Protein aging, stress, or metabolism can lead to the accumulation of numerous nonenzymatic chemical alterations that can threaten protein stability and function, particularly in long-lived proteins. Eukaryotic cells recognize these protein-damage events through repair and removal pathways, whose loss can lead to adverse effects and contribute to age-related disease pathogenesis. Here, we review recent advances in understanding the formation, repair, and removal mechanisms of posttranslational modifications arising from protein damage, including dehydroamino acids, early-stage glycation, isoaspartate, C-terminal cyclic imides, and C-terminal amides. We emphasize the emerging role of E3 ubiquitin ligases in facilitating the degradation of proteins bearing these modifications, highlight the approaches used to make these discoveries, and discuss the potential functions of these modifications beyond protein damage. Mounting evidence that protein-damage events influence cellular signaling and metabolism suggests the existence of vast undiscovered regulatory networks, creating opportunities to uncover tissue-specific repair mechanisms and their roles in development, aging, and stress responses across diverse biological contexts.
The assembly of newly synthesized proteins into functionally active oligomers has long been regarded as a posttranslational process driven by random collision of subunits. However, growing evidence indicates that, for many proteins, assembly occurs cotranslationally, tightly coupling synthesis, folding, and subunit assembly. This fundamentally different mechanism enables the spatial and temporal coordination of assembly, promotes the hierarchical formation of multisubunit assemblies, enhances the stability of involved subunits, enlarges the space of feasible protein structures including complexes with intertwined subunits, and has profound effects on protein evolution and function. In this review, we describe the molecular mechanisms, cellular requirements, and functional implications of cotranslational assembly and discuss its relevance to human disease, its evolutionary significance, and its transformative potential in synthetic biology and recombinant protein production.
Lactate's role in biochemistry and physiology has attracted considerable biochemical interest for over a century. Beyond its classical description as a glycolytic byproduct, lactate is now recognized as a central energy metabolite, a redox shuttle, and a signaling molecule. Modern advances in mass spectrometry have uncovered yet another dimension of lactate biology: lactate as a chemical modification on proteins and metabolites. Covalent conjugation of lactate to the ε-amine of a protein lysine side chain generates a posttranslational modification (lysine lactylation); in a related reaction on metabolites, conjugation of lactate to the α-amine of free amino acids produces a class of bioactive lactate-modified amino acids (the N -lactoyl amino acids). These lactate modifications represent a fundamental mechanism by which transient increases in glycolytic flux are translated into durable downstream effectors. Here, we review the detection, regulation, and function of these lactate-derived modifications in cellular and organismal homeostasis.
Our double-stranded DNA (dsDNA) genomes are famously compacted by proteins in the nuclei of our cells, resulting in meters of dsDNA being confined in micron-sized volumes. The most prevalent form of viral genomes, however, is single-stranded RNA (ssRNA), which is compacted at significantly higher density in protective protein shells with nanometer dimensions. In this review, we discuss the special nature of ssRNA that allows it to be spontaneously packaged in this way by co-self-assembly with viral capsid protein (CP). We focus on the few viruses whose nucleocapsids can be reconstituted from their purified CP and ssRNA genomes and whose CPs can spontaneously package heterologous RNA into virus-like particles (VLPs). These VLPs are then compared with their cell-synthesized versions, with lentivirus and adeno-associated virus vector particles, and with nucleocapsids formed by nonviral proteins whose messenger RNAs are put under directed evolutionary pressure to be packaged by them in cellulo.
The gut microbiome governs aspects of human growth and development. While human milk's primary purpose is metabolism, it also provides nonnutritious biologics and macromolecules. This mixture includes the human milk oligosaccharides (HMOs), which are indigestible and survive the low pH of the stomach and small intestine, reaching the large intestine intact. Here, HMOs serve as prebiotics for beneficial bacteria, providing a competitive growth advantage over potential pathogens. Upon metabolizing HMOs, commensals generate short-chain fatty acids and metabolites that enhance the gut community. Therefore, HMOs work to develop and sustain the gut microbial community as a living therapeutic that prevents illness from potential microbial pathogens and modulates development of the infant gut. The goal of this targeted review is to characterize the roles HMOs play in governing bacterial and viral members of the infant gut microbiome, describing how HMOs both define a healthy microbiota and prevent microbial dysbiosis.
