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During endoplasmic-reticulum-associated protein degradation (ERAD), misfolded proteins are polyubiquitinated, extracted from the ER membrane and degraded by the proteasome1–4. In a process called retrotranslocation, misfolded luminal proteins first need to traverse the ER membrane before ubiquitination can occur in the cytosol. It was suggested that the membrane-embedded ubiquitin ligase Hrd1 forms a retrotranslocation pore regulated by cycles of auto- and deubiquitination5–8. However, the mechanism by which auto-ubiquitination affects Hrd1 and allows polypeptides to cross the membrane and whether Hrd1 forms a membrane-spanning pore remained unknown. Here, using purified Hrd1 incorporated into different model membranes, we show that Hrd1 auto-ubiquitination leads to the opening of a pore. Substrate binding increases the pore size and its activity, whereas deubiquitination closes the pore and renders it unresponsive to substrate. We identify two binding sites for misfolded proteins in Hrd1, a low-affinity luminal site and a high-affinity cytoplasmic site formed following auto-ubiquitination of specific lysine residues in Hrd1’s RING domain. We propose that the affinity difference between the luminal and cytoplasmic binding sites provides the initial driving force for substrate movement through Hrd1.Ubiquitin ligase Hrd1 is essential for endoplasmic-reticulum-associated protein degradation. Vasic et al. demonstrate that Hrd1 forms a retrotranslocation channel controlled by auto-ubiquitination and substrate binding.

In endoplasmic reticulum-associated protein degradation (ERAD), membrane proteins are ubiquitinated, extracted from the membrane, and degraded by the proteasome. The cytosolic ATPase Cdc48 drives extraction by pulling on polyubiquitinated substrates. How hydrophobic transmembrane (TM) segments are moved from the phospholipid bilayer into cytosol, often together with hydrophilic and folded ER luminal protein parts, is not known. Using a reconstituted system with purified proteins from Saccharomyces cerevisiae, we show that the ubiquitin ligase Doa10 (Teb-4/MARCH6 in animals) is a retrotranslocase that facilitates membrane protein extraction. A substrate’s TM segment interacts with the membrane-embedded domain of Doa10 and then passively moves into the aqueous phase. Luminal substrate segments cross the membrane in an unfolded state. Their unfolding occurs on the luminal side of the membrane by cytoplasmic Cdc48 action. Our results reveal how a membrane-bound retrotranslocase cooperates with the Cdc48 ATPase in membrane protein extraction. The inside of a cell contains many different compartments called organelles, which are separated by membranes. Each organelle is composed of a unique set of proteins and performs specific roles in the cell. The endoplasmic reticulum, or ER for short, is an organelle where many proteins are produced. Most of these proteins are then released from the cell or sorted to other organelles. The ER has a strict quality control system that ensures any faulty proteins are quickly marked for the cell to destroy. However, the destruction process itself does not happen in the ER, so faulty proteins first need to leave this organelle. This is achieved by a group of proteins known as endoplasmic reticulum-associated protein degradation machinery (or ERAD for short). To extract a faulty protein from the ER, proteins of the ER and outside the ER cooperate. First, an ERAD protein called Doa10 attaches a small protein tag called ubiquitin to the faulty proteins to mark them for destruction. Then, outside of the ER, a protein called Cdc48 ‘grabs’ the ubiquitin tag and pulls. But that is only part of the story. Many of the proteins made by the ER have tethers that anchor them firmly to the membrane, making them much harder to remove. To get a better idea of how the extraction works, Schmidt et al. rebuilt the ERAD machinery in a test tube. This involved purifying proteins from yeast and inserting them into artificial membranes, allowing closer study of each part of the process. This revealed that attaching ubiquitin tags to faulty proteins is only one part of Doa10's role; it also participates in the extraction itself. Part of Doa10 resides within the membrane, and this ‘membrane-spanning domain’ can interact with faulty proteins, loosening their membrane anchors. At the same time, Cdc48 pulls from the outside. This pulling force causes the faulty proteins to unfold, allowing them to pass through the membrane. Given these findings, the next step is to find out exactly how Doa10 works by looking at its three-dimensional structure. This could have implications not only for the study of ERAD, but of similar quality control processes in other organelles too. A build-up of faulty proteins can cause diseases like neurodegeneration, so understanding how cells remove faulty proteins could help future medical research.

