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The majority of therapies that target individual proteins rely on specific activity-modulating interactions with the target protein—for example, enzyme inhibition or ligand blocking. However, several major classes of therapeutically relevant proteins have unknown or inaccessible activity profiles and so cannot be targeted by such strategies. Protein-degradation platforms such as proteolysis-targeting chimaeras (PROTACs) 1 , 2 and others (for example, dTAGs 3 , Trim-Away 4 , chaperone-mediated autophagy targeting 5 and SNIPERs 6 ) have been developed for proteins that are typically difficult to target; however, these methods involve the manipulation of intracellular protein degradation machinery and are therefore fundamentally limited to proteins that contain cytosolic domains to which ligands can bind and recruit the requisite cellular components. Extracellular and membrane-associated proteins—the products of 40% of all protein-encoding genes 7 —are key agents in cancer, ageing-related diseases and autoimmune disorders 8 , and so a general strategy to selectively degrade these proteins has the potential to improve human health. Here we establish the targeted degradation of extracellular and membrane-associated proteins using conjugates that bind both a cell-surface lysosome-shuttling receptor and the extracellular domain of a target protein. These initial lysosome-targeting chimaeras, which we term LYTACs, consist of a small molecule or antibody fused to chemically synthesized glycopeptide ligands that are agonists of the cation-independent mannose-6-phosphate receptor (CI-M6PR). We use LYTACs to develop a CRISPR interference screen that reveals the biochemical pathway for CI-M6PR-mediated cargo internalization in cell lines, and uncover the exocyst complex as a previously unidentified—but essential—component of this pathway. We demonstrate the scope of this platform through the degradation of therapeutically relevant proteins, including apolipoprotein E4, epidermal growth factor receptor, CD71 and programmed death-ligand 1. Our results establish a modular strategy for directing secreted and membrane proteins for lysosomal degradation, with broad implications for biochemical research and for therapeutics. Lysosome-targeting chimaeras—in which a small molecule or antibody is connected to a glycopeptide ligand to form a conjugate that can bind a cell-surface lysosome-shuttling receptor and a protein target—are used to achieve the targeted degradation of extracellular and membrane proteins.

Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002 as a highly transmissible pathogenic human betacoronavirus. The viral spike glycoprotein (S) utilizes angiotensin-converting enzyme 2 (ACE2) as a host protein receptor and mediates fusion of the viral and host membranes, making S essential to viral entry into host cells and host species tropism. As SARS-CoV enters host cells, the viral S is believed to undergo a number of conformational transitions as it is cleaved by host proteases and binds to host receptors. We recently developed stabilizing mutations for coronavirus spikes that prevent the transition from the pre-fusion to post-fusion states. Here, we present cryo-EM analyses of a stabilized trimeric SARS-CoV S, as well as the trypsin-cleaved, stabilized S, and its interactions with ACE2. Neither binding to ACE2 nor cleavage by trypsin at the S1/S2 cleavage site impart large conformational changes within stabilized SARS-CoV S or expose the secondary cleavage site, S2′.

Stem cells underlie tissue homeostasis, but their dynamics during ageing—and the relevance of these dynamics to organ ageing—remain unknown. Here we report that the expression of the hemidesmosome component collagen XVII (COL17A1) by epidermal stem cells fluctuates physiologically through genomic/oxidative stress-induced proteolysis, and that the resulting differential expression of COL17A1 in individual stem cells generates a driving force for cell competition. In vivo clonal analysis in mice and in vitro 3D modelling show that clones that express high levels of COL17A1, which divide symmetrically, outcompete and eliminate adjacent stressed clones that express low levels of COL17A1, which divide asymmetrically. Stem cells with higher potential or quality are thus selected for homeostasis, but their eventual loss of COL17A1 limits their competition, thereby causing ageing. The resultant hemidesmosome fragility and stem cell delamination deplete adjacent melanocytes and fibroblasts to promote skin ageing. Conversely, the forced maintenance of COL17A1 rescues skin organ ageing, thereby indicating potential angles for anti-ageing therapeutic intervention. COL17A1-driven stem cell competition and symmetric cell divisions initially govern skin homeostasis, but the same mechanisms result in skin ageing later in life.

