AI-guided pipeline for protein-protein interaction drug discovery identifies a SARS-CoV-2 inhibitor.

Protein-protein interactions (PPIs) offer great opportunities to expand the druggable proteome and therapeutically tackle various diseases, but remain challenging targets for drug discovery. Here, we provide a comprehensive pipeline that combines experimental and computational tools to identify and validate PPI targets and perform early-stage drug discovery. We have developed a machine learning approach that prioritizes interactions by analyzing quantitative data from binary PPI assays or AlphaFold-Multimer predictions. Using the quantitative assay LuTHy together with our machine learning algorithm, we identified high-confidence interactions among SARS-CoV-2 proteins for which we predicted three-dimensional structures using AlphaFold-Multimer. We employed VirtualFlow to target the contact interface of the NSP10-NSP16 SARS-CoV-2 methyltransferase complex by ultra-large virtual drug screening. Thereby, we identified a compound that binds to NSP10 and inhibits its interaction with NSP16, while also disrupting the methyltransferase activity of the complex, and SARS-CoV-2 replication. Overall, this pipeline will help to prioritize PPI targets to accelerate the discovery of early-stage drug candidates targeting protein complexes and pathways.

Polyglutamine disease proteins: Commonalities and differences in interaction profiles and pathological effects.

Currently, nine polyglutamine (polyQ) expansion diseases are known. They include spinocerebellar ataxias (SCA1, 2, 3, 6, 7, 17), spinal and bulbar muscular atrophy (SBMA), dentatorubral-pallidoluysian atrophy (DRPLA), and Huntington's disease (HD). At the root of these neurodegenerative diseases are trinucleotide repeat mutations in coding regions of different genes, which lead to the production of proteins with elongated polyQ tracts. While the causative proteins differ in structure and molecular mass, the expanded polyQ domains drive pathogenesis in all these diseases. PolyQ tracts mediate the association of proteins leading to the formation of protein complexes involved in gene expression regulation, RNA processing, membrane trafficking, and signal transduction. In this review, we discuss commonalities and differences among the nine polyQ proteins focusing on their structure and function as well as the pathological features of the respective diseases. We present insights from AlphaFold-predicted structural models and discuss the biological roles of polyQ-containing proteins. Lastly, we explore reported protein-protein interaction networks to highlight shared protein interactions and their potential relevance in disease development.

Mitochondrial fusion is required for regulation of mitochondrial DNA replication.

Mitochondrial dynamics is an essential physiological process controlling mitochondrial content mixing and mobility to ensure proper function and localization of mitochondria at intracellular sites of high-energy demand. Intriguingly, for yet unknown reasons, severe impairment of mitochondrial fusion drastically affects mtDNA copy number. To decipher the link between mitochondrial dynamics and mtDNA maintenance, we studied mouse embryonic fibroblasts (MEFs) and mouse cardiomyocytes with disruption of mitochondrial fusion. Super-resolution microscopy revealed that loss of outer mitochondrial membrane (OMM) fusion, but not inner mitochondrial membrane (IMM) fusion, leads to nucleoid clustering. Remarkably, fluorescence in situ hybridization (FISH), bromouridine labeling in MEFs and assessment of mitochondrial transcription in tissue homogenates revealed that abolished OMM fusion does not affect transcription. Furthermore, the profound mtDNA depletion in mouse hearts lacking OMM fusion is not caused by defective integrity or increased mutagenesis of mtDNA, but instead we show that mitochondrial fusion is necessary to maintain the stoichiometry of the protein components of the mtDNA replisome. OMM fusion is necessary for proliferating MEFs to recover from mtDNA depletion and for the marked increase of mtDNA copy number during postnatal heart development. Our findings thus link OMM fusion to replication and distribution of mtDNA.

TEFM regulates both transcription elongation and RNA processing in mitochondria.

