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Background Anti‐Activin Receptor Type IIA and Type IIB antibody (αActRIIA/IIB ab) is a recently developed drug class that targets the activin receptor signalling pathway. Inhibition of receptor ligands (activins, myostatin, growth differentiation factor 11, etc.) can lead to skeletal muscle hypertrophy, bone formation, and increased haematopoiesis. Despite the αActRIIA/IIB ab, bimagrumab, having progressed to clinical trials, two crucial questions about αActRIIA/IIB ab therapy remain: Does αActRIIA/IIB ab influence bone metabolism and bone strength similarly to its generic classmates (activin receptor‐based ligand traps)? Does αActRIIA/IIB ab affect red blood cell parameters, thereby increasing the risk of thromboembolism, similar to its generic classmates? Therefore, the aim of the present study was to investigate the therapeutic potential of αActRIIA/IIB ab in a mouse model of concurrent sarcopenia and osteopenia and to investigate the effect on bone and haematopoiesis in more detail. Methods In C57BL/6JRj mice, combined sarcopenia and osteopenia were induced locally by injecting botulinum toxin A into the right hindlimb, resulting in acute muscle paresis. Immediately after immobilization, mice received twice‐weekly intraperitoneal injections with αActRIIA/IIB ab (10 mg/kg) for 21 days, after which they were sacrificed. Muscle mass, skeletal muscle fibre size and Smad2 expression were analysed in the rectus femoris and gastrocnemius muscles. Bone mass and bone microstructure were analysed in the trabecular bone at the distal femoral metaphysis, while the cortical bone was analysed at the femoral mid‐diaphysis. In a substudy, the effect on haematopoiesis was explored 2 and 7 days after a single αActRIIA/IIB ab (30 mg/kg) injection in C57BL/6JRj mice. Results αActRIIA/IIB ab caused a large increase in muscle mass in both healthy (+21%) and immobilized (sarcopenic and osteopenic) (+12%) mice. Furthermore, αActRIIA/IIB ab increased trabecular bone (bone volume fraction) for both healthy (+65%) and immobilized (+44%) mice. For cortical bone, αActRIIA/IIB ab caused a small, but significant, increase in bone area (+6%) for immobilized mice, but not for healthy mice. Treatment with αActRIIA/IIB ab did not change red blood cell count, haemoglobin concentration or mean cell volume after either 2 or 7 days. Conclusions Treatment with αActRIIA/IIB ab caused a significant increase in both skeletal muscle mass and bone parameters in both healthy and immobilized mice, suggesting a potential in the treatment of concurrent osteopenia and sarcopenia. Interestingly, the bone anabolic effect of the treatment was much more pronounced on trabecular bone than on cortical bone. There was no pronounced effect of short‐term treatment on haematopoiesis.

Getting to grips with vitamin B 12 Vitamin B 12 , or cobalamin (Cbl), is an essential coenzyme in mammals that has to be taken up from the diet. Intestinal uptake of the 'extrinsic factor' Cbl is a highly specific process, dependent on gastric intrinsic factor (IF) and the ileal endocytic cubam receptor formed by cubilin and amnionless. Loss of function of any of these proteins ultimately leads to Cbl deficiency in humans. The crystal structure of the complex between IF–Cbl and the cubilin IF–Cbl-binding region (CUB 5–8 ) has now been determined at 3.3 Å resolution. The structure illuminates how multiple CUB domains collectively function as modular ligand-binding regions, and how two distant CUB domains bind the two IF domains in a Ca 2+ -dependent fashion. Vitamin B 12 (cobalamin) is an essential coenzyme in mammals, and is taken up from the diet. The proteins required for its uptake are the gastric intrinsic factor (IF) and the ileal endocytic cubam receptor, which is in turn formed from the proteins cubilin and amnionless. Here, the crystal structure is presented of the complex between IF–cobalamin and the IF–cobalamin-binding region (CUB) of cubilin. The structure illustrates how numerous CUB domains function together as modular ligand-binding regions. Cobalamin (Cbl, vitamin B 12 ) is a bacterial organic compound and an essential coenzyme in mammals, which take it up from the diet. This occurs by the combined action of the gastric intrinsic factor (IF) and the ileal endocytic cubam receptor formed by the 460-kilodalton (kDa) protein cubilin and the 45-kDa transmembrane protein amnionless 1 , 2 . Loss of function of any of these proteins ultimately leads to Cbl deficiency in man 3 , 4 . Here we present the crystal structure of the complex between IF–Cbl and the cubilin IF–Cbl-binding-region (CUB 5–8 ) 5 determined at 3.3 Å resolution. The structure provides insight into how several CUB (for ‘complement C1r/C1s, Uegf, Bmp1’) domains collectively function as modular ligand-binding regions, and how two distant CUB domains embrace the Cbl molecule by binding the two IF domains in a Ca 2+ -dependent manner. This dual-point model provides a probable explanation of how Cbl indirectly induces ligand–receptor coupling. Finally, the comparison of Ca 2+ -binding CUB domains and the low-density lipoprotein (LDL) receptor-type A modules suggests that the electrostatic pairing of a basic ligand arginine/lysine residue with Ca 2+ -coordinating acidic aspartates/glutamates is a common theme of Ca 2+ -dependent ligand–receptor interactions.

