Sub-cellular biochemistry最新文献

Electron microscopy (EM) techniques have been crucial for understanding the structure of biological specimens such as cells, tissues and macromolecular assemblies. Viruses and related viral assemblies are ideal targets for structural studies that help to define essential biological functions. Whereas conventional EM methods use chemical fixation, dehydration, and staining of the specimens, cryogenic electron microscopy (cryo-EM) preserves the native hydrated state. Combined with image processing and three-dimensional reconstruction techniques, cryo-EM provides three-dimensional maps of these macromolecular complexes from projection images, at atomic or near-atomic resolutions. Cryo-EM is also a major technique in structural biology for dynamic studies of functional complexes, which are often unstable, flexible, scarce, or transient in their native environments. State-of-the-art techniques in structural virology now extend beyond purified symmetric capsids and focus on the asymmetric elements such as the packaged genome and minor structural proteins that were previously missed. As a tool, cryo-EM also complements high-resolution techniques such as X-ray diffraction and NMR spectroscopy; these synergistic hybrid approaches provide important new information. Three-dimensional cryogenic electron tomography (cryo-ET), a variation of cryo-EM, goes further, and allows the study of pleomorphic and complex viruses not only in their physiological state but also in their natural environment in the cell, thereby bridging structural studies at the molecular and cellular levels. Cryo-EM and cryo-ET have been applied successfully in basic research, shedding light on fundamental aspects of virus biology and providing insights into threatening viruses, including SARS-CoV-2, responsible for the COVID-19 pandemic.

Icosahedral viruses exhibit elegant pathways of capsid assembly and maturation regulated by symmetry principles. Assembly is a dynamic process driven by consecutive and genetically programmed morphogenetic interactions between protein subunits. The non-symmetric capsid subunits are gathered by non-covalent contacts and interactions in assembly intermediates, which serve as blocks to build a symmetric capsid. In some virus examples, the assembly of the protein shell further requires non-symmetric interactions among intermediates to fold into specific conformations. In this chapter, the morphogenesis of some small and structurally simple icosahedral viruses, including representative members of the parvoviruses, picornaviruses, and polyomaviruses as paradigms, is described in some detail. Despite their small size, the assembly of these icosahedral viruses may follow rather complex pathways, as they may occur in different subcellular compartments, involve a panoply of cellular and viral factors, and regulatory protein post-translational modifications that challenge its comprehensive understanding. Mechanisms of viral genome encapsidation may imply direct interactions between the genome and the assembly intermediates, or active packaging into a preformed empty capsid. Further, membranes and factors at specific subcellular compartments may also be critically required for virus maturation. The high stability of intermediates and the process of viral maturation contribute to the overall irreversible character of the assembly process. These and other small, structurally less complex icosahedral viruses were pioneer models to understand basic principles of virus assembly, continue to be leading subjects of morphogenetic analyses, and have inspired ongoing studies on the assembly of larger, structurally more complex viruses as well as cellular and synthetic macromolecular complexes.

Viruses are elegant macromolecular assemblies and constitute a paradigm of the economy of genomic resources; they must use simple general principles to complete their life cycles successfully. Viruses need only one or a few different capsid structural subunits to build an infectious particle, which is possible for two reasons: extensive use of symmetry and built-in conformational flexibility. Although viruses come in many shapes and sizes, two major symmetric assemblies are found: icosahedral and helical. The enormous diversity of virus structures appears to be derived from one or a limited number of basic schemes that became more complex by consecutive incorporation of additional structural elements. The intrinsic structural polymorphism of the viral proteins results in dynamic capsids. The study of virus structures is required to understand structure-function relationships, including those related to morphogenesis and antigenicity, among many others. These structural foundations can be extended to other macromolecular complexes that control many fundamental processes in biology.

Vault ribonucleoprotein particles are naturally designed nanocages, widely found in the eukaryotic kingdom. Vaults consist of 78 copies of the major vault protein (MVP) that are organized in 2 symmetrical cup-shaped halves, of an approximate size of 70x40x40 nm, leaving a huge internal cavity which accommodates the vault poly(ADP-ribose) polymerase (vPARP), the telomerase-associated protein-1 (TEP1) and some small untranslated RNAs. Diverse hypotheses have been developed on possible functions of vaults, based on their unique capsular structure, their rapid movements and the distinct subcellular localization of the particles, implicating transport of cargo, but they are all pending confirmation. Vault particles also possess many attributes that can be exploited in nanobiotechnology, particularly in the creation of vehicles for the delivery of multiple molecular cargoes. Here we review what is known about the structure and dynamics of the vault complex and discuss a possible mechanism for the vault opening process. The recent findings in the characterization of the vaults in cells and in its natural microenvironment will be also discussed.