Regulation of replication and expression of mitochondrial DNA (mtDNA) is essential for cellular energy conversion via oxidative phosphorylation. The mitochondrial transcription elongation factor (TEFM) has been proposed to regulate the switch between transcription termination for replication primer formation and processive, near genome-length transcription for mtDNA gene expression. Here, we report that Tefm is essential for mouse embryogenesis and that levels of promoter-distal mitochondrial transcripts are drastically reduced in conditional Tefm-knockout hearts. In contrast, the promoter-proximal transcripts are much increased in Tefm knockout mice, but they mostly terminate before the region where the switch from transcription to replication occurs, and consequently, de novo mtDNA replication is profoundly reduced. Unexpectedly, deep sequencing of RNA from Tefm knockouts revealed accumulation of unprocessed transcripts in addition to defective transcription elongation. Furthermore, a proximity-labeling (BioID) assay showed that TEFM interacts with multiple RNA processing factors. Our data demonstrate that TEFM acts as a general transcription elongation factor, necessary for both gene transcription and replication primer formation, and loss of TEFM affects RNA processing in mammalian mitochondria.

SLIRP Regulates the Rate of Mitochondrial Protein Synthesis and Protects LRPPRC from Degradation.

We have studied the in vivo role of SLIRP in regulation of mitochondrial DNA (mtDNA) gene expression and show here that it stabilizes its interacting partner protein LRPPRC by protecting it from degradation. Although SLIRP is completely dependent on LRPPRC for its stability, reduced levels of LRPPRC persist in the absence of SLIRP in vivo. Surprisingly, Slirp knockout mice are apparently healthy and only display a minor weight loss, despite a 50-70% reduction in the steady-state levels of mtDNA-encoded mRNAs. In contrast to LRPPRC, SLIRP is dispensable for polyadenylation of mtDNA-encoded mRNAs. Instead, deep RNA sequencing (RNAseq) of mitochondrial ribosomal fractions and additional molecular analyses show that SLIRP is required for proper association of mRNAs to the mitochondrial ribosome and efficient translation. Our findings thus establish distinct functions for SLIRP and LRPPRC within the LRPPRC-SLIRP complex, with a novel role for SLIRP in mitochondrial translation. Very surprisingly, our results also demonstrate that mammalian mitochondria have a great excess of transcripts under basal physiological conditions in vivo.

Bioenergetic roles of mitochondrial fusion.

Mitochondria are bioenergetic hotspots, producing the bulk of ATP by the oxidative phosphorylation process. Mitochondria are also structurally dynamic and undergo coordinated fusion and fission to maintain their function. Recent studies of the mitochondrial fusion machinery have provided new evidence in detailing their role in mitochondrial metabolism. Remarkably, mitofusin 2, in addition to its role in fusion, is important for maintaining coenzyme Q levels and may be an integral player in the mevalonate synthesis pathway. Here, we review the bioenergetic roles of mitochondrial dynamics and emphasize the importance of the in vitro growth conditions when evaluating mitochondrial respiration. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016,' edited by Prof. Paolo Bernardi.

Delineation of functional subdomains of Huntingtin protein and their interaction with HAP40.

The huntingtin (HTT) protein plays critical roles in numerous cellular pathways by functioning as a scaffold for its many interaction partners and HTT knock out is embryonic lethal. Interrogation of HTT function is complicated by the large size of this protein so we studied a suite of structure-rationalized subdomains to investigate the structure-function relationships within the HTT-HAP40 complex. Protein samples derived from the subdomain constructs were validated using biophysical methods and cryo-electron microscopy, revealing they are natively folded and can complex with validated binding partner, HAP40. Derivatized versions of these constructs enable protein-protein interaction assays in vitro, with biotin tags, and in cells, with luciferase two-hybrid assay-based tags, which we use in proof-of-principle analyses to further interrogate the HTT-HAP40 interaction. These open-source biochemical tools enable studies of fundamental HTT biochemistry and biology, will aid the discovery of macromolecular or small-molecule binding partners and help map interaction sites across this large protein.

Schizophrenia risk candidate protein ZNF804A interacts with STAT2 and influences interferon-mediated gene transcription in mammalian cells.