Protein–lipid crosstalk is fundamental to homeostasis in the endoplasmic reticulum (ER). The ER-associated degradation (ERAD) pathway, a branch of the ubiquitin–proteasome system, maintains ER membrane properties by degrading lipid metabolic enzymes. However, the ERAD components that sense membrane properties and their mechanisms remain poorly defined. Using reconstituted systems with purified ERAD factors, we show that membrane composition modulates the ubiquitination cascade at multiple levels. The membrane-anchored E2 UBE2J2 acts as a sensor for lipid packing: in loosely packed membranes, UBE2J2 becomes inactive due to membrane association that impedes ubiquitin loading, while tighter packing promotes its active conformation and interaction with E1. UBE2J2 activity directs ubiquitin transfer by the E3 ligases RNF145, MARCHF6, and RNF139, targeting both themselves and the substrate squalene monooxygenase. Additionally, RNF145 senses cholesterol, altering its oligomerization and activity. These findings reveal that ERAD integrates multiple lipid signals, with UBE2J2 relaying and extending the effect of lipid signals through its cooperation with multiple E3 ligases. ER membranes tune protein degradation to lipid composition. Using reconstitution approaches, the authors show that the ubiquitin conjugating enzyme UBE2J2 senses lipid packing, modulating its own and partner enzyme activities; together, they integrate lipid saturation and cholesterol signals.

Summary Advances in biological engineering and systems biology have provided new approaches and tools for the industrialization of biology. In the next decade, advanced biocatalytic systems will increasingly be used for the production of chemicals that cannot be made by current processes and/or where the use of enzyme catalysts is more resource efficient with a much reduced environmental impact. We expect that in the future, manufacture of chemicals and materials will utilize both biocatalytic and chemical synthesis synergistically. The realization of such advanced biomanufacturing processes currently faces a number of major challenges. Ready‐to‐deploy portfolios of biocatalysts for design to production must be created from biological diverse sources and through protein engineering. Robust and efficient multi‐step enzymatic reaction cascades must be developed that can operate simultaneously in one‐pot. For this to happen, bio‐orthogonal strategies for spatial and temporal control of biocatalyst activities must be developed. Promising approaches and technologies are emerging that will eventually lead to the design of in vitro biocatalytic systems that mimic the metabolic pathways and networks of cellular systems which will be discussed in this roadmap. This contribution outlines a roadmap for the next decade of industrial biocatalysis. Current challenges are identified and promising technologies and strategies are discussed for the development of industrially relevant enzyme cascades for in vitro biocatalysis.

Whale sharks are a declining species for which little biological data is available. While these animals are protected in many parts of their range, they are fished legally and illegally in some countries. Baseline biological and ecological data are needed to allow the formulation of an effective conservation plan for whale sharks. It is not known, for example, whether the whale shark is represented by a single worldwide panmictic population or by numerous, reproductively isolated populations. Genetic analysis of population structure is one essential component of the baseline data required for whale shark conservation. We have identified 8 polymorphic microsatellites in the whale shark and used these markers to assess genetic variation and population structure in a panel of whale sharks covering a broad geographic region. This is the first record of microsatellite loci in the whale shark, which displayed an average of 9 alleles per locus and mean H(o) = 0.66 and H(e) = 0.69. All but one of the eight loci meet the expectations of Hardy-Weinberg equilibrium. Analysis of these loci in whale sharks representing three major portions of their range, the Pacific (P), Caribbean (C), and Indian (I) Oceans, determined that there is little population differentiation between animals sampled in different geographic regions, indicating historical gene flow between populations. F(ST) values for inter-ocean comparisons were low (PxC = 0.0387, CxI = 0.0296 and PxI = -0.0022), and only CxI approached statistical significance (p = 0.0495). We have shown only low levels of genetic differentiation between geographically distinct whale shark populations. Existing satellite tracking data have revealed both regional and long-range migration of whale sharks throughout their range, which supports the finding of gene flow between populations. Whale sharks traverse geographic and political boundaries during their life history and interbreed with animals from distant populations; conservation efforts must therefore target international protection for this species.

