In the final stages of apoptosis, apoptotic cells can generate a variety of membrane-bound vesicles known as apoptotic extracellular vesicles (ApoEVs). Apoptotic bodies (ApoBDs), a major subset of ApoEVs, are formed through a process termed apoptotic cell disassembly characterised by a series of tightly regulated morphological steps including plasma membrane blebbing, apoptotic membrane protrusion formation and fragmentation into ApoBDs. To better characterise the properties of ApoBDs and elucidate their function, a number of methods including differential centrifugation, filtration and fluorescence-activated cell sorting were developed to isolate ApoBDs. Furthermore, it has become increasingly clear that ApoBD formation can contribute to various biological processes such as apoptotic cell clearance and intercellular communication. Together, recent literature demonstrates that apoptotic cell disassembly and thus, ApoBD formation, is an important process downstream of apoptotic cell death. In this chapter, we discuss the current understandings of the molecular mechanisms involved in regulating apoptotic cell disassembly, techniques for ApoBD isolation, and the functional roles of ApoBDs in physiological and pathological settings.

Transcription is a highly regulated process that supplies living cells with coding and non-coding RNA molecules. Failure to properly regulate transcription is associated with human pathologies, including cancers. RNA polymerase II is the enzyme complex that synthesizes messenger RNAs that are then translated into proteins. In spite of its complexity, RNA polymerase requires a plethora of general transcription factors to be recruited to the transcription start site as part of a large transcription pre-initiation complex, and to help it gain access to the transcribed strand of the DNA. This chapter reviews the structure and function of these eukaryotic transcription pre-initiation complexes, with a particular emphasis on two of its constituents, the multisubunit complexes TFIID and TFIIH. We also compare the overall architecture of the RNA polymerase II pre-initiation complex with those of RNA polymerases I and III, involved in transcription of ribosomal RNA and non-coding RNAs such as tRNAs and snRNAs, and discuss the general, conserved features that are applicable to all eukaryotic RNA polymerasesystems.

With an increasingly ageing population that is expected to double by 2050 in the U.S., it is paramount that we further understand the neurological changes that occur during ageing. This is relevant not only in the context of "pathological" ageing, where the development of many neurodegenerative disorders is typically a feature of only the older population (and indeed, age is the primary risk factor for many conditions such as Alzheimer's disease), but also for what is considered to be "normal" or "healthy" ageing. Specifically, a significant proportion of the older population are affected by "age-related cognitive decline" (ARCD), which is both independent of dementia and has an incidence 70% higher than dementia alone. However, whilst it is reported that there are pathogenic and phenotypic overlaps between healthy and pathological ageing, it is clear that there is a need to identify the pathways and understand the mechanisms that contribute to this loss of cognitive function with normal ageing, particularly in light of the increasing life expectancy of the global population. Importantly, there is an increasing body of evidence implicating zinc homeostasis as a key player in learning and memory and also potentially ARCD. Further research will ultimately contribute to the development of targeted therapeutics that will promote successful brain ageing. In this chapter we will explore the notion of ARCD, with a perspective on potential key neurochemical pathways that can be targeted for future intervention.

The periplasm of Gram-negative bacteria contains a specialized chaperone network that facilitates the transport of unfolded membrane proteins to the outer membrane as its primary functional role. The network, involving the chaperones Skp and SurA as key players and potentially additional chaperones, is indispensable for the survival of the cell. Structural descriptions of the apo forms of these molecular chaperones were initially provided by X-ray crystallography. Subsequently, a combination of experimental biophysical methods including solution NMR spectroscopy provided a detailed understanding of full-length chaperone-client complexes . The data showed that conformational changes and dynamic re-organization of the chaperones upon client binding, as well as client dynamics on the chaperone surface are crucial for function. This chapter gives an overview of the structure-function relationship of the dynamic conformational rearrangements that regulate the functional cycles of the periplasmic molecular chaperones Skp and SurA.

p97 belongs to the functional diverse superfamily of AAA+ (ATPases Associated with diverse cellular Activities) ATPases and is characterized by an N-terminal regulatory domain and two stacked hexameric ATPase domains forming a central protein conducting channel. p97 is highly versatile and has key functions in maintaining protein homeostasis including protein quality control mechanisms like the ubiquitin proteasome system (UPS) and autophagy to disassemble polyubiquitylated proteins from chromatin, membranes, macromolecular protein complexes and aggregates which are either degraded by the proteasome or recycled. p97 can use energy derived from ATP hydrolysis to catalyze substrate unfolding and threading through its central channel. The function of p97 in a large variety of different cellular contexts is reflected by its simultaneous association with different cofactors, which are involved in substrate recognition and processing, thus leading to the formation of transient multi-protein complexes. Dysregulation in protein homeostasis and proteotoxic stress are often involved in the development of cancer and neurological diseases and targeting the UPS including p97 in cancer is a well-established pharmacological strategy. In this chapter we will describe structural and functional aspects of the p97 interactome in regulating diverse cellular processes and will discuss the role of p97 in targeted cancer therapy.