Cancer cells acquire hallmark behaviors through adaptations that extend beyond genetic and epigenetic changes. Proteostasis-the biochemical network governing protein synthesis, folding, trafficking, and degradation-is a fundamental, yet underappreciated, mediator of these adaptations that merits consideration as a hallmark-enabling mechanism. Metabolic alterations impose proteotoxic stress, globally rewire protein homeostasis, and selectively modulate key oncogenic and tumor suppressive proteins. A unifying framework is proposed wherein metabolic deregulation of proteostasis operates throughout carcinogenesis: early, by enhancing accumulation of premalignant clones bearing cancer-driving somatic mutations in response to environmental and systemic metabolic stress, and later, by buffering proteotoxic stress to sustain malignant growth in hostile tissue environments. This perspective connects cancer risk with genetic background, diet, microbiome-derived metabolites, and metabolic disease, introduces metabolic bypass of tumor suppression as an alternative to classical genetic models, and highlights the metabolism-proteostasis interface as a promising target for cancer prevention and therapy.
The autophagy core machinery carries out the fundamental reactions of autophagosome biogenesis across all forms of bulk and selective macroautophagy. In humans, the core complexes consist of the ULK1 complex (ULK1C), the class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1), the ATG8 proteins and the ATG8ylation machinery, the phosphatidylinositol 3-phosphate (PI3P)-sensing WIPI proteins, the lipid transporter ATG2, and the lipid scramblase and initiation scaffold ATG9. These complexes form a web of interactions that can be initiated by clustering of the FIP200 subunit of ULK1C but also by PI3KC3-C1 or WIPI2. Upon autophagy induction, these interactions are intensified by feed-forward signaling loops. These loops are amplified by WIPI-PI3P interactions and the conjugation of ATG8 proteins to the membrane by the ATG12-ATG5-ATG16L1 complex. Autophagosomes are seeded by ATG9 vesicles, which accrue initiation machinery on their surface and dock onto a PI3P-positive domain of the endoplasmic reticulum known as the omegasome. The omegasome contact site is the focal point for autophagosome growth, which is fed by lipid transport through the ATG2 bridge-like lipid transporter. The core complexes function in a dynamic manner, which makes autophagy vulnerable to stalling when dynamism fails. Disassembly and dissociation of the machinery, which is promoted at least in part by ULK1, is likely to be as important as assembly.
Massachusetts Institute of Technology Professor Emerita JoAnne Stubbe was the chair of Mary O'Reilly's thesis committee. Over a decade later, they became neighbors, and then a global pandemic made them friends. Here they attempt to summarize JoAnne's life and her life's work—a story of passion, curiosity, and persistence, all for the thrill of discovery. Though, it may be premature: She is not done yet.
I studied medicine to become an endocrinologist but ended up in biochemistry. As a graduate student, I studied tumor mitochondria and discovered the malate-aspartate shuttle (the major route in animal cells for shuttling reducing equivalents into mitochondria). As a postdoc in New York, I switched to the replication of RNA bacteriophages, and on my return to Amsterdam, I started on mitochondrial biogenesis and discovered the circular mitochondrial DNAs (mtDNAs) of animal mitochondria and yeast. I also tackled trypanosomatids, major parasites of humans, in which we characterized their exotic mtDNA networks and discovered the glycosome, an organelle containing most of the glycolytic system. We helped to unravel the mechanism of antigenic variation in African trypanosomes and even discovered a new base, base J, in the DNA of trypanosomatids.After moving to the Netherlands Cancer Institute, I combined the trypanosomatids with a project on mechanisms of multidrug resistance in cancer cells. In particular, studying mice with one or more disrupted ABC transporter genes resulted in interesting findings in drug pharmacokinetics and in the elucidation of the cause of two inborn errors.
Apurinic/apyrimidinic (AP) sites, also known as abasic sites, are among the most frequent DNA lesions, arising spontaneously or as intermediates in base excision repair. Their structural impediment to DNA replication fork progression, lack of coding information, and conversion into strand breaks constitute a threat and can lead to genome instability if not properly managed. This review examines the impact of AP sites on DNA replication, detailing mechanisms of lesion bypass, including translesion synthesis, template switching, and repriming of DNA synthesis. We highlight protective pathways that shield AP sites from nucleolytic attack and explore how endogenous processes such as uracil excision, cytosine methylation, and oxidative damage generate these lesions. By integrating biochemical and cellular perspectives, we present a comprehensive view of how cells replicate their DNA in the presence of AP sites and how their mismanagement contributes to replication stress, mutagenesis, and disease.