Previously evidence was presented that the single-nucleotide polymorphism rs1344706 located in an intronic region of the ZNF804A gene is associated with reduced transcript levels in fetal brains. This genetic variation in the gene encoding the zinc-finger protein ZNF804A is associated with schizophrenia (SZ) and bipolar disorder. Currently, the molecular and cellular function of ZNF804A is unclear. Here, we generated a high-confidence protein-protein interaction (PPI) network for ZNF804A using a combination of yeast two-hybrid and bioluminescence-based PPI detection assays, directly linking 15 proteins to the disease-associated target protein. Among the top hits was the signal transducer and activator of transcription 2 (STAT2), an interferon-regulated transcription factor. Detailed mechanistic studies revealed that STAT2 binds to the unstructured N terminus of ZNF804A. This interaction is mediated by multiple short amino acid motifs in ZNF804A but not by the conserved C2H2 zinc-finger domain, which is also located at the N terminus. Interestingly, investigations in HEK293 cells demonstrated that ZNF804A and STAT2 both co-translocate from the cytoplasm into the nucleus upon interferon (IFN) treatment. Furthermore, a concentration-dependent effect of ZNF804A overproduction on STAT2-mediated gene expression was observed using a luciferase reporter, which is under the control of an IFN-stimulated response element (ISRE). Together these results indicate the formation of ZNF804A:STAT2 protein complex and its translocation from the cytoplasm into the nucleus upon IFN stimulation, suggesting that it may function as a signal transducer that activates IFN-mediated gene expression programs.

MitoRibo-Tag Mice Provide a Tool for In Vivo Studies of Mitoribosome Composition.

Mitochondria harbor specialized ribosomes (mitoribosomes) necessary for the synthesis of key membrane proteins of the oxidative phosphorylation (OXPHOS) machinery located in the mitochondrial inner membrane. To date, no animal model exists to study mitoribosome composition and mitochondrial translation coordination in mammals in vivo. Here, we create MitoRibo-Tag mice as a tool enabling affinity purification and proteomics analyses of mitoribosomes and their interactome in different tissues. We also define the composition of an assembly intermediate formed in the absence of MTERF4, necessary for a late step in mitoribosomal biogenesis. We identify the orphan protein PUSL1, which interacts with a large subunit assembly intermediate, and demonstrate that it is an inner-membrane-associated mitochondrial matrix protein required for efficient mitochondrial translation. This work establishes MitoRibo-Tag mice as a powerful tool to study mitoribosomes in vivo, enabling future studies on the mitoribosome interactome under different physiological states, as well as in disease and aging.

Análise toxicológica da fase I do exsudato extraído do fruto de Mangifera indica L. / Análisis toxicológico de la fase I del exudado del fruto de Mangifera indica L. / Toxilogical analysis phase I of the exudates from Mangifera indica L. fruit

Introdução: as plantas representam ainda uma importante fonte de materia prima para a confecção de medicamentos. Mas, apesar do grande número de espécies vegetais no Brasil, apenas uma pequena parcela tem sido estudada adequadamente. A Mangifera indica, hoje integrante da flora brasileira, tem em suas diferentes partes, substâncias ativas importantes, embora não haja na literatura informações sobre os efeitos dos componentes existentes no exsudato do pedúnculo do fruto, principalmente das suas fases de separação espontânea. Objetivo: avaliação das respostas espontâneas em Rattus novergicus albinus, adultos, sob o efeito da fração hidrofílica do exsudato do pedúnculo do fruto de Mangifera indica. Métodos: a obtenção do exsudato do pedúnculo dos frutos de Mangifera indica de espécimes obtidos do Campus da Universidade Federal Rural de Pernambuco foi providenciada. Após um período de decantação de seis dias, espontaneamente, foram visualizadas quatro fases distintas. Da fase um, após os devidos processos de separação e pesagens, alíquotas de 0,5 mL foram administradas, intraperitonealmente, em cada animal, sendo a atividade espontânea observada por 100 min. Resultados: em relação ao grupo controle, foram observadas respostas comportamentais sugestivas, tais como tremores mandibulares, variações importantes nos ritmos de ventilação pulmonar, micção, cauda apoiada à superfície, catatonia, locomoção retrógrada, movimentos mandibulares, contração do focinho, inércia a estímulos para marcha, dificuldade de locomoção, hipnose, contração abdominal, piloereção na cauda e analgesia. Estas respostas apresentaram intervalos temporais de ocorrência característicos. Não ocorreram óbitos. Conclusões: as respostas observadas sugerem que importantes mecanismos fisiológicos sejam oportunamente acionados quando os animais estão sob os efeitos dos constituintes químicos da fase 1, hidrofílica, do exsudato de Mangifera indica; podendo, a partir de estudos posteriores, proporcionar a obtenção de drogas importantes para a medicina.