The brain uptake of biotherapeutics for brain diseases is hindered by the blood–brain barrier (BBB). The BBB selectively regulates the transport of large molecules into the brain and thereby maintains brain homeostasis. Receptor-mediated transcytosis (RMT) is one mechanism to deliver essential proteins into the brain parenchyma. Receptors expressed in the brain endothelial cells have been explored to ferry therapeutic antibodies across the BBB in bifunctional antibody formats. In this study, we generated and characterized monoclonal antibodies (mAbs) binding to the basigin receptor, which recently has been proposed as a target for RMT across the BBB. Antibody binding properties such as affinity have been demonstrated to be important factors for transcytosis capability and efficiency. Nevertheless, studies of basigin mAb properties' effect on RMT are limited. Here we characterize different basigin mAbs for their ability to associate with and subsequently internalize human brain endothelial cells. The mAbs were profiled to determine whether receptor binding epitope and affinity affected receptor-mediated uptake efficiency. By competitive epitope binning studies, basigin mAbs were categorized into five epitope bins. mAbs from three of the epitope bins demonstrated properties required for RMT candidates judged by binding characteristics and their superior level of internalization in human brain endothelial cells.

CD163, a macrophage-specific receptor, plays a critical role in scavenging hemoglobin released during hemolysis, protecting against oxidative effects of heme iron. In the bloodstream, hemoglobin is bound by haptoglobin, leading to its immediate endocytosis by CD163. While haptoglobin’s structure and function are well understood, CD163’s structure and its interaction with the haptoglobin-hemoglobin complex have remained elusive. Here, we present the cryo-electron microscopy structure of the entire extracellular domain of human CD163 in complex with haptoglobin-hemoglobin. The structure reveals that CD163 assembles into trimers (and to some extent dimers), binding haptoglobin-hemoglobin in their center. Key acidic residues in CD163 interact with lysine residues from both haptoglobin and hemoglobin. Calcium-binding sites located near the haptoglobin-hemoglobin interface in CD163 provide explanation for the calcium dependence of the interaction. Furthermore, we show that the interaction facilitating CD163 oligomerization mimics ligand binding and is also calcium dependent. This structural insight into CD163 advances our understanding of its role in hemoglobin scavenging as well as its broader relevance to structurally related scavenger receptors. CD163, a macrophage receptor, is essential for clearing hemoglobin during hemolysis to prevent oxidative damage. Here, the authors reveal the cryo-electron microscopy structure of CD163 bound to haptoglobin-hemoglobin, uncovering calcium-dependent interactions critical for its function and oligomerization.

