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Moyamoya disease is a rare cause of stroke, radiologically characterised by progressive stenosis of the terminal portion of the internal carotid arteries and compensatory capillary collaterals. The discovery that RNF213, which encodes an unconventional E3 ubiquitin ligase, is the major susceptibility gene for moyamoya disease in people from east Asia has opened new avenues for investigation into the mechanisms of disease and potential treatment targets. The Arg4810Lys variant of the gene is most strongly associated with moyamoya disease, but the penetrance is lower than 1%, suggesting a synergistic relationship with additional environmental and genetic risk factors. White people carry less common non-Arg4810Lys variants of RNF213, which partly explains the lower prevalence of moyamoya disease in European countries and in the USA than in east Asian countries. Several monogenic moyamoya syndromes possess the radiological characteristics of moyamoya disease and have been associated with multiple genes and pathways involved in moyamoya angiopathy pathogenesis. Further clarification of the genetic and environmental factors that contribute to the emergence of moyamoya angiopathy could enable development of new treatment strategies for moyamoya disease.

RNF213 is the major susceptibility factor for Moyamoya disease, a progressive cerebrovascular disorder that often leads to brain stroke in adults and children. Characterization of disease-associated mutations has been complicated by the enormous size of RNF213. Here, we present the cryo-EM structure of mouse RNF213. The structure reveals the intricate fold of the 584 kDa protein, comprising an N-terminal stalk, a dynein-like core with six ATPase units, and a multidomain E3 module. Collaboration with UbcH7, a cysteine-reactive E2, points to an unexplored ubiquitin-transfer mechanism that proceeds in a RING-independent manner. Moreover, we show that pathologic MMD mutations cluster in the composite E3 domain, likely interfering with substrate ubiquitination. In conclusion, the structure of RNF213 uncovers a distinct type of an E3 enzyme, highlighting the growing mechanistic diversity in ubiquitination cascades. Our results also provide the molecular framework for investigating the emerging role of RNF213 in lipid metabolism, hypoxia, and angiogenesis. Moyamoya disease is a genetic disorder affecting both adults and children. It is characterized by narrowing of the blood vessels in the brain, which can lead to strokes. Moyamoya patients often have mutations in the gene for a protein called RNF213. This protein is linked to multiple processes in the body, including the development of blood vessels. Despite this, its role in Moyamoya disease is still something of a mystery. RNF213 is known to fall into two protein ‘classes’. First, it is an E3 enzyme. This type of protein tags unwanted or defective proteins for disposal by the cell. Second, it is a motor protein. Motor proteins contain tiny molecular ‘engines’, called ATPases, that normally convert chemical energy to movement. No other human protein combines these two activities, making RNF213 unique. RNF213 is also an extremely large protein, which means it is difficult to manipulate in the laboratory and thus hard to study. Scientists still need more detailed information on RNF213’s structure and chemical activity before we can understand what the mutant protein might be doing in Moyamoya disease. Ahel et al. therefore set out to make the RNF213 protein and ‘dissect’ it in a test tube. Electron microscopy experiments using the mouse-version of RNF213 revealed that it consisted of a single, giant molecule, folded up to form three regions with distinct structures. These were a long ‘arm’ at one end, a ring-shaped part in the middle, containing the ATPase ‘motor’, and the E3 enzyme module at the other end. Further chemical analysis showed that RNF213’s ATPase and E3 modules worked in unexpected ways. Although the ATPase did resemble another well-known motor protein, in RNF213 it did not generate movement but rather appeared to act like an intricate molecular ‘switch’. The E3 module of RNF213 ‘tagged’ other molecules as expected but did not contain an additional structure that all other known E3 enzymes need to work properly. This suggests that RNF213 represents a distinct class of E3 enzymes. Biochemical tests of the mutation most commonly found in Moyamoya patients revealed that it left RNF213’s overall structure, ATPase motor and E3 module intact. That is, the disease-causing mutation appeared to hinder interactions with other partner proteins, rather than disrupting RNF213 itself. By providing the first detailed molecular description of the architecture of RNF213, Ahel et al. hope that these findings will help future investigations of both this giant protein’s biological role in the cell and its contribution to Moyamoya disease.

