Sub-cellular biochemistry最新文献

A multifaceted biological process of ageing culminates in the gradual decline of tissue and organ functions, escalating vulnerability to age-related diseases. Stem cell therapies, standing at the frontier of regenerative medicine, hold the potential to mitigate the challenges induced by ageing. By harnessing the unique regenerative capabilities of stem cells, these therapies aim to renew and heal ageing or damaged cells and tissues, thereby bolstering their function. In this chapter, we explore the potential of stem cell-based interventions against age-related degeneration, emphasising their underlying mechanisms, challenges, and future possibilities. As elucidated by the Buck Institute for Research on Aging, ageing is characterised by an accrual of macromolecular damage, genomic instability, and loss of heterochromatin (Campisi et al. Nature 571:183-192, 2019). These aspects culminate in stem cell fatigue and a dwindling tissue regenerative capacity. However, with the advent of stem cell therapy and regenerative medicine, we now hold the tools to reverse these age-induced changes by rejuvenating stem cells, the keystones of tissue regeneration, and fostering their proliferation and differentiation.

The ageing process involves a gradual decline of chromosome integrity throughout an organism's lifespan. Telomeres are protective DNA-protein complexes that cap the ends of linear chromosomes in eukaryotic organisms. Telomeric DNA consists of long stretches of short "TTAGGG" repeats that are conserved across most eukaryotes including humans. Telomeres shorten progressively with each round of DNA replication due to the inability of conventional DNA polymerase to completely replicate the chromosome ends, known as the "end-replication problem". The telomerase enzyme counteracts the telomeric DNA loss by de novo addition of telomeric repeats onto chromosomal ends. Germline and stem cells maintain significant levels of telomerase activity to maintain telomere length and can divide almost indefinitely. However, the differentiation of stem cells accompanies the inactivation of telomerase gene expression, resulting in the progressive shortening of telomeres in somatic cells over successive divisions. Critically short telomeres elicit and sustain a persistent DNA damage response leading to permanent growth arrest of cells known as cellular senescence, a hallmark of cellular ageing. The accumulation of senescent cells in tissues and organs contributes to organismal ageing. Thus, the prevention of telomere shortening is a promising means to delay or even reverse cellular ageing. In this chapter, we summarize potential anti-ageing interventions that mitigate telomere shortening through increasing telomerase level or activity and discuss these strategies' risks, benefits, and future outlooks.

Virus particles (VPs) are naturally evolved nanomachines. Their outstanding molecular structures, physical and chemical properties, and biological activities make them potentially useful for many biomedical or technological applications. Natural VPs such as virions or capsids must, however, be modified by genetic and/or chemical engineering in order to become adequate for many specific uses. We present first a general overview of the methods used for obtaining virions and viral capsids, and of genetic and chemical engineering approaches to suitably modify VPs. In the second part of the chapter, we present an updated overview on current or developing applications of engineered VPs as tools, materials, reagents, or nanodevices in biomedicine, biotechnology, or nanotechnology.

With ageing comes some of life's best and worst moments. Those lucky enough to live out into the seventh, eighth, and nineth decades and perhaps beyond have more opportunities to experience the wonders and joys of the world. As the world's population shifts towards more and more of these individuals, this is something to be celebrated. However, it is not without negative consequences. Advanced age also ushers in health decline and the burden of non-communicable diseases such as cancer, heart disease, stroke, and organ function decay. Thus, alleviating or at least dampening the severity of ageing as a whole, as well as these individual age-related disorders will enable the improvement in lifespan and healthspan. In the following chapter, we delve into hypothesised causes of ageing and experimental interventions that can be taken to slow their progression. We also highlight cellular and subcellular mechanisms of ageing with a focus on protein thiol oxidation and posttranslational modifications that impact cellular homeostasis and the advent and progression of ageing-related cancers. By having a better understanding of the mechanisms of ageing, we can hopefully develop effective, safe, and efficient therapeutic modalities that can be used prophylactically and/or concurrent to the onset of ageing.

The global emergence of multidrug resistance (MDR) in gram-negative bacteria has become a matter of worldwide concern. MDR in these pathogens is closely linked to the overexpression of certain efflux pumps, particularly the resistance-nodulation-cell division (RND) efflux pumps. Inhibition of these pumps presents an attractive and promising strategy to combat antibiotic resistance, as the efflux pump inhibitors can effectively restore the potency of existing antibiotics. AcrAB-TolC is one well-studied RND efflux pump, which transports a variety of substrates, therefore providing resistance to a broad spectrum of antibiotics. To develop effective pump inhibitors, a comprehensive understanding of the structural aspect of the AcrAB-TolC efflux pump is imperative. Previous studies on this pump's structure have been limited to individual components or in vitro determination of fully assembled pumps. Recent advancements in cellular cryo-electron tomography (cryo-ET) have provided novel insights into this pump's assembly and functional mechanism within its native cell membrane environment. Here, we present a summary of the structural data regarding the AcrAB-TolC efflux pump, shedding light on its assembly pathway and operational mechanism.

