Incremental value of myocardial global longitudinal strain in predicting major adverse cardiac events among patients with hypertrophic cardiomyopathy.

OBJECTIVES: Endocardial global longitudinal strain (endo-GLS) measured with echocardiography (echo) has been demonstrated to be associated with myocardial fibrosis (MF) and is a prognostic predictor in patients with hypertrophic cardiomyopathy (HCM). Late gadolinium enhancement cardiac magnetic resonance (LGE-CMR) imaging showed that MF is primarily located in the myocardial layer of the extremely hypertrophic septal or ventricular wall. We hypothesized that GLS of the myocardial layer (myo-GLS) is more strongly correlated with the extent of LGE (%LGE) and is a more powerful prognostic factor than endo-GLS. METHODS: A total of 177 inpatients (54.0 [IQR: 43.0, 64.0] years, female 37.3%) with HCM were retrospectively included from May 2019 to April 2021. Among them, 162 patients underwent echocardiographic examination and contrast-enhanced CMR within 7 days. Myo-GLS and %LGE were blindly assessed in a core laboratory. All the patients were followed after they were discharged. RESULTS: During a mean follow-up of 33.77 [IQR 30.05, 35.40] months, 14 participants (7.91%) experienced major adverse cardiac events (MACE). The MACE (+) group showed lower absolute endo-GLS and myo-GLS than the MACE (-) group. Myo-GLS was more associated with %LGE (r = -.68, P < .001) than endo-GLS (r = -.64, P < .001). Cox multivariable analysis indicated that absolute myo-GLS was independently associated with MACE (adjusted hazard ratio = .75, P < .05). Myo-GLS was better than endo-GLS at detecting MACE (+) patients (-8.64%, AUC .939 vs. - 16.375%, AUC .898, P < .05). CONCLUSIONS: Myo-GLS is a stronger predictor of MACE than endo-GLS in patients with HCM and is highly correlated with %LGE.

Mechanistic Insights into the Protection Effect of Argonaute-RNA Complex on the HCV Genome.

While host miRNA usually plays an antiviral role, the relentless tides of viral evolution have carved out a mechanism to recruit host miRNA as a viral protector. By complementing miR-122 at the 5' end of the genome, the hepatitis C virus (HCV) gene can form a complex with Argonaute 2 (Ago2) protein to protect the 5' end of HCV RNA from exonucleolytic attacks. Experiments showed that the disruption of the stem-loop 1(SL1) structure and the 9th nucleotide (T9) of HCV site 1 RNA could enhance the affinity of the Ago2 protein to the HCV site 1 RNA (target RNA). However, the underlying mechanism of how the conformation and dynamics of the Ago2: miRNA: target RNA complex is affected by the SL1 and T9 remains unclear. To address this, we performed large-scale molecular dynamics simulations on the AGO2-miRNA complex binding with the WT target, T9-abasic target and SL1-disruption target, respectively. The results revealed that the T9 and SL1 structures could induce the departing motion of the PAZ, PIWI and N domains, propping up the mouth of the central groove which accommodates the target RNA, causing the instability of the target RNA and disrupting the Ago2 binding. The coordinated motion among the PAZ, PIWI and N domains were also weakened by the T9 and SL1 structures. Moreover, we proposed a new model wherein the Ago2 protein could adopt a more constraint conformation with the proximity and more correlated motions of the PAZ, N and PIWI domains to protect the target RNA from dissociation. These findings reveal the mechanism of the Ago2-miRNA complex's protective effect on the HCV genome at the atomic level, which will offer guidance for the design of drugs to confront the protection effect and engineering of Ago2 as a gene-regulation tool.

Mechanistic insights into the clinical Y96D mutation with acquired resistance to AMG510 in the KRASG12C.

