tRNase ZL consists of related but diverged amino and carboxy domains connected by a flexible linker (also referred to as a flexible tether) and functions as a monomer. The amino domain retains the flexible arm responsible for substrate recognition and binding while the carboxy domain retains the active site. The linker region was explored by Ala-scanning through two conserved regions of D. melanogaster tRNase Z: NdomTprox, located at the carboxy end of the amino domain proximal to the linker, and Tflex, a flexible site in the linker. Periodic substitutions in a hydrophobic patch (F329 and L332) at the carboxy end of NdomTprox show 2,700 and 670-fold impairment relative to wild type, respectively, accompanied by reduced linker flexibility at N-T inside the Ndom- linker boundary. The Ala substitution for N378 in the Tflex region has 10-fold higher catalytic efficiency than wild type and locally decreased flexibility, while the Ala substitution at R382 reduces catalytic efficiency ~50-fold. These changes in pre-tRNA processing kinetics and protein flexibility are interpreted in light of a recent crystal structure for S. cerevisiae tRNase Z, suggesting transmission of local changes in hydrophobicity into the skeleton of the amino domain

Linker interactions with two skeletal β-twisted sheets in the amino domain of tRNase Z^L.

<p>As illustrated using the crystal structure of <i>S</i>. <i>cerevisiae</i> Trz1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186277#pone.0186277.ref011" target="_blank">11</a>], short β strands in the flexible linker are incorporated by polar backbone contacts into the two β twisted sheets which provide the structural core of the amino domain tRNase Z^L. (A) Overview of the <i>S</i>. <i>cerevisiae</i> Trz1 structure (PDB 5MTZ) with the two β twisted sheets in the amino domain highlighted. (B) Isolated view of the β twisted sheet (β7-β13) rotated for optimal viewing of the β strands. The flexible arm is extruded from the body of tRNase Z between β9 (ascending) and β10 (descending). In the linker, residue H₃₉₂ in β13 (cyan) forms polar backbone contacts (dashed lines) with H₃₁₅ and I₃₁₇ in β12, the neighboring parallel strand. Hydrophobic interactions between bulky hydrophobic residues in α8 of N_domT_prox and β13 of T_flex, shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186277#pone.0186277.g009" target="_blank">Fig 9</a>, are also presented here. (C) View of the second β twisted sheet (β14-15-1-6), showing antiparallel polar backbone contacts between β14, β15, and β1 (dashed lines). N₄₁₅ in β15 forms backbone polar contacts with T₄₀₁ in β14. Two residues in β14, V₄₀₀ and F₄₀₂, form backbone polar contacts with F₄ and F₂ in β1, respectively.</p