The endocytic receptor cubam formed by the 460-kDa protein cubilin and the 45-kDa transmembrane protein amnionless (AMN), is essential for intestinal vitamin B 12 (B 12 ) uptake and for protein (e.g. albumin) reabsorption from the kidney filtrate. Loss of function of any of the two components ultimately leads to serious B 12 deficiency and urinary protein loss in humans (Imerslund-Gräsbeck’s syndrome, IGS). Here, we present the crystal structure of AMN in complex with the amino-terminal region of cubilin, revealing a sophisticated assembly of three cubilin subunits combining into a single intertwined β-helix domain that docks to a corresponding three-faced β-helix domain in AMN. This β-helix-β-helix association thereby anchors three ligand-binding cubilin subunits to the transmembrane AMN. Electron microscopy of full-length cubam reveals a 700–800 Å long tree-like structure with the potential of dimerization into an even larger complex. Furthermore, effects of known human mutations causing IGS are explained by the structural information. Cubilin and the transmembrane protein amnionless (AMN) form the endocytic receptor cubam that is essential for intestinal vitamin B 12 uptake. Here the authors present the 2.3 Å crystal structure of AMN in complex with the amino-terminal region of cubilin and discuss cubam architecture and disease causing mutations.

Cutibacterium acnes is an abundant skin commensal with several proposed mutualistic functions. A protein with strong antioxidant activity was recently identified from the C . acnes secretome. This protein, termed RoxP, facilitated aerobic bacterial growth in vitro and ex vivo . As reducing events naturally occurred outside of the bacterial cell, it was further hypothesized that RoxP could also serve to modulate redox status of human skin. The biological function of RoxP was here assessed in vitro and in vivo , through oxidatively stressed cell cultures and through protein quantification from skin affected by oxidative disease (actinic keratosis and basal cell carcinoma), respectively. 16S rDNA amplicon deep sequencing and single locus sequence typing was used to correlate bacterial prevalence to cutaneous RoxP abundances. We show that RoxP positively influence the viability of monocytes and keratinocytes exposed to oxidative stress, and that a congruent concentration decline of RoxP can be observed in skin affected by oxidative disease. Basal cell carcinoma was moreover associated with microbial dysbiosis, characterized by reduced C . acnes prevalence. C . acnes ’s secretion of RoxP, an exogenous but naturally occurring antioxidant on human skin, is likely to positively influence the human host. Results furthermore attest to its prospective usability as a biopharmaceutical.

Cutibacterium acnes is a predominant bacterium on human skin and is generally regarded as commensal. Recently, the abundantly secreted protein produced by C. acnes , RoxP, was shown to alleviate radical-induced cell damage, presumably via antioxidant activity, which could potentially be harnessed to fortify skin barrier function. The aim of this study was to determine the structure of RoxP and elucidate the mechanisms behind its antioxidative effect. Here, we present the solution structure of RoxP revealing a compact immunoglobulin-like domain containing a long flexible loop which, in concert with the core domain, forms a positively charged groove that could function as a binding site for cofactors or substrates. Although RoxP shares structural features with cell-adhesion proteins, we show that it does not appear to be responsible for adhesion of C. acnes bacteria to human keratinocytes. We identify two tyrosine-containing stretches located in the flexible loop of RoxP, which appear to be responsible for the antioxidant activity of RoxP.

