Annual review of biochemistry最新文献

The visualization and manipulation of proteins in live cells are critical for studying complex biological processes. Self-labeling proteins do so by enabling the specific and covalent attachment of synthetic probes, offering unprecedented flexibility in the chemical labeling of proteins in live cells and in vivo. By combining the excellent photophysical properties of synthetic dyes with genetic targetability, these tags provide a modular and innovative toolbox for live-cell and high-resolution fluorescence imaging. In this review, we explore the development and diverse applications of the key self-labeling protein technologies, HaloTag7, SNAP-tag, and CLIP-tag, as well as the covalent trimethoprim (TMP)-tag. We discuss recent innovations in both protein engineering and substrate design that have introduced new functionalities to enable multiplexed imaging, super-resolution microscopy, and the design of novel biosensors and recorders.

Gene expression is essential for life and development, allowing the cell to modulate mRNA production in response to intrinsic and extracellular cues. Initiation of gene transcription requires a highly regulated molecular process to assemble multisubunit complexes into the preinitiation complex (PIC). Attempts to visualize these processes have been driven largely by electron microscopy, with near atomic-level resolution producing static snapshots complemented by low-resolution fluorescence cell imaging. Here, we review how new advances in superresolution single-molecule imaging in live cells can track transcription across vast spatiotemporal scales. We discuss how recent imaging research has fundamentally recast our understanding of PIC assembly from a stable, ordered process to one constantly in flux, dominated by multivalent weak interactions. We also discuss future advancements that will further expand our ability to measure PIC assembly in concert with cellular behavior, predict complex interactions computationally, and target undruggable transcription factors to treat human disease.

Heme oxygenase (HO)-like metalloenzymes are an emerging protein superfamily diverse in reaction outcome and mechanism. Found primarily in bacterial biosynthetic pathways, members conserve a flexible protein scaffold shared with the heme catabolic enzyme, HO, and a set of metal-binding residues. Most HO-like metalloenzymes assemble a diiron cluster, although manganese-iron and mononuclear iron cofactors can also be accommodated. In the canonical HO-like diiron oxygenases/oxidases (HDOs), an Fe₂(II/II) complex reacts with O₂ to form a peroxo-Fe₂(III/III) intermediate (P), common to all HDOs studied to date. The HO-like scaffold confers both distinctive metal-binding properties, allowing for dynamic cofactor assembly and disassembly, and unusual reactivity to its associated metallocofactor. These features may prove to be important in HDO-mediated catalysis of the fragmentation and rearrangement reactions that remain unprecedented among other dinuclear iron enzymes. Much of the sequence space in the HO-like metalloenzyme superfamily remains unexplored, offering exciting opportunities for the discovery of new mechanisms and reactivities.

Metabolism and gene regulation are vital processes that need to be tightly coordinated to maintain homeostasis or to enable growth and development. Recent research has begun to reveal the surprisingly interlaced relationship between metabolism and gene expression control. Because key metabolites are cofactors or cosubstrates of chromatin-modifying enzymes, changes in their concentrations can modulate chromatin states and gene expression. Additionally, an increasing number of key metabolic enzymes are found to directly regulate chromatin and transcription in response to changes in metabolic state. These include enzymes that fuel chromatin-associated metabolism and moonlighting enzymes that function as transcription factors, independent of their enzymatic activity. Conversely, accumulating evidence suggests that chromatin itself serves key metabolic functions, independent of transcriptional regulation. Here, we discuss the bidirectional interface between metabolism and chromatin and its corruption in cancer cells.

Viruses must egress from the cells in which they have replicated to spread and propagate. Historically, viruses have been classified into enveloped and nonenveloped forms: Enveloped viruses exploit cellular membrane-trafficking pathways to egress while maintaining cell integrity, and nonenveloped viruses, i.e., those lacking a membrane around their capsids, lytically egress. Here, we make the compelling case that all animal and plant and many archaeal and bacterial viruses egress through nonlytic pathways. Most of these nonlytic pathways can be separated into those that enable viruses to spread without leaving the confines of cell bodies and those that traffic them to the extracellular space in enveloped membrane-bound forms. Nonlytic egress pathways bestow viruses with distinct transmission advantages including high multiplicity of infection, quality control over transmitting infectious units, and evasion of innate and adaptive antiviral immune defense mechanisms.