During metazoan development, immune surveillance and cancer dissemination, cells migrate in complex three-dimensional microenvironments 1 – 3 . These spaces are crowded by cells and extracellular matrix, generating mazes with differently sized gaps that are typically smaller than the diameter of the migrating cell 4 , 5 . Most mesenchymal and epithelial cells and some—but not all—cancer cells actively generate their migratory path using pericellular tissue proteolysis 6 . By contrast, amoeboid cells such as leukocytes use non-destructive strategies of locomotion 7 , raising the question how these extremely fast cells navigate through dense tissues. Here we reveal that leukocytes sample their immediate vicinity for large pore sizes, and are thereby able to choose the path of least resistance. This allows them to circumnavigate local obstacles while effectively following global directional cues such as chemotactic gradients. Pore-size discrimination is facilitated by frontward positioning of the nucleus, which enables the cells to use their bulkiest compartment as a mechanical gauge. Once the nucleus and the closely associated microtubule organizing centre pass the largest pore, cytoplasmic protrusions still lingering in smaller pores are retracted. These retractions are coordinated by dynamic microtubules; when microtubules are disrupted, migrating cells lose coherence and frequently fragment into migratory cytoplasmic pieces. As nuclear positioning in front of the microtubule organizing centre is a typical feature of amoeboid migration, our findings link the fundamental organization of cellular polarity to the strategy of locomotion. Geometrically defined microenvironments are used to show that leukocytes migrate along chemokine gradients using the nucleus as a mechanical gauge to sample potential paths and identify the path of least resistance.

Complex multicellular organisms evolved on Earth in an oxygen-rich atmosphere 1 ; their tissues, including stem-cell niches, require continuous oxygen provision for efficient energy metabolism 2 . Notably, the maintenance of the pluripotent state of animal stem cells requires hypoxic conditions, whereas higher oxygen tension promotes cell differentiation 3 . Here we demonstrate, using a combination of genetic reporters and in vivo oxygen measurements, that plant shoot meristems develop embedded in a low-oxygen niche, and that hypoxic conditions are required to regulate the production of new leaves. We show that hypoxia localized to the shoot meristem inhibits the proteolysis of an N-degron-pathway 4 , 5 substrate known as LITTLE ZIPPER 2 (ZPR2)—which evolved to control the activity of the class-III homeodomain-leucine zipper transcription factors 6 – 8 —and thereby regulates the activity of shoot meristems. Our results reveal oxygen as a diffusible signal that is involved in the control of stem-cell activity in plants grown under aerobic conditions, which suggests that the spatially distinct distribution of oxygen affects plant development. In molecular terms, this signal is translated into transcriptional regulation by the N-degron pathway, thereby linking the control of metabolic activity to the regulation of development in plants. Hypoxia in the shoot meristem of Arabidopsis links the regulation of metabolic activity to development by inhibiting proteolysis of a substrate of the N-degron pathway, which controls class-III homeodomain-leucine zipper transcription factors.

Genome sequencing of environmental bacteria allows identification of biosynthetic gene clusters encoding unusual combinations of enzymes that produce unknown natural products. We identified a pathway in which a ribosomally synthesized small peptide serves as a scaffold for nonribosomal peptide extension and chemical modification. Amino acids are transferred to the carboxyl terminus of the peptide through adenosine triphosphate and amino acyl-tRNA–dependent chemistry that is independent of the ribosome. Oxidative rearrangement, carboxymethylation, and proteolysis of a terminal cysteine yields an amino acid–derived small molecule. Microcrystal electron diffraction demonstrates that the resulting product is isosteric to glutamate. We show that a similar peptide extension is used during the biosynthesis of the ammosamides, which are cytotoxic pyrroloquinoline alkaloids. These results suggest an alternative paradigm for biosynthesis of amino acid–derived natural products.

A short, 14-amino-acid segment called SP1, located in the Gag structural protein 1 , has a critical role during the formation of the HIV-1 virus particle. During virus assembly, the SP1 peptide and seven preceding residues fold into a six-helix bundle, which holds together the Gag hexamer and facilitates the formation of a curved immature hexagonal lattice underneath the viral membrane 2 , 3 . Upon completion of assembly and budding, proteolytic cleavage of Gag leads to virus maturation, in which the immature lattice is broken down; the liberated CA domain of Gag then re-assembles into the mature conical capsid that encloses the viral genome and associated enzymes. Folding and proteolysis of the six-helix bundle are crucial rate-limiting steps of both Gag assembly and disassembly, and the six-helix bundle is an established target of HIV-1 inhibitors 4 , 5 . Here, using a combination of structural and functional analyses, we show that inositol hexakisphosphate (InsP6, also known as IP 6 ) facilitates the formation of the six-helix bundle and assembly of the immature HIV-1 Gag lattice. IP 6 makes ionic contacts with two rings of lysine residues at the centre of the Gag hexamer. Proteolytic cleavage then unmasks an alternative binding site, where IP 6 interaction promotes the assembly of the mature capsid lattice. These studies identify IP 6 as a naturally occurring small molecule that promotes both assembly and maturation of HIV-1. Inositol hexakisphosphate, which is found in all mammalian cells, binds to two separate sites to promote the assembly and maturation of HIV-1 virus particles.