Integral membrane proteins play critical roles in mammalian cells, ranging from mediating cell-cell interactions to regulating apoptosis. These increasingly diverse functions necessitated the evolution of membrane proteins with more complex biophysical properties and architectures. In turn, specialized complexes called insertases have coevolved to integrate these proteins into the appropriate lipid bilayer. Notably, key sites of membrane protein biogenesis such as the endoplasmic reticulum and the outer and inner membranes of the mitochondria rely on distinct sets of insertases that work in concert, each specializing in membrane protein segments with particular features or properties. Here, we describe recent discoveries that shed light both on the molecular mechanisms of these insertases and on the many distinct pathways required for the insertion and folding of the mammalian membrane proteome.
Radical chemistry, once deemed too reactive for biological systems, is delicately controlled within metalloenzymes to catalyze challenging chemical transformations under physiological conditions. This review explores the diverse strategies employed by metalloenzymes to generate, stabilize, and utilize highly reactive radical intermediates. We discuss amino acid-based radicals (tyrosyl, tryptophan, cysteinyl, glycyl, and DOPA), radicals derived from molecular oxygen (in heme, manganese, copper, and nonheme iron enzymes), and cofactor-based radicals (specifically adenosylcobalamin and radical S-adenosylmethionine enzymes). We devote special attention to ribonucleotide reductases as a prime example of evolutionary convergence of radical mechanisms. The interplay among redox-active cofactors, metal ions, and protein scaffolds highlights nature's ingenuity in generating, controlling, and utilizing radicals. We also discuss emerging themes and open questions, emphasizing how advances in structural and spectroscopic techniques continue to deepen our understanding of these complex and vital enzymatic processes.
To protect their delicate, carefully curated contents from the world, bacteria encase themselves within a protective envelope made up of sugars, lipids, and proteins. Cell envelopes give bacteria their characteristic shapes, provide rigidity and mechanical stability, and form a selective antechamber-granting access only to a desirable subset of environmental substances. Yet this protective layer is a double-edged sword: Its effectiveness at keeping things out also makes it difficult for things to leave, including the proteins required to interface with the outside world and form the envelope itself. Bacteria have solved this problem by constructing an array of proteinaceous nanomachines that expend energy to selectively shuttle proteins and other building blocks to their intended destinations. Here, we present an overview of our current understanding of how these transporters work, focusing on the major, conserved machines that ferry proteins across the cell envelope throughout the domain Bacteria. The emphasis is on recent discoveries and open questions, with the hope that answering these will provide new avenues to help combat the rising threat of antimicrobial resistance and the rapidly expanding list of diseases linked to human microbiome composition.
Damage to mitochondria imparts multifaceted cellular stress that extends beyond bioenergetic deficit. One newly emerged example is mitochondrial precursor overaccumulation stress (mPOS). mPOS is marked by impaired mitochondrial protein import, causing the toxic accumulation and aggregation of unimported mitochondrial precursor proteins in the cytosol. Analogous to the well-studied endoplasmic reticulum stress, which blocks proteins from leaving the cell, mPOS can impose a drastic proteostatic burden in the cytosol and closely interconnects with cell signaling pathways. Here, we review how researchers discovered mPOS and discuss its central importance in several major mitochondria-induced stress signaling pathways. We then focus on the emerging field of mPOS in cell demise and human disease, and we present recent evidence that mPOS can affect cell fitness and survival independent of bioenergetics. Looking forward, mPOS may provide a complementary or alternative pathogenic mechanism to bioenergetic deficit for classic mitochondriopathy and many aging-associated degenerative diseases involving mitochondrial stress.
The ability of cells to transmit information encoded in the genome, and its organization into chromatin across cell generations, is a cornerstone of eukaryotic life. Chromatin replication, the copying of the mammalian genome in its structural and functional chromatin context to maintain cell identity and fate, is fundamental to lifelong health and has important implications for cancer and aging. Here, we review the major breakthroughs in our understanding of chromatin dynamics during DNA replication, critical for genome and epigenome inheritance. We discuss how chromatin is disrupted at the replication fork and how the replication machinery ensures transmission of parental histones with their modifications to daughter DNA strands with high fidelity. We highlight how incorporation of new histones is integrated into this process to maintain chromatin integrity and functionality. Finally, we consider how these processes maintain gene expression programs and thus cellular identity and function across cell division throughout the organismal life span.
