Deciphering the Cofilin Oligomers via Intermolecular Disulfide Bond Formation: A Coarse-Grained Molecular Dynamics Approach to Understanding Cofilin's Regulation on Actin Filaments.

Cofilin, a key actin-binding protein, orchestrates the dynamics of the actomyosin network through its actin-severing activity and by promoting the recycling of actin monomers. Recent experiments suggest that cofilin forms functionally distinct oligomers via thiol post-translational modifications (PTMs) that promote actin nucleation and assembly. Despite these advances, the structural conformations of cofilin oligomers that modulate actin activity remain elusive because there are combinatorial ways to oxidize thiols in cysteines to form disulfide bonds rapidly. This study employs molecular dynamics simulations to investigate human cofilin 1 as a case study for exploring cofilin dimers via disulfide bond formation. Utilizing a biasing scheme in simulations, we focus on analyzing dimer conformations conducive to disulfide bond formation. Additionally, we explore potential PTMs arising from the examined conformational ensemble. Using the free energy profiling, our simulations unveil a range of probable cofilin dimer structures not represented in current Protein Data Bank entries. These candidate dimers are characterized by their distinct population distributions and relative free energies. Of particular note is a dimer featuring an interface between cysteines 139 and 147 residues, which demonstrates stable free energy characteristics and intriguingly symmetrical geometry. In contrast, the experimentally proposed dimer structure exhibits a less stable free energy profile. We also evaluate frustration quantification based on the energy landscape theory in the protein-protein interactions at the dimer interfaces. Notably, the 39-39 dimer configuration emerges as a promising candidate for forming cofilin tetramers, as substantiated by frustration analysis. Additionally, docking simulations with actin filaments further evaluate the stability of these cofilin dimer-actin complexes. Our findings thus offer a computational framework for understanding the role of thiol PTM of cofilin proteins in regulating oligomerization, and the subsequent cofilin-mediated actin dynamics in the actomyosin network.

Protein quality control and aggregation in the endoplasmic reticulum: From basic to bedside.

Endoplasmic reticulum (ER) is the largest membrane-bound compartment in all cells and functions as a key regulator in protein biosynthesis, lipid metabolism, and calcium balance. Mammalian endoplasmic reticulum has evolved with an orchestrated protein quality control system to handle defective proteins and ensure endoplasmic reticulum homeostasis. Nevertheless, the accumulation and aggregation of misfolded proteins in the endoplasmic reticulum may occur during pathological conditions. The inability of endoplasmic reticulum quality control system to clear faulty proteins and aggregates from the endoplasmic reticulum results in the development of many human disorders. The efforts to comprehensively understand endoplasmic reticulum quality control network and protein aggregation will benefit the diagnostics and therapeutics of endoplasmic reticulum storage diseases. Herein, we overview recent advances in mammalian endoplasmic reticulum protein quality control system, describe protein phase transition model, and summarize the approaches to monitor protein aggregation. Moreover, we discuss the therapeutic applications of enhancing endoplasmic reticulum protein quality control pathways in endoplasmic reticulum storage diseases.

CRISPR-Based Therapeutic Gene Editing for Duchenne Muscular Dystrophy: Advances, Challenges and Perspectives.

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disease arising from loss-of-function mutations in the dystrophin gene and characterized by progressive muscle degeneration, respiratory insufficiency, cardiac failure, and premature death by the age of thirty. Albeit DMD is one of the most common types of fatal genetic diseases, there is no curative treatment for this devastating disorder. In recent years, gene editing via the clustered regularly interspaced short palindromic repeats (CRISPR) system has paved a new path toward correcting pathological mutations at the genetic source, thus enabling the permanent restoration of dystrophin expression and function throughout the musculature. To date, the therapeutic benefits of CRISPR genome-editing systems have been successfully demonstrated in human cells, rodents, canines, and piglets with diverse DMD mutations. Nevertheless, there remain some nonignorable challenges to be solved before the clinical application of CRISPR-based gene therapy. Herein, we provide an overview of therapeutic CRISPR genome-editing systems, summarize recent advancements in their applications in DMD contexts, and discuss several potential obstacles lying ahead of clinical translation.

Nonmuscle myosin 2 regulates cortical stability during sprouting angiogenesis.