Protein turnover is a tightly controlled process that is crucial for the removal of aberrant polypeptides and for cellular signalling. Whereas ubiquitin marks eukaryotic proteins for proteasomal degradation, a general tagging system for the equivalent bacterial Clp proteases is not known. Here we describe the targeting mechanism of the ClpC–ClpP proteolytic complex from Bacillus subtilis . Quantitative affinity proteomics using a ClpP-trapping mutant show that proteins phosphorylated on arginine residues are selectively targeted to ClpC–ClpP. In vitro reconstitution experiments demonstrate that arginine phosphorylation by the McsB kinase is required and sufficient for the degradation of substrate proteins. The docking site for phosphoarginine is located in the amino-terminal domain of the ClpC ATPase, as resolved at high resolution in a co-crystal structure. Together, our data demonstrate that phosphoarginine functions as a bona fide degradation tag for the ClpC–ClpP protease. This system, which is widely distributed across Gram-positive bacteria, is functionally analogous to the eukaryotic ubiquitin–proteasome system. In Gram-positive bacteria, arginine phosphorylation by the McsB kinase functions as a general post-translational marker for Clp-mediated proteolysis. Degradation signal for the ClpC–ClpP proteolytic complex Protein ubiquitination can mark proteins for degradation by the proteasome in eukaryotic cells, but it is unknown whether such a tagging system for the equivalent bacterial Clp proteases exists. Here, Tim Clausen and colleagues report that arginine phosphorylation by the McsB kinase functions as a general post-translational marker for proteasomal degradation in Gram-positive bacteria, tagging at least 25% of all proteins degraded by Clp. The phosphoarginine degradation pathway is essential to cope with proteotoxic stress in vivo .

The Ndc80 complex is the key microtubule‐binding element of the kinetochore. In contrast to the well‐characterized interaction of Ndc80‐Nuf2 heads with microtubules, little is known about how the Spc24‐25 heterodimer connects to centromeric chromatin. Here, we present molecular details of Spc24‐25 in complex with the histone‐fold protein Cnn1/CENP‐T illustrating how this connection ultimately links microtubules to chromosomes. The conserved Ndc80 receptor motif of Cnn1 is bound as an α helix in a hydrophobic cleft at the interface between Spc24 and Spc25. Point mutations that disrupt the Ndc80–Cnn1 interaction also abrogate binding to the Mtw1 complex and are lethal in yeast. We identify a Cnn1‐related motif in the Dsn1 subunit of the Mtw1 complex, necessary for Ndc80 binding and essential for yeast growth. Replacing this region with the Cnn1 peptide restores viability demonstrating functionality of the Ndc80‐binding module in different molecular contexts. Finally, phosphorylation of the Cnn1 N‐terminus coordinates the binding of the two competing Ndc80 interaction partners. Together, our data provide structural insights into the modular binding mechanism of the Ndc80 complex to its centromere recruiters. The yeast kinetochore receptors Cnn1 and Dsn1 employ a related peptide motif to recruit the microtubule‐binding Ndc80 complex in a competitive, phosphorylation‐regulated manner.