Coronaviruses (family Coronaviridae, order Nidovirales) include major human and animal pathogens. They have exceptionally large RNA genomes and use complex strategies to replicate and express these genomes. Intensive research activities in recent years have significantly advanced our knowledge of the molecular mechanisms involved in coronavirus RNA synthesis. Here, we briefly review these mechanisms and focus in particular on the structures and functions of the core replication-transcription complex (RTC) and other enzyme functions that can be recruited to this complex to fulfil additional functions, for example, in the context of 5' capping of viral mRNAs or in the context of mechanisms that control the processivity, replication fidelity, and backtracking of RTCs. Some of these recent studies provided fundamentally new insight into specific roles of previously identified genetic markers of coronaviruses and other nidoviruses, including specific functions in an unconventional RNA capping mechanism and potential roles in proofreading and discontinuous negative-strand RNA synthesis.
The eleven known zinc-dependent histone deacetylases (HDACs) catalyze the deacetylation or deacylation of myriad protein and small molecule substrates throughout the cell. The biological functions of HDACs are much more diverse than the name HDAC implies, but this name is nonetheless retained for historical purposes. The chemical mechanism of catalysis is generally conserved among HDAC isozymes: Electrophilic activation of the substrate is achieved by zinc coordination and hydrogen bonding, and nucleophilic activation of a zinc-bound water molecule is enhanced by a general base. Since aberrant activity is observed for specific HDAC isozymes in certain diseases, the development of isozyme-selective inhibitors is a current priority in worldwide medicinal chemistry campaigns. In this review, the biological functions and chemical mechanisms of the HDACs are discussed to establish the molecular context of catalysis and inhibition, particularly as the chemistry of catalysis is harnessed in the development of mechanism-based inhibitors.
Axon degeneration is a tightly regulated process that plays a central role in the pathogenesis of many neurodegenerative diseases. Three core mediators, DLK (dual leucine zipper kinase), NMNAT2 (nicotinamide mononucleotide adenylyltransferase 2), and SARM1 (sterile alpha and Toll/interleukin-1 receptor motif-containing 1) form a molecular axis that orchestrates axonal self-destruction. Upon stress, DLK initiates mitogen-activated protein kinase signaling, which triggers the expression of prodegenerative genes. NMNAT2, an essential nicotinamide adenine dinucleotide biosynthetic enzyme, is rapidly depleted following injury. Loss of NMNAT2 leads to the accumulation of its substrate, nicotinamide mononucleotide, which in turn activates SARM1, a central executioner of axon degeneration. Together, these proteins constitute a coordinated signaling axis that monitors cellular stress and metabolic cues to regulate axonal integrity. In this review, we provide an overview of the biochemical and cellular mechanisms of DLK, NMNAT2, and SARM1 signaling and discuss how targeting these factors offers opportunities for therapeutic intervention in a broad range of neurodegenerative disorders.
A signature feature of transcription on most genes in multicellular animals is that RNA polymerase II (RNAPII) piles up approximately 50 bases downstream of the start site at the promoter-proximal pause (PPP). Promoter-proximal pausing is controlled by positive and negative elongation factors that associate with RNAPII. There are two major outcomes for promoter-proximally paused RNAPII complexes: release into the gene body and premature termination. Here we discuss how RNAPII dynamics at the PPP function in a quality control checkpoint and in regulation of RNAPII flux through genes. We propose a pause release-attenuation model to describe RNAPII dynamics at the PPP.
Three fundamental classes of gene regulatory elements were identified in Escherichia coli in the 1960s: operators bound by repressor proteins, promoters bound by the basic transcription machinery, and enhancers bound by activator proteins. Promoters mediate constitutive gene expression, whereas operators and enhancers regulate expression in response to physiological conditions. As discovered in the 1980s, interactions between proteins associated with spatially separated elements can loop out the intervening DNA and regulate transcription. Eukaryotic gene regulation is mediated by the same three classes of genetic elements, but due to semantic confusion, it is often believed the eukaryotic enhancers activate transcription from long distances via enhancer-promoter loops. However, eukaryotic enhancers mediate only local changes in chromatin, and they stimulate transcription via directional and short-range interactions with the basic RNA polymerase II machinery at promoters. Enhancer action at a distance is mediated by loops between proximal and distal enhancers that bring activator proteins associated with distal enhancers in proximity to promoters. Thus, Jacques Monod's 1954 conjecture that "what is true for E. coli is true for the elephant, only more so" has proven correct.