Ribosomes are biological nanomachine that synthesise all proteins within a cell. It took decades to reveal the architecture of this essential cellular component. To understand the structure -function relationship of this nanomachine needed the utilisisation of different biochemical, biophysical and structural techniques. Structural studies combined with mutagenesis of the different ribosomal complexes comprising various RNAs and proteins enabled us to understand how this machine works inside a cell. Nowadays quite a number of ribosomal structures were published that confirmed biochemical studies on particular steps of protein synthesis by the ribosome . Four major steps were identified: initiation , elongation, termination and recycling. These steps lead us to the important question how the ribosome function can be regulated. Advances in technology for cryo electron microscopy: sample preparations, image recording, developments in algorithms for image analysis and processing significantly helped in revelation of structural details of the ribosome . We now have a library of ribosome structures from prokaryotes to eukaryotes that enable us to understand the complex mechanics of this nanomachine. As this structural library continues to grow, we gradually improve our understanding of this process and how it can be regulated and how the specific ribosomes can be stalled or activated, or completely disabled. This article provides a comprehensive overview of ribosomal structures that represent structural snapshots of the ribosome at its different functional states. Better understanding rises more particular questions that have to be addressed by determination structures of more complexes.Synopsis: Structural biology of the ribosome.

Integration of the reverse-transcribed viral cDNA into the host's genome is a critical step in the lifecycle of all retroviruses. Retrovirus integration is carried out by integrase (IN), a virus-encoded enzyme that forms an oligomeric 'intasome' complex with both ends of the linear viral DNA to catalyze their concerted insertions into the backbones of the host's DNA. IN also forms a complex with host proteins, which guides the intasome to the host's chromosome. Recent structural studies have revealed remarkable diversity as well as conserved features among the architectures of the intasome assembly from different genera of retroviruses. This chapter will review how IN oligomerizes to achieve its function, with particular focus on alpharetrovirus including the avian retrovirus Rous sarcoma virus. Another chapter (Craigie) will focus on the structure and function of IN from HIV-1.

Understanding how the human gut microbiota might influence ageing is challenging. The gut microbiota is a hugely complex ecology of organisms that varies greatly with individuals and time, making age-related changes difficult to measure. However, elderly and younger populations do show differences in gut microbe composition. The key question is whether these differences only reflect age-related changes in host physiology and diet, or if microbes can drive host ageing? Model organisms allow this question to be addressed. Longitudinal analyses in the fruit fly Drosophila melanogaster show that changes in microbial composition precedes intestinal and host ageing, and antibiotic treatment increases lifespan, implicating microbes in accelerating ageing. Antibiotics also extend the lifespan of middle-aged killifish but additional transplantation of gut microbes from young killifish extends lifespan further, suggesting a positive effect of microbes associated with young animals. Microbes from old, but not young, mice induce inflammation when added to germ-free mice suggesting that microbes become more harmful to the host with age. These studies implicate broad classes of bacteria, particularly members of the phylum Proteobacteria, as drivers of ageing in a feed-forward loop with intestinal degradation and inflammation. The nematode Caenorhabditis elegans can be associated with single strains of genetically-tractable bacteria, and this simplified system has revealed specific interventions in bacterial metabolism, such as inhibition of bacterial folate synthesis, that extend animal lifespan. Transferring this understanding to the human microbiota is challenging but promises to reveal how manipulation of the gut microbiota might be a route to maintain health in old age.

Purification technologies are one of the working horses in organelle proteomics studies as they guarantee the separation of organelle-specific proteins from the background contamination by other subcellular compartments. The development of methods for the separation of organelles was a major prerequisite for the initial detection and characterization of peroxisome as a discrete entity of the cell. Since then, isolated peroxisomes fractions have been used in numerous studies in order to characterize organelle-specific enzyme functions, to allocate the peroxisome-specific proteome or to unravel the organellar membrane composition. This review will give an overview of the fractionation methods used for the isolation of peroxisomes from animals, plants and fungi. In addition to "classic" centrifugation-based isolation methods, relying on the different densities of individual organelles, the review will also summarize work on alternative technologies like free-flow-electrophoresis or flow field fractionation which are based on distinct physicochemical parameters. A final chapter will further describe how different separation methods and quantitative mass spectrometry have been used in proteomics studies to assign the proteome of PO.

One hundred years has passed since the discovery of vitamin D as the active component of cod-liver oil which cured the bone disease rickets. Since then our knowledge of vitamin D has expanded tremendously and has included recognition of the importance of UV radiation as a source of the vitamin as well as the discovery of the vitamin as a nutrient, a pro-hormone and a potent steroid hormone with a major role in calcium and bone metabolism. In the last 25years or so, the discovery of the vitamin D receptor in over 30 different body tissues together with the existence of the alpha-1-hydroxylase enzyme in these tissues provided evidence of a pleiotropic role of vitamin D outside its classical role in the skeleton. These important discoveries have provided the basis for the increasing interest in vitamin D in the context of nutritional requirements for health including the prevention of chronic diseases of ageing. The recent publication of the Dietary Reference Intake report on vitamin D and calcium by the North American Institute of Medicine (IOM) is the most comprehensive report to date on the basis for setting nutritional requirements for vitamin D. This chapter will summarize the nutritional aspects of vitamin D and discuss the changes in vitamin D metabolism and requirements with ageing. It will summarize key evidence on the relationship between vitamin D status and some of the main ageing related health outcomes including bone, muscle and cognitive health as well as survival focusing on the published literature in very-old adults (those >= 85 years of age).