Eukaryotes have evolved various quality control mechanisms to promote proteostasis in the endoplasmic reticulum (ER). Selective removal of certain ER domains via autophagy (termed as ER-phagy) has emerged as a major quality control mechanism. However, the degree to which ER-phagy is employed by other branches of ER-quality control remains largely elusive. Here, we identify a cytosolic protein, C53, that is specifically recruited to autophagosomes during ER-stress, in both plant and mammalian cells. C53 interacts with ATG8 via a distinct binding epitope, featuring a shuffled ATG8 interacting motif (sAIM). C53 senses proteotoxic stress in the ER lumen by forming a tripartite receptor complex with the ER-associated ufmylation ligase UFL1 and its membrane adaptor DDRGK1. The C53/UFL1/DDRGK1 receptor complex is activated by stalled ribosomes and induces the degradation of internal or passenger proteins in the ER. Consistently, the C53 receptor complex and ufmylation mutants are highly susceptible to ER stress. Thus, C53 forms an ancient quality control pathway that bridges selective autophagy with ribosome-associated quality control in the ER. For cells to survive they need to be able to remove faulty or damaged components. The ability to recycle faulty parts is so crucial that some of the molecular machinery responsible is the same across the plant and animal kingdoms. One of the major recycling pathways cells use is autophagy, which labels damaged proteins with molecular tags that say 'eat-me'. Proteins called receptors then recognize these tags and move the faulty component into vesicles that transport the cargo to a specialized compartment that recycles broken parts. Cells make and fold around 40% of their proteins at a site called the endoplasmic reticulum, or ER for short. However, the process of folding and synthesizing proteins is prone to errors. For example, when a cell is under stress this can cause a ‘stall’ in production, creating a build-up of faulty, partially constructed proteins that are toxic to the cell. There are several quality control systems which help recognize and correct these errors in production. Yet, it remained unclear how autophagy and these quality control mechanisms are linked together. Here, Stephani, Picchianti et al. screened for receptors that regulate the recycling of faulty proteins by binding to the ‘eat-me’ tags. This led to the identification of a protein called C53, which is found in both plant and animal cells. Microscopy and protein-protein interaction tests showed that C53 moves into transport vesicles when the ER is under stress and faulty proteins start to build-up. Once there, C53 interacts with two proteins embedded in the wall of the endoplasmic reticulum. These proteins form part of the quality control system that senses stalled protein production, labelling the stuck proteins with ‘eat-me’ tags. Together with C53, they identify and remove half-finished proteins before they can harm the cell. The fact that C53 works in the same way in both plant and human cells suggests that many species might use this receptor to recycle stalled proteins. This has implications for a wide range of research areas, from agriculture to human health. A better understanding of C53 could be beneficial for developing stress-resilient crops. It could also aid research into human diseases, such as cancer and viral infections, that have been linked to C53 and its associated proteins.

The serine protease HTRA1 utilizes a \"disintegration\" mechanism involving its flexible PDZ domains to first loosen tau amyloid fibrils and subsequently disintegrating the fibrillar core structure for efficient proteolytic degradation. Excessive aggregation of proteins has a major impact on cell fate and is a hallmark of amyloid diseases in humans. To resolve insoluble deposits and to maintain protein homeostasis, all cells use dedicated protein disaggregation, protein folding and protein degradation factors. Despite intense recent research, the underlying mechanisms controlling this key metabolic event are not well understood. Here, we analyzed how a single factor, the highly conserved serine protease HTRA1, degrades amyloid fibrils in an ATP-independent manner. This PDZ protease solubilizes protein fibrils and disintegrates the fibrillar core structure, allowing productive interaction of aggregated polypeptides with the active site for rapid degradation. The aggregate burden in a cellular model of cytoplasmic tau aggregation is thus reduced. Mechanistic aspects of ATP-independent proteolysis and its implications in amyloid diseases are discussed.

Myosin motors are critical for diverse motility functions, ranging from cytokinesis and endocytosis to muscle contraction. The UNC-45 chaperone controls myosin function mediating the folding, assembly, and degradation of the muscle protein. Here, we analyze the molecular mechanism of UNC-45 as a hub in myosin quality control. We show that UNC-45 forms discrete complexes with folded and unfolded myosin, forwarding them to downstream chaperones and E3 ligases. Structural analysis of a minimal chaperone:substrate complex reveals that UNC-45 binds to a conserved FX 3 HY motif in the myosin motor domain. Disrupting the observed interface by mutagenesis prevents myosin maturation leading to protein aggregation in vivo. We also show that a mutation in the FX 3 HY motif linked to the Freeman Sheldon Syndrome impairs UNC-45 assisted folding, reducing the level of functional myosin. These findings demonstrate that a faulty myosin quality control is a critical yet unexplored cause of human myopathies. The quality control of muscle myosin relies on the chaperone UNC-45. The chaperone binds folded and misfolded myosin and channel them into folding and degradation pathways. Interaction with UNC-45 is mediated by a conserved FX 3 HY motif, carrying myopathy mutations that can cause myosin to aggregate.

