A C-Degron Structure-Based Approach for the Development of Ligands Targeting the E3 Ligase TRIM7.

TRIM7 is a ubiquitin E3 ligase with key regulatory functions, mediating viral infection, tumor biology, innate immunity, and cellular processes, such as autophagy and ferroptosis. It contains a PRYSPRY domain that specifically recognizes degron sequences containing C-terminal glutamine. Ligands that bind to the TRIM7 PRYSPRY domain may have applications in the treatment of viral infections, as modulators of inflammation, and in the design of a new class of PROTACs (PROteolysis TArgeting Chimeras) that mediate the selective degradation of therapeutically relevant proteins (POIs). Here, we developed an assay toolbox for the comprehensive evaluation of TRIM7 ligands. Using TRIM7 degron sequences together with a structure-based design, we developed the first series of peptidomimetic ligands with low micromolar affinity. The terminal carboxylate moiety was required for ligand activity but prevented cell penetration. A prodrug strategy using an ethyl ester resulted in enhanced permeability, which was evaluated using confocal imaging.

Crystal structure and mutational analysis of the human TRIM7 B30.2 domain provide insights into the molecular basis of its binding to glycogenin-1.

Tripartite motif (TRIM)7 is an E3 ubiquitin ligase that was first identified through its interaction with glycogenin-1 (GN1), the autoglucosyltransferase that initiates glycogen biosynthesis. A growing body of evidence indicates that TRIM7 plays an important role in cancer development, viral pathogenesis, and atherosclerosis and, thus, represents a potential therapeutic target. TRIM family proteins share a multidomain architecture with a conserved N-terminal TRIM and a variable C-terminal domain. Human TRIM7 contains the canonical TRIM motif and a B30.2 domain at the C terminus. To contribute to the understanding of the mechanism of action of TRIM7, we solved the X-ray crystal structure of its B30.2 domain (TRIM7B30.2) in two crystal forms at resolutions of 1.6 Å and 1.8 Å. TRIM7B30.2 exhibits the typical B30.2 domain fold, consisting of two antiparallel ß-sheets of seven and six strands, arranged as a distorted ß-sandwich. Furthermore, two long loops partially cover the concave face of the ß-sandwich defined by the ß-sheet of six strands, thus forming a positively charged cavity. We used sequence conservation and mutational analyses to provide evidence of a putative binding interface for GN1. These studies showed that Leu423, Ser499, and Cys501 of TRIM7B30.2 and the C-terminal 33 amino acids of GN1 are critical for this binding interaction. Molecular dynamics simulations also revealed that hydrogen bond and hydrophobic interactions play a major role in the stability of a modeled TRIM7B30.2-GN1 C-terminal peptide complex. These data provide useful information that could be used to target this interaction for the development of potential therapeutic agents.

Dynamics of the Gene Regulatory Network of HIV-1 and the Role of Viral Non-coding RNAs on Latency Reversion.

The use of latency reversing agents (LRAs) is currently a promising approach to eliminate latent reservoirs of HIV-1. However, this strategy has not been successful in vivo. It has been proposed that cellular post-transcriptional mechanisms are implicated in the underperformance of LRAs, but it is not clear whether proviral regulatory elements like viral non-coding RNAs (vncRNAs) are also implicated. In order to visualize the complexity of the HIV-1 gene expression, we used experimental data to construct a gene regulatory network (GRN) of latent proviruses in resting CD4+ T cells. We then analyzed the dynamics of this GRN using Boolean and continuous mathematical models. Our simulations predict that vncRNAs are able to counteract the activity of LRAs, which may explain the failure of these compounds to reactivate latent reservoirs of HIV-1. Moreover, our results also predict that using inhibitors of histone methyltransferases, such as chaetocin, together with releasers of the positive transcription elongation factor (P-TEFb), like JQ1, may increase proviral reactivation despite self-repressive effects of vncRNAs.

Multidimensional study of orofacial chronic neuropathic pain: an experimental study in rats

Orofacial neuropathic chronic pain (NCP) is frequently attributed to lesions caused by orofacial surgeries and dental treatments. there are many experimental models available to study orofacial NCP, however, many are extremely painful for the animal due to the amplitude of the innervated region. a previously proposed mental nerve constriction model, mNC, was used in this project. forty wistar rats were randomly divided into two groups: one group included rats with mNC (n=20), and another rats with sham lesions (n=20). through the use of the fixed ratio program and the progressive program, a decrease of motivation for a sweet substance, caused by the lesion, was evaluated. the possibility of alterations in cognitive learning and adaptation abilities was also assessed using the go/no-go behavioral task. the mNC group showed low induced and spontaneously evoked pain responses, as well as a decrease in the motivation for sucrose, a sign of anhedonia. this decrease does not depend on taste processing. finally, although no alterations in the learning-memory process were observed, the mNC group did show alterations when adapting to a new rule.

