Intrinsically disordered proteins (IDPs) are proteins that lack rigid 3D structure. Hence, they are often misconceived to present a challenge to Anfinsen's dogma. However, IDPs exist as ensembles that sample a quasi-continuum of rapidly interconverting conformations and, as such, may represent proteins at the extreme limit of the Anfinsen postulate. IDPs play important biological roles and are key components of the cellular protein interaction network (PIN). Many IDPs can interconvert between disordered and ordered states as they bind to appropriate partners. Conformational dynamics of IDPs contribute to conformational noise in the cell. Thus, the dysregulation of IDPs contributes to increased noise and “promiscuous” interactions. This leads to PIN rewiring to output an appropriate response underscoring the critical role of IDPs in cellular decision making. Nonetheless, IDPs are not easily tractable experimentally. Furthermore, in the absence of a reference conformation, discerning the energy landscape representation of the weakly funneled IDPs in terms of reaction coordinates is challenging. To understand conformational dynamics in real time and decipher how IDPs recognize multiple binding partners with high specificity, several sophisticated knowledge-based and physics-based in silico sampling techniques have been developed. Here, using specific examples, we highlight recent advances in energy landscape visualization and molecular dynamics simulations to discern conformational dynamics and discuss how the conformational preferences of IDPs modulate their function, especially in phenotypic switching. Finally, we discuss recent progress in identifying small molecules targeting IDPs underscoring the potential therapeutic value of IDPs. Understanding structure and function of IDPs can not only provide new insight on cellular decision making but may also help to refine and extend Anfinsen's structure/function paradigm.

Intrinsically disordered proteins: Ensembles at the limits of Anfinsen's dogma

Neurodegenerative diseases are prominent causes of pain, suffering, and death worldwide. Traditional approaches modelling neurodegenerative diseases are deficient, and therefore, improved strategies that effectively recapitulate the pathophysiological conditions of neurodegenerative diseases are the need of the hour. The generation of human-induced pluripotent stem cells (iPSCs) has transformed our ability to model neurodegenerative diseases in vitro and provide an unlimited source of cells (including desired neuronal cell types) for cell replacement therapy. Recently, CRISPR/Cas9-based genome editing has also been gaining popularity because of the flexibility they provide to generate and ablate disease phenotypes. In addition, the recent advancements in CRISPR/Cas9 technology enables researchers to seamlessly target and introduce precise modifications in the genomic DNA of different human cell lines, including iPSCs. CRISPR-iPSC-based disease modelling, therefore, allows scientists to recapitulate the pathological aspects of most neurodegenerative processes and investigate the role of pathological gene variants in healthy non-patient cell lines. This review outlines how iPSCs, CRISPR/Cas9, and CRISPR-iPSC-based approaches accelerate research on neurodegenerative diseases and take us closer to a cure for neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, Amyotrophic Lateral Sclerosis, and so forth. 

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CRISPR and iPSCs: Recent Developments and Future Perspectives in Neurodegenerative Disease Modelling, Research, and Therapeutics

https://doi.org/10.1016/j.psj.2022.102057

Emergence of plasmid-mediated tigecycline, β-lactam and florfenicol resistance genes tet(X), blaOXA-347 and floR in Riemerella anatipestifer isolated in China

Octamer-binding transcription factor 4 (OCT4) is vital for early embryonic development and is a master regulator of pluripotency in embryonic stem cells. Notably, OCT4 is a key reprogramming factor to derive induced pluripotent stem cells, which have tremendous prospects in regenerative medicine. In the current study, we report heterologous expression and purification of human OCT4 in E. coli to produce pure recombinant protein under native conditions. To achieve this, the 1083 bp coding sequence of the human OCT4 gene was codon-optimized for heterologous expression in E. coli. The codon-optimized sequence was fused with fusion tags, namely a cell-penetrating peptide sequence for intracellular delivery, a nuclear localization sequence for intranuclear delivery, and a His-tag for affinity purification. Subsequently, the codon-optimized sequence and the fusion tags were cloned in the protein expression vector, pET28a(+), and transformed into E. coli strain BL21(DE3) for expression. The recombinant OCT4 protein was purified from the soluble fraction under native conditions using immobilized metal ion affinity chromatography in a facile manner, and its identity was confirmed by Western blotting and mass spectrometry. Furthermore, the secondary structure of the recombinant protein was analyzed using far ultraviolet circular dichroism spectroscopy, which confirmed that the purified fusion protein maintained a secondary structure conformation, and it predominantly composed of α-helices. Next, the recombinant OCT4 protein was applied to human cells, and was found that it was able to enter the cells and translocate to the nucleus. Furthermore, the biological activity of the transduced OCT4 protein was also demonstrated on human cells. This recombinant tool can substitute for genetic and viral forms of OCT4 to enable the derivation of integration-free pluripotent cells. It can also be used to elucidate its biological role in various cellular processes and diseases and for structural and biochemical studies.