All organisms have to monitor the folding state of cellular proteins precisely. The heat-shock protein DegP is a protein quality control factor in the bacterial envelope that is involved in eliminating misfolded proteins and in the biogenesis of outer-membrane proteins. Here we describe the molecular mechanisms underlying the regulated protease and chaperone function of DegP from Escherichia coli . We show that binding of misfolded proteins transforms hexameric DegP into large, catalytically active 12-meric and 24-meric multimers. A structural analysis of these particles revealed that DegP represents a protein packaging device whose central compartment is adaptable to the size and concentration of substrate. Moreover, the inner cavity serves antagonistic functions. Whereas the encapsulation of folded protomers of outer-membrane proteins is protective and might allow safe transit through the periplasm, misfolded proteins are eliminated in the molecular reaction chamber. Oligomer reassembly and concomitant activation on substrate binding may also be critical in regulating other HtrA proteases implicated in protein-folding diseases. Basis for DegP chaperone function DegP is a protease-chaperone in the bacterial envelope that is involved in protein quality control and outer membrane protein (OMP) biogenesis. In this study Clausen and colleagues show that binding of misfolded proteins transforms hexameric DegP into 12- and 24-meric multimers. Structural analysis of these particles uncovered a protein-packaging device whose central compartment is adaptable to the size and concentration of substrate. Moreover, the inner cavity of DegP serves antagonistic functions ensuring the safe transit of folded OMP protomers through the periplasm, while eliminating misfolded proteins.

Tripartite motif 52 (TRIM52) exhibits strong positive selection in humans, yet is lost in many other mammals. In contrast to what one would expect for such a non-conserved factor, TRIM52 loss compromises cell fitness. We set out to determine the cellular function of TRIM52. Genetic and proteomic analyses revealed TRIM52 physically and functionally interacts with the DNA repair machinery. Our data suggest that TRIM52 limits topoisomerase 2 adducts, thereby preventing cell-cycle arrest. Consistent with a fitness-promoting function, TRIM52 is upregulated in various cancers, prompting us to investigate its regulatory pathways. We found TRIM52 to be targeted for ultra-rapid proteasomal degradation by the giant E3 ubiquitin ligases BIRC6, HUWE1, and UBR4/KCMF1. BIRC6 mono-ubiquitinates TRIM52, with subsequent extension by UBR4/KCMF1. These findings suggest a role for TRIM52 in maintaining genome integrity, and regulation of its own abundance through multi-ligase degradation. Despite not conserved in all mammals, TRIM52 is crucial for fitness in human cells. Here, the authors show that TRIM52 interacts with the DNA repair machinery to prevent cell-cycle arrest, and that TRIM52 is rapidly degraded by multiple giant E3 ligases. These findings link TRIM52 to genome integrity control.

Antimicrobial resistance is a global health threat that requires the development of new treatment concepts. These should not only overcome existing resistance but be designed to slow down the emergence of new resistance mechanisms. Targeted protein degradation, whereby a drug redirects cellular proteolytic machinery towards degrading a specific target, is an emerging concept in drug discovery. We are extending this concept by developing proteolysis targeting chimeras active in bacteria (BacPROTACs) that bind to ClpC1, a component of the mycobacterial protein degradation machinery. The anti- Mycobacterium tuberculosis ( Mtb ) BacPROTACs are derived from cyclomarins which, when dimerized, generate compounds that recruit and degrade ClpC1. The resulting Homo-BacPROTACs reduce levels of endogenous ClpC1 in Mycobacterium smegmatis and display minimum inhibitory concentrations in the low micro- to nanomolar range in mycobacterial strains, including multiple drug-resistant Mtb isolates. The compounds also kill Mtb residing in macrophages. Thus, Homo-BacPROTACs that degrade ClpC1 represent a different strategy for targeting Mtb and overcoming drug resistance. Antimicrobial resistance is a global health threat and the development of alternative strategies to overcome it is of high interest. Here, the authors report proteolysis targeting chimeras active in bacteria (BacPROTACs) that bind to ClpC1, a component of the mycobacterial protein degradation machinery, and apply them for targeting a range of mycobacterial strains, including antibiotic-resistant ones.