Special oncogenic mutations in the RAS proteins lead to the aberrant activation of RAS and its downstream signaling pathways. AMG510, the first approval drug for KRAS, covalently binds to the mutated cysteine 12 of KRASG12C protein and has shown promising antitumor activity in clinical trials. Recent studies have reported that the clinically acquired Y96D mutation could severely affect the effectiveness of AMG510. However, the underlying mechanism of the drug-resistance remains unclear. To address this, we performed multiple microsecond molecular dynamics simulations on the KRASG12C-AMG510 and KRASG12C/Y96D-AMG510 complexes at the atomic level. The direct interaction between the residue 96 and AMG510 was impaired owing to the Y96D mutation. Moreover, the mutation yielded higher flexibility and more coupled motion of the switch II and α3-helix, which led to the departing motion of the switch II and α3-helix. The resulting departing motion impaired the interaction between the switch II and α3-helix and subsequently induced the opening and loosening of the AMG510 binding pocket, which further disrupted the interaction between the key residues in the pocket and AMG510 and induced an increased solvent exposure of AMG510. These findings reveal the resistance mechanism of AMG510 to KRASG12C/Y96D, which will help to offer guidance for the development of KRAS targeted drugs to overcome acquired resistance.

Mechanistic Insights into the Long-range Allosteric Regulation of KRAS Via Neurofibromatosis Type 1 (NF1) Scaffold Upon SPRED1 Loading.

Allosteric regulation is the most direct and efficient way of regulating protein function, wherein proteins transmit the perturbations at one site to another distinct functional site. Deciphering the mechanism of allosteric regulation is of vital importance for the comprehension of both physiological and pathological events in vivo as well as the rational allosteric drug design. However, it remains challenging to elucidate dominant allosteric signal transduction pathways, especially for large and multi-component protein machineries where long-range allosteric regulation exits. One of the quintessential examples having long-range allosteric regulation is the ternary complex, SPRED1-RAS-neurofibromin type 1 (NF1, a RAS GTPase-activating protein), in which SPRED1 facilitates RAS-GTP hydrolysis by interacting with NF1 at a distal, allosteric site from the RAS binding site. To address the underlying mechanism, we performed extensive Gaussian accelerated molecular dynamics simulations and Markov state model analysis of KRAS-NF1 complex in the presence and absence of SPRED1. Our findings suggested that SPRED1 loading allosterically enhanced KRAS-NF1 binding, but hindered conformational transformation of the NF1 catalytic center for RAS hydrolysis. Moreover, we unveiled the possible allosteric pathways upon SPRED1 binding through difference contact network analysis. This study not only provided an in-depth mechanistic insight into the allosteric regulation of KRAS by SPRED1, but also shed light on the investigation of long-range allosteric regulation among complex macromolecular systems.

Elucidation of the conformational dynamics and assembly of Argonaute-RNA complexes by distinct yet coordinated actions of the supplementary microRNA.

Argonaute (AGO) proteins, the core of RNA-induced silencing complex, are guided by microRNAs (miRNAs) to recognize target RNA for repression. The miRNA-target RNA recognition forms initially through pairing at the seed region while the additional supplementary pairing can enhance target recognition and compensate for seed mismatch. The extension of miRNA lengths can strengthen the target affinity when pairing both in the seed and supplementary regions. However, the mechanism underlying the effect of the supplementary pairing on the conformational dynamics and the assembly of AGO-RNA complex remains poorly understood. To address this, we performed large-scale molecular dynamics simulations of AGO-RNA complexes with different pairing patterns and miRNA lengths. The results reveal that the additional supplementary pairing can not only strengthen the interaction between miRNA and target RNA, but also induce the increased plasticity of the PAZ domain and enhance the domain connectivity among the PAZ, PIWI, N domains of the AGO protein. The strong community network between these domains tightens the mouth of the supplementary chamber of AGO protein, which prevents the escape of target RNA from the complex and shields it from solvent water attack. Importantly, the inner stronger matching pairs between the miRNA and target RNA can compensate for weaker mismatches at the edge of supplementary region. These findings provide guidance for the design of miRNA mimics and anti-miRNAs for both clinical and experimental use and open the way for further engineering of AGO proteins as a new tool in the field of gene regulation.