Generation of biologically active recombinant human OCT4 protein from E. coli

Maintenance of stemness is tightly linked to cell cycle regulation through protein phosphorylation by cyclin‐dependent kinases (CDKs). However, how this process is reversed during differentiation is unknown. We report here that exit from stemness and differentiation of pluripotent cells along the neural lineage are controlled by CDC14, a CDK‐counteracting phosphatase whose function in mammals remains obscure. Lack of the two CDC14 family members, CDC14A and CDC14B, results in deficient development of the neural system in the mouse and impairs neural differentiation from embryonic stem cells (ESCs). Mechanistically, CDC14 directly dephosphorylates specific proline‐directed Ser/Thr residues of undifferentiated embryonic transcription Factor 1 (UTF1) during the exit from stemness, triggering its proteasome‐dependent degradation. Multiomic single‐cell analysis of transcription and chromatin accessibility in differentiating ESCs suggests that increased UTF1 levels in the absence of CDC14 prevent the proper firing of bivalent promoters required for differentiation. CDC14 phosphatases are dispensable for mitotic exit, suggesting that CDC14 phosphatases have evolved to control stemness rather than cell cycle exit and establish the CDK‐CDC14 axis as a critical molecular switch for linking cell cycle regulation and self‐renewal.

Mammalian CDC14 phosphatases control exit from stemness in pluripotent cells

The stem cell-specific SOX2 transcription factor is critical for early embryonic development and the maintenance of embryonic and neural stem cell identity. It is also crucial for the generation of induced pluripotent and neural stem cells, thus providing immense prospect in patient-specific therapies. Here, we report soluble expression and purification of human SOX2 protein under native conditions from a bacterial system. To generate this macromolecule, we codon-optimized the protein-coding sequence and fused it to a nuclear localization signal, a protein transduction domain, and a His-tag. This was then cloned into a protein expression vector and was expressed in Escherichia coli. Subsequently, we have screened and identified the optimal expression conditions to obtain recombinant fusion protein in a soluble form and studied its expression concerning the position of fusion tags at either terminal. Furthermore, we purified two versions of recombinant SOX2 fusion proteins to homogeneity under native conditions and demonstrated that they maintained their secondary structure. This molecular tool can substitute genetic and viral forms of SOX2 to facilitate the derivation of integration-free induced pluripotent and neural stem cells. Furthermore, it can be used in elucidating its role in stem cells, various cellular processes and diseases, and for structural and biochemical studies.

Generation of a Recombinant Stem Cell-Specific Human SOX2 Protein from Escherichia coli Under Native Conditions

The curiosity to understand the mechanisms regulating transcription in pluripotent cells resulted in identifying a unique transcription factor named Undifferentiated embryonic cell transcription factor 1 (UTF1) This proline-rich, nuclear protein is highly conserved among placental mammals with prominent expression observed in pluripotent, germ, and cancer cells In pluripotent and germ cells, its role has been implicated primarily in proper cell differentiation, whereas in cancer, it shows tissue-specific function, either as an oncogene or a tumor suppressor gene Furthermore, UTF1 is crucial for germ cell development, spermatogenesis, and maintaining male fertility in mice In addition, recent studies have demonstrated the importance of UTF1 in the generation of high quality induced Pluripotent Stem Cells (iPSCs) and as an excellent biomarker to identify bona fide iPSCs Functionally, UTF1 aids in establishing a favorable chromatin state in embryonic stem cells, reducing "transcriptional noise" and possibly functions similarly in re-establishing this state in differentiated cells upon their reprogramming to generate mature iPSCs This review highlights the multifaceted roles of UTF1 and its implication in development, spermatogenesis, stem, and cancer cells

An Insight into the Role of UTF1 in Development, Stem Cells, and Cancer

Induced pluripotent stem cells (iPSCs) derived using integration-free reprogramming approaches have improved the prospects of these cells for various biomedical applications. Typically, iPSCs are generated by introducing a specific cohort of reprogramming factors into adult somatic cells. The initial studies identified two reprogramming factor combinations [Oct4, Sox2, Klf4, and c-Myc (also known as Yamanaka factors) and Oct4, Sox2, Nanog, and Lin28 (also known as Thomson factors)] to derive iPSCs. Subsequently, various studies have identified numerous genes playing a vital role in the generation of bona fide mouse and human iPSCs in a variety of reprogramming factor combinations. Further exploring the role of these genes would provide a holistic understanding of the reprogramming process and will immensely contribute toward realizing the full potential of these cells. In this chapter, we present a comprehensive overview of the crucial auxiliary genes, and this knowledge will pave the way for establishing strategies for safe and efficient reprogramming.

S Sudhagar

Papers

Generation of a Recombinant Stem Cell-Specific Human SOX2 Protein from Escherichia coli Under Native Conditions

An Insight into the Role of UTF1 in Development, Stem Cells, and Cancer

Auxiliary pluripotency-associated genes and their contributions in the generation of induced pluripotent stem cells

Generation of biologically active recombinant human OCT4 protein from E. coli