Enhancement by GOSPEL protein of GAPDH aggregation induced by nitric oxide donor and its inhibition by NAD(.).

Glyceraldehyde-3-phosphate dehydrogenase's (GAPDH's) competitor of Siah Protein Enhances Life (GOSPEL) is the protein that competes with Siah1 for binding to GAPDH under NO-induced stress conditions preventing Siah1-bound GAPDH nuclear translocation and subsequent apoptosis. Under these conditions, GAPDH may also form amyloid-like aggregates proposed to be involved in cell death. Here, we report the in vitro enhancement by GOSPEL of NO-induced GAPDH aggregation resulting in the formation GOSPEL-GAPDH co-aggregates with some amyloid-like properties. Our findings suggest a new function for GOSPEL, contrasting with its helpful role against the apoptotic nuclear translocation of GAPDH. NAD(+) inhibited both GAPDH aggregation and co-aggregation with GOSPEL, a hitherto undescribed effect of the coenzyme against the consequences of oxidative stress.

Criticality is an emergent property of genetic networks that exhibit evolvability.

Accumulating experimental evidence suggests that the gene regulatory networks of living organisms operate in the critical phase, namely, at the transition between ordered and chaotic dynamics. Such critical dynamics of the network permits the coexistence of robustness and flexibility which are necessary to ensure homeostatic stability (of a given phenotype) while allowing for switching between multiple phenotypes (network states) as occurs in development and in response to environmental change. However, the mechanisms through which genetic networks evolve such critical behavior have remained elusive. Here we present an evolutionary model in which criticality naturally emerges from the need to balance between the two essential components of evolvability: phenotype conservation and phenotype innovation under mutations. We simulated the Darwinian evolution of random Boolean networks that mutate gene regulatory interactions and grow by gene duplication. The mutating networks were subjected to selection for networks that both (i) preserve all the already acquired phenotypes (dynamical attractor states) and (ii) generate new ones. Our results show that this interplay between extending the phenotypic landscape (innovation) while conserving the existing phenotypes (conservation) suffices to cause the evolution of all the networks in a population towards criticality. Furthermore, the networks produced by this evolutionary process exhibit structures with hubs (global regulators) similar to the observed topology of real gene regulatory networks. Thus, dynamical criticality and certain elementary topological properties of gene regulatory networks can emerge as a byproduct of the evolvability of the phenotypic landscape.

Intraluminal tracheal occlusion using a modified 8-mm Z-stent in a sheep model of left-sided congenital diaphragmatic hernia.

OBJECTIVE: To evaluate pulmonary growth and development after fetoscopic intraluminal tracheal occlusion (FITO) using a modified 8-mm Z-stent in an ovine model of congenital left-sided diaphragmatic hernia (CDH). METHODS: Thirty-three time-dated ewes were studied: Group I: healthy controls; Group II: CDH controls (untreated); Group III: CDH treated with FITO. CDH was created in Groups II and III at 70-80 days' gestation. FITO was performed at 100-110 days. Left lung histological, morphometric, immunohistochemical and biochemical studies were conducted after delivery and euthanasia at 138 days. RESULTS: Fifteen (45%) animals (Group I: 3; Group II: 5; Group III: 7) were available for analysis. The left lung parenchymal volume to fetal weight ratios were similar between Groups I and III (p = 0.24), and higher than Group II (p < 0.05III (79 versus 75%, p = 0.26), compared to 41% in Group II (p < 0.05). Pulmonary hypoplasia occurred in 1/7 (16%) in the FITO group, compared to 100% in Group II and 0% in Group I (p = .003). DNA and protein were significantly increased in Group III (p < 0.001). The concentration of type II pneumocytes was similar between healthy controls and the FITO group, and was paradoxically increased in untreated hernia fetuses. There was no histological evidence of tracheal injury. CONCLUSION: FITO with a modified 8-mm Z-stent is associated with lung growth and maturation similar to controls without obvious deleterious effects. A phase I clinical trial of FITO with the modified 8-mm Z-stent in severe CDH patients seems warranted.