Minichromosome maintenance proteins (MCMs) are DNA-dependent ATPases that bind to replication origins and license them to support a single round of DNA replication. A large excess of MCM2–7 assembles on chromatin in G1 phase as pre-replication complexes (pre-RCs), of which only a fraction become the productive CDC45–MCM–GINS (CMG) helicases that are required for genome duplication 1 – 4 . It remains unclear why cells generate this surplus of MCMs, how they manage to sustain it across multiple generations, and why even a mild reduction in the MCM pool compromises the integrity of replicating genomes 5 , 6 . Here we show that, for daughter cells to sustain error-free DNA replication, their mother cells build up a nuclear pool of MCMs both by recycling chromatin-bound (parental) MCMs and by synthesizing new (nascent) MCMs. Although all MCMs can form pre-RCs, it is the parental pool that is inherently stable and preferentially matures into CMGs. By contrast, nascent MCM3–7 (but not MCM2) undergo rapid proteolysis in the cytoplasm, and their stabilization and nuclear translocation require interaction with minichromosome-maintenance complex-binding protein (MCMBP), a distant MCM paralogue 7 , 8 . By chaperoning nascent MCMs, MCMBP safeguards replicating genomes by increasing chromatin coverage with pre-RCs that do not participate on replication origins but adjust the pace of replisome movement to minimize errors during DNA replication. Consequently, although the paucity of pre-RCs in MCMBP-deficient cells does not alter DNA synthesis overall, it increases the speed and asymmetry of individual replisomes, which leads to DNA damage. The surplus of MCMs therefore increases the robustness of genome duplication by restraining the speed at which eukaryotic cells replicate their DNA. Alterations in physiological fork speed might thus explain why even a minor reduction in MCM levels destabilizes the genome and predisposes to increased incidence of tumour formation. Mother cells recycle parental MCMs and simultaneously synthesize nascent MCMs, both of which are inherited by daughter cells, in which the former are preferentially used to form active replisomes and the latter adjust the pace of replisome movement to minimize errors during DNA replication.

Thyroglobulin (TG) is the protein precursor of thyroid hormones, which are essential for growth, development and the control of metabolism in vertebrates 1 , 2 . Hormone synthesis from TG occurs in the thyroid gland via the iodination and coupling of pairs of tyrosines, and is completed by TG proteolysis 3 . Tyrosine proximity within TG is thought to enable the coupling reaction but hormonogenic tyrosines have not been clearly identified, and the lack of a three-dimensional structure of TG has prevented mechanistic understanding 4 . Here we present the structure of full-length human thyroglobulin at a resolution of approximately 3.5 Å, determined by cryo-electron microscopy. We identified all of the hormonogenic tyrosine pairs in the structure, and verified them using site-directed mutagenesis and in vitro hormone-production assays using human TG expressed in HEK293T cells. Our analysis revealed that the proximity, flexibility and solvent exposure of the tyrosines are the key characteristics of hormonogenic sites. We transferred the reaction sites from TG to an engineered tyrosine donor–acceptor pair in the unrelated bacterial maltose-binding protein (MBP), which yielded hormone production with an efficiency comparable to that of TG. Our study provides a framework to further understand the production and regulation of thyroid hormones. The cryo-electron microscopy structure of human thyroglobulin reveals that proximity, flexibility and solvent exposure are key characteristics of its hormonogenic tyrosine pairs, and provides a framework for understanding the formation of thyroid hormones.

Reprogramming of mitochondria provides cells with the metabolic flexibility required to adapt to various developmental transitions such as stem cell activation or immune cell reprogramming, and to respond to environmental challenges such as those encountered under hypoxic conditions or during tumorigenesis 1 – 3 . Here we show that the i -AAA protease YME1L rewires the proteome of pre-existing mitochondria in response to hypoxia or nutrient starvation. Inhibition of mTORC1 induces a lipid signalling cascade via the phosphatidic acid phosphatase LIPIN1, which decreases phosphatidylethanolamine levels in mitochondrial membranes and promotes proteolysis. YME1L degrades mitochondrial protein translocases, lipid transfer proteins and metabolic enzymes to acutely limit mitochondrial biogenesis and support cell growth. YME1L-mediated mitochondrial reshaping supports the growth of pancreatic ductal adenocarcinoma (PDAC) cells as spheroids or xenografts. Similar changes to the mitochondrial proteome occur in the tumour tissues of patients with PDAC, suggesting that YME1L is relevant to the pathophysiology of these tumours. Our results identify the mTORC1–LIPIN1–YME1L axis as a post-translational regulator of mitochondrial proteostasis at the interface between metabolism and mitochondrial dynamics. Under conditions such as hypoxia or starvation, an mTORC1-lipid signalling pathway initiates mitochondrial proteolysis by YME1L.