Valosin-containing protein (VCP), also known as p97, is an evolutionarily conserved AAA+ ATPase essential for cellular homeostasis. Cooperating with different sets of cofactors, VCP is involved in multiple cellular processes through either the ubiquitin-proteasome system (UPS) or the autophagy/lysosomal route. Pathogenic mutations frequently found at the interface between the NTD domain and D1 ATPase domain have been shown to cause malfunction of VCP, leading to degenerative disorders including the inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD), amyotrophic lateral sclerosis (ALS), and cancers. Therefore, VCP has been considered as a potential therapeutic target for neurodegeneration and cancer. Most of previous studies found VCP predominantly exists and functions as a hexamer, which unfolds and extracts ubiquitinated substrates from protein complexes for degradation. However, recent studies have characterized a new VCP dodecameric state and revealed a controlling mechanism of VCP oligomeric states mediated by the D2 domain nucleotide occupancy. Here, we summarize our recent knowledge on VCP oligomerization, regulation, and potential implications of VCP in cellular function and pathogenic progression.

Atomic force microscopy (AFM) makes it possible to obtain images at nanometric resolution, and to accomplish the manipulation and physical characterization of specimens, including the determination of their mechanical and electrostatic properties. AFM has an ample range of applications, from materials science to biology. The specimen, supported on a solid surface, can be imaged and manipulated while working in air, ultra-high vacuum or, most importantly for virus studies, in liquid. The adaptability of AFM is also favored by the large variety of specimens of very different sizes that it can deal with, such as atoms, molecules, and molecular complexes including viruses and cells. AFM allows, in addition, the possibility to observe dynamics in real time. Indeed, AFM facilitates single molecule experiments enabling not only to see but also to touch the material under study (i.e., mechanical manipulations) and constitutes a fundamental source of information for materials characterization. In particular, the study of the mechanical properties of viruses and other biomolecular aggregates at the nanoscale is providing humongous information This helps to elaborate mechano-chemical structure/function models of complex protein aggregates, expanding and complementing the information obtained by other techniques.

Understanding the dynamic processes involving virus structural components within host cells is crucial for comprehending viral infection, as viruses rely entirely on host cells for replication. Viral infection involves various intracellular stages, including cell entry, genome uncoating, replication, transcription and translation, assembly of new virus particles in a complex morphogenetic process, and the release of new virions from the host cell. These events are dynamic and scarce and can be obscured by other cellular processes, necessitating novel approaches for their in situ characterization. Among these methods, correlative microscopy integrates the labeling, localization, and functional characterization of events of interest through visible light microscopy, complemented by the structural insights provided by high-resolution imaging techniques. This correlative approach enables a comprehensive exploration of subcellular events within the cellular context, including those related to viral morphogenesis. This chapter provides an introduction to correlative three-dimensional imaging methods, specifically designed to study viral morphogenesis and other intracellular stages of the viral cycle under conditions closely resembling their native environment. The integration of whole-cell imaging and high-resolution structural biology techniques is emphasized as essential for unraveling the mechanisms by which viruses generate and disseminate their progeny.

Nuclear magnetic resonance (NMR) is a spectroscopic technique based on the absorption of radiofrequency radiation by atomic nuclei in the presence of an external magnetic field. NMR has followed a "bottom-up" approach to solve the structures of isolated domains of viral proteins, including capsid protein subunits, or to provide information about other macromolecular partners with which such proteins interact. NMR has been instrumental in describing conformational changes in viral proteins and nucleic acids, showing the presence of dynamic equilibria which are thought to be important at different stages of the virus life cycle. In this sense, NMR is also the only technique currently available to describe, in atomic detail, the conformational preferences of intrinsically disordered viral proteins. Furthermore, NMR can provide insights into the thermodynamic parameters governing binding reactions between different viral macromolecules. NMR has also complemented X-ray crystallography and has been combined with electron microscopy to obtain pseudo-atomic models of entire virus capsids. Finally, the joint use of liquid and solid-state NMR has allowed the identification of conformational changes in viral capsids upon insertion into host membranes.

Telomeres at the end of eukaryotic chromosomes are extended by a specialized set of enzymes and telomere-associated proteins, collectively termed here the telomere "replisome." The telomere replisome acts on a unique replicon at each chromosomal end of the telomeres, the 3' DNA overhang. This telomere replication process is distinct from the replisome mechanism deployed to duplicate the human genome. The G-rich overhang is first extended before the complementary C-strand is filled in. This overhang is extended by telomerase, a specialized ribonucleoprotein and reverse transcriptase. The overhang extension process is terminated when telomerase is displaced by CTC1-STN1-TEN1 (CST), a single-stranded DNA-binding protein complex. CST then recruits DNA polymerase α-primase to complete the telomere replication process by filling in the complementary C-strand. In this chapter, the recent structure-function insights into the human telomere C-strand fill-in machinery (DNA polymerase α-primase and CST) will be discussed.