Discovered in 1993, inositol pyrophosphates are evolutionarily conserved signaling metabolites whose versatile modes of action are being increasingly appreciated. These include their emerging roles as energy regulators, phosphodonors, steric/allosteric regulators, and G protein-coupled receptor messengers. Through studying enzymes that metabolize inositol pyrophosphates, progress has also been made in elucidating the various cellular and physiological functions of these pyrophosphate-containing, energetic molecules. The two main forms of inositol pyrophosphates, 5-IP₇ and IP₈, synthesized respectively by inositol-hexakisphosphate kinases (IP6Ks) and diphosphoinositol pentakisphosphate kinases (PPIP5Ks), regulate phosphate homeostasis, ATP synthesis, and several other metabolic processes ranging from insulin secretion to cellular energy utilization. Here, we review the current understanding of the catalytic and regulatory mechanisms of IP6Ks and PPIP5Ks, as well as their counteracting phosphatases. We also highlight the genetic and cellular evidence implicating inositol pyrophosphates as essential mediators of mammalian metabolic homeostasis.

The nicotinic acetylcholine receptor has served, since its biochemical identification in the 1970s, as a model of an allosteric ligand-gated ion channel mediating signal transition at the synapse. In recent years, the application of X-ray crystallography and high-resolution cryo-electron microscopy, together with molecular dynamic simulations of nicotinic receptors and homologs, have opened a new era in the understanding of channel gating by the neurotransmitter. They reveal, at atomic resolution, the diversity and flexibility of the multiple ligand-binding sites, including recently discovered allosteric modulatory sites distinct from the neurotransmitter orthosteric site, and the conformational dynamics of the activation process as a molecular switch linking these multiple sites. The model emerging from these studies paves the way for a new pharmacology based, first, upon the occurrence of an original mode of indirect allosteric modulation, distinct from a steric competition for a single and rigid binding site, and second, the design of drugs that specifically interact with privileged conformations of the receptor such as agonists, antagonists, and desensitizers. Research on nicotinic receptors is still at the forefront of understanding the mode of action of drugs on the nervous system.

RAF family protein kinases are a key node in the RAS/RAF/MAP kinase pathway, the signaling cascade that controls cellular proliferation, differentiation, and survival in response to engagement of growth factor receptors on the cell surface. Over the past few years, structural and biochemical studies have provided new understanding of RAF autoregulation, RAF activation by RAS and the SHOC2 phosphatase complex, and RAF engagement with HSP90-CDC37 chaperone complexes. These studies have important implications for pharmacologic targeting of the pathway. They reveal RAF in distinct regulatory states and show that the functional RAF switch is an integrated complex of RAF with its substrate (MEK) and a 14-3-3 dimer. Here we review these advances, placing them in the context of decades of investigation of RAF regulation. We explore the insights they provide into aberrant activation of the pathway in cancer and RASopathies (developmental syndromes caused by germline mutations in components of the pathway).

Writing a career retrospective for this prestigious series is a huge challenge. Is my story really of that much interest? One thing that is different about my life in science is the heavy influence of the turmoil of the past century. Born in the US, raised in East Germany, and returning to the US relatively late in life, I experienced research under both suboptimal and privileged conditions. My scientific story, like the political winds that blew me from one continent to the next, involved shifts into different fields. For advice to young scientists, I would suggest: Don't be afraid to start something new, it pays to be persistent, and science is a passion. In addition to telling my own story, this article also provides the opportunity to express my gratitude to my trainees and colleagues and to convey my conviction that we have the best job on earth.

During the last ten years, developments in cryo-electron microscopy have transformed our understanding of eukaryotic ribosome assembly. As a result, the field has advanced from a list of the vast array of ribosome assembly factors toward an emerging molecular movie in which individual frames are represented by structures of stable ribosome assembly intermediates with complementary biochemical and genetic data. In this review, we discuss the mechanisms driving the assembly of yeast and human small and large ribosomal subunits. A particular emphasis is placed on the most recent findings that illustrate key concepts of ribosome assembly, such as folding of preribosomal RNA, the enforced chronology of assembly, enzyme-mediated irreversible transitions, and proofreading of preribosomal particles.