Among the three nonmuscle myosin 2 (NM2) paralogs, NM 2A and 2B, but not 2C, are detected in endothelial cells. To study the role of NM2 in vascular formation, we ablate NM2 in endothelial cells in mice. Ablating NM2A, but not NM2B, results in reduced blood vessel coverage and increased vascular branching in the developing mouse skin and coronary vasculature. NM2B becomes essential for vascular formation when NM2A expression is limited. Mice ablated for NM2B and one allele of NM2A develop vascular abnormalities similar to those in NM2A ablated mice. Using the embryoid body angiogenic sprouting assay in collagen gels reveals that NM2A is required for persistent angiogenic sprouting by stabilizing the endothelial cell cortex, and thereby preventing excessive branching and ensuring persistent migration of the endothelial sprouts. Mechanistically, NM2 promotes focal adhesion formation and cortical protrusion retraction during angiogenic sprouting. Further studies demonstrate the critical role of Rho kinase-activated NM2 signaling in the regulation of angiogenic sprouting in vitro and in vivo.

Folic Acid Supports Pluripotency and Reprogramming by Regulating LIF/STAT3 and MAPK/ERK Signaling.

Pluripotent stem cells act as an excellent cell source for disease therapy because of its specific characteristics of self-renewal and differentiation. Pluripotent stem cells are heterogeneous, consisting of naive stem cells as well as primed epiblast stem cells. However, the strategies and mechanisms of maintaining naive pluripotent stem cells remain unclear. In this study, we found that folic acid (FA) sustained mouse embryonic stem cell (ESC) pluripotency and enabled long-term maintenance of the naive state of ESCs under CHIR99021 conditions. Mechanistic experiments showed that STAT3 pathway partially mediated the effect of FA after which the interaction between STAT3 and importin α5 was enhanced. Meanwhile, MEK/ERK signaling also acted downstream of FA in maintaining ESC pluripotency. Furthermore, FA significantly promoted mouse somatic cell reprogramming. Overall, our study identified an effective chemical condition for maintaining homogeneous ESCs and highlighted the important roles of LIF/STAT3 and MEK/ERK signaling in naive ESC pluripotency.

An HDAC2-TET1 switch at distinct chromatin regions significantly promotes the maturation of pre-iPS to iPS cells.

The maturation of induced pluripotent stem cells (iPS) is one of the limiting steps of somatic cell reprogramming, but the underlying mechanism is largely unknown. Here, we reported that knockdown of histone deacetylase 2 (HDAC2) specifically promoted the maturation of iPS cells. Further studies showed that HDAC2 knockdown significantly increased histone acetylation, facilitated TET1 binding and DNA demethylation at the promoters of iPS cell maturation-related genes during the transition of pre-iPS cells to a fully reprogrammed state. We also found that HDAC2 competed with TET1 in the binding of the RbAp46 protein at the promoters of maturation genes and knockdown of TET1 markedly prevented the activation of these genes. Collectively, our data not only demonstrated a novel intrinsic mechanism that the HDAC2-TET1 switch critically regulates iPS cell maturation, but also revealed an underlying mechanism of the interplay between histone acetylation and DNA demethylation in gene regulation.

Pwp1 is required for the differentiation potential of mouse embryonic stem cells through regulating Stat3 signaling.

Leukemia inhibitory factor/Stat3 signaling is critical for maintaining the self-renewal and differentiation potential of mouse embryonic stem cells (mESCs). However, the upstream effectors of this pathway have not been clearly defined. Here, we show that periodic tryptophan protein 1 (Pwp1), a WD-40 repeat-containing protein associated with histone H4 modification, is required for the exit of mESCs from the pluripotent state into all lineages. Knockdown (KD) of Pwp1 does not affect mESC proliferation, self-renewal, or apoptosis. However, KD of Pwp1 impairs the differentiation potential of mESCs both in vitro and in vivo. PWP1 chromatin immunoprecipitation-seq results revealed that the PWP1-occupied regions were marked with significant levels of H4K20me3. Moreover, Pwp1 binds to sites in the upstream region of Stat3. KD of Pwp1 decreases the level of H4K20me3 in the upstream region of Stat3 gene and upregulates the expression of Stat3. Furthermore, Pwp1 KD mESCs recover their differentiation potential through suppressing the expression of Stat3 or inhibiting the tyrosine phosphorylation of STAT3. Together, our results suggest that Pwp1 plays important roles in the differentiation potential of mESCs.

MiR-495 suppresses mesendoderm differentiation of mouse embryonic stem cells via the direct targeting of Dnmt3a.