The present work delves into the enigmatic world of mitochondrial alpha-keto acid dehydrogenase complexes discussing their metabolic significance, enzymatic operation, moonlighting activities, and pathological relevance with links to underlying structural features. This ubiquitous family of related but diverse multienzyme complexes is involved in carbohydrate metabolism (pyruvate dehydrogenase complex), the citric acid cycle (α-ketoglutarate dehydrogenase complex), and amino acid catabolism (branched-chain α-keto acid dehydrogenase complex, α-ketoadipate dehydrogenase complex); the complexes all function at strategic points and also participate in regulation in these metabolic pathways. These systems are among the largest multienzyme complexes with at times more than 100 protein chains and weights ranging up to ~10 million Daltons. Our chapter offers a wealth of up-to-date information on these multienzyme complexes for a comprehensive understanding of their significance in health and disease.

Eukaryotic cells coordinate available nutrients with their growth through the mechanistic target of rapamycin complex 1 (mTORC1) pathway, in which numerous evolutionarily conserved protein complexes survey and transmit nutrient inputs toward mTORC1. mTORC1 integrates these inputs and activates downstream anabolic or catabolic programs that are in tune with cellular needs, effectively maintaining metabolic homeostasis. The GAP activity toward Rags-1 (GATOR1) protein complex is a critical negative regulator of the mTORC1 pathway and, in the absence of amino acid inputs, is activated to turn off mTORC1 signaling. GATOR1-mediated inhibition of mTORC1 signaling is tightly regulated by an ensemble of protein complexes that antagonize or promote its activity in response to the cellular nutrient environment. Structural, biochemical, and biophysical studies of the GATOR1 complex and its interactors have advanced our understanding of how it regulates cellular metabolism when amino acids are limited. Here, we review the current research with a focus on GATOR1 structure, its enzymatic mechanism, and the growing group of proteins that regulate its activity. Finally, we discuss the implication of GATOR1 dysregulation in physiology and human diseases.

Lipoprotein lipase (LPL) is a critical enzyme in humans that provides fuel to peripheral tissues. LPL hydrolyzes triglycerides from the cores of lipoproteins that are circulating in plasma and interacts with receptors to mediate lipoprotein uptake, thus directing lipid distribution via catalytic and non-catalytic functions. Functional losses in LPL or any of its myriad of regulators alter lipid homeostasis and potentially affect the risk of developing cardiovascular disease-either increasing or decreasing the risk depending on the mutated protein. The extensive LPL regulatory network tunes LPL activity to allocate fatty acids according to the energetic needs of the organism and thus is nutritionally responsive and tissue dependent. Multiple pharmaceuticals in development manipulate or mimic these regulators, demonstrating their translational importance. Another facet of LPL biology is that the oligomeric state of the enzyme is also central to its regulation. Recent structural studies have solidified the idea that LPL is regulated not only by interactions with other binding partners but also by self-associations. Here, we review the complexities of the protein-protein and protein-lipid interactions that govern LPL structure and function.

Cellular senescence is recognised as a contributor to the ageing process and the development of multiple age-related conditions. Researchers have launched efforts to identify compounds capable to selectively kill senescent cells, known as senolytics, without affecting non senescent cells. As of now, over 40 compounds have demonstrated senolytic properties, offering promising prospects for reversing or ameliorating age-related conditions in preclinical studies.This chapter presents the most recent developments in senolytic drug research, encompassing investigations spanning basic science, preclinical trials, and clinical studies. While many of these investigations have generated encouraging results in the realm of age-related interventions, this chapter also addresses potential challenges and pitfalls.

With advancing age, achievement of dietary adequacy for all nutrients is increasingly difficult and this is particularly so for minerals. Various factors impede mineral acquisition and absorption including reduced appetite, depressed gastric acid production and dysregulation across a range of signalling pathways in the intestinal mucosa. Minerals are required in sufficient levels since they are critical for the proper functioning of metabolic processes in cells and tissues, including energy metabolism, DNA and protein synthesis, immune function, mobility, and skeletal integrity. When uptake is diminished or loss exceeds absorption, alternative approaches are required to enable individuals to maintain adequate mineral levels. Currently, supplementation has been used effectively in populations for the restoration of levels of some minerals like iron, zinc, and calcium, but these may not be without inherent challenges. Therefore, in this chapter we review the current understanding around the effectiveness of mineral supplementation for the minerals most clinically relevant for the elderly.

Ageing is an inevitable phenomenon that remains under control of a plethora of signalling pathways and regulatory mechanisms. Slowing of cellular homeostasis and repair pathways, declining genomic and proteomic integrity, and deficient stress regulatory machinery may cause accumulating damage triggering initiation of pathways leading to ageing-associated changes. Multiple genetic studies in small laboratory organisms focused on the manipulation of proteasomal activities have shown promising results in delaying the age-related decline and improving the lifespan. In addition, a number of studies indicate a prominent role of small molecule-based proteasome activators showing positive results in ameliorating the stress conditions, protecting degenerating neurons, restoring cognitive functions, and extending life span of organisms. In this chapter, we provide a brief overview of the multi-enzyme proteasome complex, its structure, subunit composition and variety of cellular functions. We also highlight the strategies applied in the past to modulate the protein degradation efficiency of proteasome and their impact on rebalancing the proteostasis defects. Finally, we provide a descriptive account of proteasome activation mechanisms and small molecule-based strategies to improve the overall organismal health and delay the development of age-associated pathologies.