The west Greenland shelf is a dynamic marine environment influenced by various physicochemical and biological processes. This study provides a comprehensive overview of the main factors affecting the distribution of macronutrients, carbonate system parameters, and dissolved trace elements during July. Key drivers include major ocean currents, melting sea ice, and terrestrial freshwater runoff, each contributing uniquely to the cycling and spatial distribution of chemical constituents. Major ocean currents, such as the southward-moving Baffin Island Current (BIC) and the northward-moving West Greenland Current (WGC), introduce water masses with distinct chemical signatures that shape the chemical composition of shelf waters. Melting sea ice serves as an important source of freshwater and dissolved constituents for the marine environment. During the study period, we were able to capture a distinct nutrient gradient following the east-to-west direction of sea ice retreat, with low nutrient levels in highly productive shelf waters and high nutrient levels in areas with prolonged ice cover. This process also influenced the carbonate system, leading to changes in pH and aragonite saturation states, both of which are is critical to the health of marine organisms. Terrestrial freshwater runoff, particularly from the Greenland Ice Sheet (GIS), replenishes macronutrients in the photic zone, stimulating primary production and creating important CO2 sinks. However, coastal surface waters become more susceptible to acidification by the input of poorly buffered glacial freshwater. Understanding these key drivers is essential for predicting future changes in the marine chemistry and ecosystem dynamics on the west Greenland shelf, especially in the context of ongoing climate change within this high-latitude region.

Background The motor‐cognitive syndrome apraxia is a common stroke sequela and severely affects the outcome after stroke by impairing activities of daily living. Notably, like in many health conditions, there is a massive backlog regarding studies on sex differences in patients with apraxia despite common knowledge that sex influences praxis performance in healthy participants. We investigated putative sex differences in apraxic stroke patients at the behavioral and neural levels. Methods We retrospectively analysed the data of a cohort of 102 left‐hemisphere stroke patients in the (sub)acute phase who were apraxic according to the Cologne Apraxia Screening (KAS). We conducted voxel‐based lesion–symptom mapping (VLSM) to elucidate the lesion patterns. Further, in an age‐matched subsample (tolerance of 5 years) with equal numbers of men and women, behavioral comparisons and a VLSM analysis were conducted to explore differential sex‐related lesion patterns. Results Overall, apraxic deficits were associated with lesions in the parietal, temporal, and frontal regions in the cohort of 102 left‐hemisphere stroke patients. The age‐matched cohort consisted of 30 women and 30 men and showed no significant differences in demographic and clinical characteristics. There were no performance differences between men and women at the behavioral level regarding praxis functions. In contrast, VLSM revealed differential lesion patterns by sex. Male compared to female apraxic stroke patients significantly more often showed lesions that affected the left inferior frontal gyrus. Conclusion The data suggest a differential organization of the praxis system in men and women, warranting further exploration.

While the role of cortical regions in cognitive control processes is well accepted, the contribution of subcortical structures (e.g., the striatum), especially to the control of response interference, remains controversial. Therefore, the present study aimed to investigate the cortical and particularly subcortical neural mechanisms of response interference control (including selective inhibition). Thirteen healthy young participants underwent event-related functional magnetic resonance imaging while performing a unimanual version of the Simon task. In this task, successful performance required the resolution of stimulus–response conflicts in incongruent trials by selectively inhibiting interfering response tendencies. The behavioral results show an asymmetrical Simon effect that was more pronounced in the contralateral hemifield. Contrasting incongruent trials with congruent trials (i.e., the overall Simon effect) significantly activated clusters in the right anterior cingulate cortex, the right posterior insula, and the caudate nucleus bilaterally. Furthermore, a region of interest analysis based on previous patient studies revealed that activation in the bilateral caudate nucleus significantly co-varied with a parameter of selective inhibition derived from distributional analyses of response times. Our results corroborate the notion that the cognitive control of response interference is supported by a fronto-striatal circuitry, with a functional contribution of the caudate nucleus to the selective inhibition of interfering response tendencies.