Embryonic stem cells (ESCs) are promising resources for clinical therapies due to their potential to generate multiple cell types. The dynamic expression of de novo methyltransferases (Dnmt3a and Dnmt3b) is essential to ESCs; however, the regulatory mechanism of Dnmt3a or Dnmt3b expression in ESCs is still poorly understood. Here, we reported that decreased expression of microRNA-495 (miR-495) in the first 2days of embryoid body (EB) formation was required for mouse embryonic stem cell (mESC) differentiation because repressed mesoderm and endoderm lineages were detected in ectopic miR-495 expression mESCs. This effect was reversed by the function blockade of miR-495. We identified Dnmt3a as a functional target of miR-495 and showed that endogenous miR-495 repressed the expression of Dnmt3a in mESCs. Furthermore, the effect of miR-495 on mESCs could be eliminated by Dnmt3a overexpression. Moreover, miR-495 had no effect on the expression of Dnmt3b despite the findings obtained from previous studies that mainly focused on the common characteristics of the regulatory mechanisms of Dnmt3a and Dnmt3b expression. Thus, our studies not only uncovered a previously uncharacterized function of miR-495 in mESC differentiation but also generated a new idea to explore the mechanisms governing the functional difference between Dnmt3a and Dnmt3b.

Lysine acetyltransferase GCN5 potentiates the growth of non-small cell lung cancer via promotion of E2F1, cyclin D1, and cyclin E1 expression.

The lysine acetyltransferases play crucial but complex roles in cancer development. GCN5 is a lysine acetyltransferase that generally regulates gene expression, but its role in cancer development remains largely unknown. In this study, we report that GCN5 is highly expressed in non-small cell lung cancer tissues and that its expression correlates with tumor size. We found that the expression of GCN5 promotes cell growth and the G1/S phase transition in multiple lung cancer cell lines. Further study revealed that GCN5 regulates the expression of E2F1, cyclin D1, and cyclin E1. Our reporter assays indicated that the expression of GCN5 enhances the activities of the E2F1, cyclin D1, and cyclin E1 promoters. ChIP experiments suggested that GCN5 binds directly to these promoters and increases the extent of histone acetylation within these regions. Mechanistic studies suggested that GCN5 interacts with E2F1 and is recruited by E2F1 to the E2F1, cyclin D1, and cyclin E1 promoters. The function of GCN5 in lung cancer cells is abrogated by the knockdown of E2F1. Finally, we confirmed that GCN5 regulates the expression of E2F1, cyclin D1, and cyclin E1 and potentiates lung cancer cell growth in a mouse tumor model. Taken together, our results demonstrate that GCN5 specifically potentiates lung cancer growth by directly promoting the expression of E2F1, cyclin D1, and cyclin E1 in an E2F1-dependent manner. Our study identifies a specific and novel function of GCN5 in lung cancer development and suggests that the GCN5-E2F1 interaction represents a potential target for lung cancer treatment.

Critical regulation of miR-200/ZEB2 pathway in Oct4/Sox2-induced mesenchymal-to-epithelial transition and induced pluripotent stem cell generation.

Fibroblasts can be reprogrammed to induced pluripotent stem cells (iPSCs) by application of transcription factors octamer-binding protein 4 (Oct4), SRY-box containing gene 2 (Sox2), Kruppel-like factor 4 (Klf4), and c-Myelocytomatosis oncogene (c-Myc) (OSKM), but the underlying mechanisms remain unclear. Here, we report that exogenous Oct4 and Sox2 can bind at the promoter regions of mir-141/200c and mir-200a/b/429 cluster, respectively, and induce the transcription activation of miR-200 family during the OSKM-induced reprogramming. Functional suppression of miR-200s with specific inhibitors significantly represses the OSKM-caused mesenchymal-to-epithelial transition (MET, an early event in reprogramming of fibroblasts to iPSCs) and iPSC generation, whereas overexpression of miR-200s promotes the MET and iPSC generation. Mechanistic studies showed that miR-200s significantly repress the expression of zinc finger E-box binding homeobox 2 (ZEB2) through directly targeting its 3' UTR and direct inhibition of ZEB2 can mimic the effects of miR-200s on iPSC generation and MET process. Moreover, the effects of miR-200s during iPSC generation can be blocked by ZEB2 overexpression. Collectively, our findings not only reveal that members of the miR-200 family are unique mediators of the reprogramming factors Oct4/Sox2, but also demonstrate that the miR-200/ZEB2 pathway as one critical mechanism of Oct4/Sox2 to induce somatic cell reprogramming at the early stage.