Accurate Description of Protein–Protein Recognition and Protein Aggregation with the Implicit-Solvent-Based PACSAB Protein Model

We used the PACSAB protein model, based on the implicit solvation approach, to simulate protein–protein recognition and study the effect of helical structure on the association of aggregating peptides. After optimization, the PACSAB force field was able to reproduce correctly both the correct binding interface in ubiquitin dimerization and the conformational ensemble of the disordered protein activator for hormone and retinoid receptor (ACTR). The PACSAB model allowed us to predict the native binding of ACTR with its binding partner, reproducing the refolding upon binding mechanism of the disordered protein.

RASAL1 and ROS1 Gene Variants in Hereditary Breast Cancer

Breast cancer (BC) is the second leading cause of death in women. BC patients with family history or clinical features suggestive of inherited predisposition are candidate to genetic testing to determine whether a hereditary cancer syndrome is present. We aimed to identify new predisposing variants in familial BC patients using next-generation sequencing approaches. We performed whole exome sequencing (WES) in first-degree cousin pairs affected by hereditary BC negative at the BRCA1/2 (BReast CAncer gene 1/2) testing. Targeted analysis, for the genes resulting mutated via WES, was performed in additional 131 independent patients with a suspected hereditary predisposition (negative at the BRCA1/2 testing). We retrieved sequencing data for the mutated genes from WES of 197 Italian unrelated controls to perform a case-controls collapsing analysis. We found damaging variants in NPL (N-Acetylneuraminate Pyruvate Lyase), POLN (DNA Polymerase Nu), RASAL1 (RAS Protein Activator Like 1) and ROS1 (ROS Proto-Oncogene 1, Receptor Tyrosine Kinase), shared by the corresponding cousin pairs. We demonstrated that the splice site alterations identified in NPL and ROS1 (in two different pairs, respectively) impaired the formation of the correct transcripts. Target analysis in additional patients identified novel and rare damaging variants in RASAL1 and ROS1, with a significant allele frequency increase in cases. Moreover, ROS1 achieved a significantly higher proportion of variants among cases in comparison to our internal control database of Italian subjects (p = 0.0401). Our findings indicate that germline variants in ROS1 and RASAL1 might confer susceptibility to BC.

Cocaine-mediated circadian reprogramming in the striatum through dopamine D2R and PPARγ activation

Substance abuse disorders are linked to alteration of circadian rhythms, although the molecular and neuronal pathways implicated have not been fully elucidated. Addictive drugs, such as cocaine, induce a rapid increase of dopamine levels in the brain. Here, we show that acute administration of cocaine triggers reprogramming in circadian gene expression in the striatum, an area involved in psychomotor and rewarding effects of drugs. This process involves the activation of peroxisome protein activator receptor gamma (PPARγ), a nuclear receptor involved in inflammatory responses. PPARγ reprogramming is altered in mice with cell-specific ablation of the dopamine D2 receptor (D2R) in the striatal medium spiny neurons (MSNs) (iMSN-D2RKO). Administration of a specific PPARγ agonist in iMSN-D2RKO mice elicits substantial rescue of cocaine-dependent control of circadian genes. These findings have potential implications for development of strategies to treat substance abuse disorders.

Accelerated evolution of a minimal 63–amino acid dual transcription factor

Transcription factors control gene expression in all life. This raises the question of what is the smallest protein that can support such activity. In nature, Cro from bacteriophage λ is one of the smallest known repressors (66 amino acids), and activators are typically much larger (e.g., λ cI, 237 amino acids). Previous efforts to engineer a minimal activator from λ Cro resulted in no activity in vivo in cells. In this study, we show that directed evolution results in a new Cro activator-repressor that functions as efficiently as λ cI in vivo. To achieve this, we develop phagemid-assisted continuous evolution (PACEmid). We find that a peptide as small as 63 amino acids functions efficiently as an activator and/or repressor. To our knowledge, this is the smallest protein activator that enables polymerase recruitment, highlighting the capacity of transcription factors to evolve from very short peptide sequences.

Type 2 and interferon inflammation strongly regulate SARS-CoV-2 related gene expression in the airway epithelium

Coronavirus disease 2019 (COVID-19) outcomes vary from asymptomatic infection to death. This disparity may reflect different airway levels of the SARS-CoV-2 receptor, ACE2, and the spike protein activator, TMPRSS2. Here we explore the role of genetics and co-expression networks in regulating these genes in the airway, through the analysis of nasal airway transcriptome data from 695 children. We identify expression quantitative trait loci (eQTL) for both ACE2 and TMPRSS2, that vary in frequency across world populations. Importantly, we find TMPRSS2 is part of a mucus secretory network, highly upregulated by T2 inflammation through the action of interleukin-13, and that interferon response to respiratory viruses highly upregulates ACE2 expression. Finally, we define airway responses to coronavirus infections in children, finding that these infections upregulate IL6 while also stimulating a more pronounced cytotoxic immune response relative to other respiratory viruses. Our results reveal mechanisms likely influencing SARS-CoV-2 infectivity and COVID-19 clinical outcomes.

Xpo7 negatively regulates Hedgehog signaling by exporting Gli2 from the nucleus

Dynamic bidirectional transport between the nucleus and the cytoplasm is critical for the regulation of many transcription factors, whose levels inside the nucleus must be tightly controlled. Efficient shuttling across the nuclear membrane is especially crucial with regard to the Hedgehog (Hh) pathway, where the transcriptional signal depends on the fine balance between the amounts of Gli protein activator and repressor forms in the nucleus. The nuclear export machinery prevents the unchecked nuclear accumulation of Gli proteins, but the mechanistic insight into this process is limited. We show that the atypical exportin Xpo7 functions as a major nuclear export receptor that actively excludes Gli2 from the nucleus and controls the outcome of Hh signaling. We show that Xpo7 interacts with several domains of Gli2 and that this interaction is dependent on SuFu, a key negative regulator of Hh signaling. Our data pave the way for a more complete understanding of the nuclear shuttling of Gli proteins and the regulation of their transcriptional activity.