Introducción: las plantas representan aún una importante fuente de materia prima para la confección de medicamentos. Pero, a pesar del gran número de especies vegetales de Brasil, apenas una pequeña parte ha sido estudiada adecuadamente. Mangifera indica, integrante en la actualidad de la flora brasileña, tiene en sus diferentes partes sustancias activas importantes, sin embargo, no se encuentra en la literatura información sobre los efectos de los componentes existentes en el exudado del pedúnculo del fruto, principalmente en sus fases de separación espontánea. Objetivo: evaluar las respuestas espontáneas en Rattus novergicus albinus adultos, bajo el efecto de la fracción hidrofílica del exudado del pedúnculo del fruto de Mangifera indica. Métodos: se logró la obtención del exudado del pedúnculo de los frutos de Mangifera indica a partir de especímenes obtenidos del Campus de la Universidad Federal Rural de Pernambuco. Tras un período de decantación de 6 d, espontáneamente, se pudieron visualizar 4 fases diferentes. De la fase uno, tras los debidos procesos de separación de pesaje, se administraron alícuotas de 0,5 mL, intraperitonealmente, en cada animal y se observó la actividad espontánea durante 100 min. Resultados: comparado con el grupo de control, se observaron respuestas conductuales sugestivas, como temblores mandibulares, variaciones importantes en los ritmos de ventilación pulmonar, micción, cola apoyada en la superficie, catatonia, locomoción retrógrada, movimientos mandibulares, contracción del hocico, inercia a estímulos para marchar, dificultades en la locomoción, hipnosis, contracción abdominal, piloerección de la cola y analgesia. Estas respuestas presentaron intervalos temporales de ocurrencia característicos. No se produjeron muertes. Conclusiones: las respuestas observadas sugieren que importantes mecanismos fisiológicos son oportunamente accionados cuando los animales están bajo los efectos de los constituyentes químicos de la fase 1 del exudado hidrofílico de Mangifera indica. Sobre la base de estudios posteriores pudiera lograrse la obtención de drogas importantes para la medicina.

Introduction: the plants still represent an important source of raw material for making medicines. Despite the large number of plant species in Brazil, only a small portion has been adequately studied. Mangifera indica L, a member of the Brazilian flora, has important active substances in its various parts, although there is information in the literature on the effects of existing components in the exudate of the fruit peduncle, especially the spontaneous separation of phases. Objective: evaluation of spontaneous responses in Rattus novergicus albinus, adults under the influence of the hydrophilic fraction of the exudates from the stalk of the Mangifera indica fruit. Methods: the exudate obtained from the stalk of Mangifera indica fruits specimens obtained from the Campus UFRPE was provided. After a settling period of six days, four distinct phases were spontaneously seen. In phase one, after the separation and weighing processes, 0.5 mL aliquots were administered intraperitoneally in each animal, and the spontaneous activity was observed for 100 min. Results: compared with the control group, suggestive behavioral responses were observed such as mandibular tremors, important variations in pulmonary ventilation rates, urination, tail supported at the surface, catatonia, retrograde movement, jaw movements, contraction of the muzzle, inertia to gait stimulation, difficult walking, hypnosis, abdominal contraction, piloerection on the tail, and analgesia. These responses presented characteristic time intervals for occurrence. There were no deaths. Conclusions: the responses suggested that important physiological mechanisms are appropriately triggered when the animals are under the effects of the chemical constituents included in the first phase. Based on further studies, Mangifera indica L. hydrophilic exudate could allow obtaining important medical drugs.