This study reports the crystal structure of porcine haptoglobin in complex with haemoglobin at 2.9 Å resolution; this provides a structural basis of haptoglobin-mediated recognition of haemoglobin, and insight into the protective role of haptoglobin at the atomic level. How haptoglobin neutralizes free haemoglobin The release of extracellular haemoglobin into the plasma is potentially hazardous because the exposed haem group is highly reactive and therefore toxic. The circulating protein haptoglobin counters this by soaking up free haemoglobin in a stable complex that is cleared from the blood through its binding to the macrophage scavenger receptor CD163. This paper presents the 2.9-ångström crystal structure of the dimeric porcine haptoglobin–haemoglobin complex. The structure provides a mechanism for haptoglobin-mediated recognition of haemoglobin. Red cell haemoglobin is the fundamental oxygen-transporting molecule in blood, but also a potentially tissue-damaging compound owing to its highly reactive haem groups. During intravascular haemolysis, such as in malaria and haemoglobinopathies 1 , haemoglobin is released into the plasma, where it is captured by the protective acute-phase protein haptoglobin. This leads to formation of the haptoglobin–haemoglobin complex, which represents a virtually irreversible non-covalent protein–protein interaction 2 . Here we present the crystal structure of the dimeric porcine haptoglobin–haemoglobin complex determined at 2.9 Å resolution. This structure reveals that haptoglobin molecules dimerize through an unexpected β-strand swap between two complement control protein (CCP) domains, defining a new fusion CCP domain structure. The haptoglobin serine protease domain forms extensive interactions with both the α- and β-subunits of haemoglobin, explaining the tight binding between haptoglobin and haemoglobin. The haemoglobin-interacting region in the αβ dimer is highly overlapping with the interface between the two αβ dimers that constitute the native haemoglobin tetramer. Several haemoglobin residues prone to oxidative modification after exposure to haem-induced reactive oxygen species are buried in the haptoglobin–haemoglobin interface, thus showing a direct protective role of haptoglobin. The haptoglobin loop previously shown to be essential for binding of haptoglobin–haemoglobin to the macrophage scavenger receptor CD163 (ref. 3 ) protrudes from the surface of the distal end of the complex, adjacent to the associated haemoglobin α-subunit. Small-angle X-ray scattering measurements of human haptoglobin–haemoglobin bound to the ligand-binding fragment of CD163 confirm receptor binding in this area, and show that the rigid dimeric complex can bind two receptors. Such receptor cross-linkage may facilitate scavenging and explain the increased functional affinity of multimeric haptoglobin–haemoglobin for CD163 (ref. 4 ).

BACKGROUNDProteinuria is considered an unfavorable clinical condition that accelerates renal and cardiovascular disease. However, it is not clear whether all forms of proteinuria are damaging. Mutations in CUBN cause Imerslund-Gräsbeck syndrome (IGS), which is characterized by intestinal malabsorption of vitamin B12 and in some cases proteinuria. CUBN encodes for cubilin, an intestinal and proximal tubular uptake receptor containing 27 CUB domains for ligand binding.METHODSWe used next-generation sequencing for renal disease genes to genotype cohorts of patients with suspected hereditary renal disease and chronic proteinuria. CUBN variants were analyzed using bioinformatics, structural modeling, and epidemiological methods.RESULTSWe identified 39 patients, in whom biallelic pathogenic variants in the CUBN gene were associated with chronic isolated proteinuria and early childhood onset. Since the proteinuria in these patients had a high proportion of albuminuria, glomerular diseases such as steroid-resistant nephrotic syndrome or Alport syndrome were often the primary clinical diagnosis, motivating renal biopsies and the use of proteinuria-lowering treatments. However, renal function was normal in all cases. By contrast, we did not found any biallelic CUBN variants in proteinuric patients with reduced renal function or focal segmental glomerulosclerosis. Unlike the more N-terminal IGS mutations, 37 of the 41 proteinuria-associated CUBN variants led to modifications or truncations after the vitamin B12-binding domain. Finally, we show that 4 C-terminal CUBN variants are associated with albuminuria and slightly increased GFR in meta-analyses of large population-based cohorts.CONCLUSIONCollectively, our data suggest an important role for the C-terminal half of cubilin in renal albumin reabsorption. Albuminuria due to reduced cubilin function could be an unexpectedly common benign condition in humans that may not require any proteinuria-lowering treatment or renal biopsy.FUNDINGATIP-Avenir program, Fondation Bettencourt-Schueller (Liliane Bettencourt Chair of Developmental Biology), Agence Nationale de la Recherche (ANR) Investissements d'avenir program (ANR-10-IAHU-01) and NEPHROFLY (ANR-14-ACHN-0013, to MS), Steno Collaborative Grant 2018 (NNF18OC0052457, to TSA and MS), Heisenberg Professorship of the German Research Foundation (KO 3598/5-1, to AK), Deutsche Forschungsgemeinschaft (DFG) Collaborative Research Centre (SFB) KIDGEM 1140 (project 246781735, to CB), and Federal Ministry of Education and Research (BMB) (01GM1515C, to CB).