The amyloid precursor protein (APP) and its neurotoxic cleavage product Aβ are key players in the development of Alzheimer's disease (AD) and appear to be essential for neuronal development and cell homeostasis. Proteolytic processing of APP and its physiological function depend on its interaction with heparin and are influenced by the binding of metal ions and sorLA. We created various mutations of metal binding site M1 residing within the extracellular E2 domain of APP. Using isothermal titration calorimetry and circular dichroism spectroscopy, we analyzed the binding of Cu(2+) and Zn(2+) to APP E2 and identified two mutations that are most suited for functional studies to dissect ion specific effects of metal binding. The H313A mutation abrogates only copper-based effects, whereas the H382A mutation weakens any metal binding at M1 of APP E2. Subsequently, we tested the effect of Cu(2+) and Zn(2+) on the binding of heparin and sorLA to APP E2 using a chromatographic technique and surface plasmon resonance. We show that Zn(2+) and to a larger degree also Cu(2+) enhance the binding of heparin to APP E2, consistent with an extracellular regulation of the function of APP by both metal ions. In contrast, neither ion seemed to affect the interaction between APP E2 and sorLA. This supports an intracellular interaction between the latter two partners that would not sense extracellular variations of metal ions upon synaptic activity.

Acne vulgaris is a very common disease of the pilosebaceous unit of the human skin. The pathological processes of acne are not fully understood. To gain further insight sebaceous follicular casts were extracted from 18 healthy and 20 acne-affected individuals by cyanoacrylate-gel biopsies and further processed for mass spectrometry analysis, aiming at a proteomic analysis of the sebaceous follicular casts. Human as well as bacterial proteins were identified. Human proteins enriched in acne and normal samples were detected, respectively. Normal follicular casts are enriched in proteins such as prohibitins and peroxiredoxins which are involved in the protection from various stresses, including reactive oxygen species. By contrast, follicular casts extracted from acne-affected skin contained proteins involved in inflammation, wound healing and tissue remodeling. Among the most distinguishing proteins were myeloperoxidase, lactotransferrin, neutrophil elastase inhibitor and surprisingly, vimentin. The most significant biological process among all acne-enriched proteins was 'response to a bacterium'. Identified bacterial proteins were exclusively from Propionibacterium acnes. The most abundant P. acnes proteins were surface-exposed dermatan sulphate adhesins, CAMP factors, and a so far uncharacterized lipase in follicular casts extracted from normal as well as acne-affected skin. This is a first proteomic study that identified human proteins together with proteins of the skin microbiota in sebaceous follicular casts.

The lymphatic vessel endothelial receptor LYVE-1 is implicated in the uptake of hyaluronan (HA) and trafficking of leukocytes to draining lymph nodes. Yet LYVE-1 has only weak affinity for hyaluronan and depends on receptor clustering and higher order ligand organization for durable binding in lymphatic endothelium. An unusual feature of LYVE-1 not found in other HA receptors is the potential to form disulfide-linked homodimers. However, their influence on function has not been investigated. Here we show LYVE-1 homodimers are the predominant configuration in lymphatic endothelium in vitro and in vivo, and formation solely requires the unpaired cysteine residue Cys-201 within the membrane-proximal domain, yielding a 15-fold higher HA binding affinity and an ∼67-fold slower off-rate than the monomer. Moreover, we show non-dimerizing LYVE-1 mutants fail to bind HA even when expressed at high densities in lymphatic endothelial cells or artificially cross-linked with antibody. Consistent with these findings, small angle X-ray scattering (SAXS) indicates the Cys-201 interchain disulfide forms a hinge that maintains the homodimer in an "open scissors" conformation, likely allowing arrangement of the two HA binding domains for mutual engagement with ligand. Finally, we demonstrate the Cys-201 interchain disulfide is highly labile, and selective reduction with TCEP-HCl disrupts LYVE-1 homodimers, ablating HA binding. These findings reveal binding is dependent not just on clustering but also on the biochemical properties of LYVE-1 homodimers. They also mark LYVE-1 as the first Link protein superfamily member requiring covalent homodimerization for function and suggest the interchain disulfide acts as a redox switch in vivo.

Here, we describe a new strategy that allows the rapid and efficient engineering of mono and multispecific trivalent antibodies. By fusing single-domain antibodies from camelid heavy-chain-only immunoglobulins (VHHs) to the N-terminus of a human collagen XVIII trimerization domain (TIE(XVIII)) we produced monospecific trimerbodies that were efficiently secreted as soluble functional proteins by mammalian cells. The purified VHH-TIE(XVIII) trimerbodies were trimeric in solution and exhibited excellent antigen binding capacity. Furthermore, by connecting with two additional glycine-serine-based linkers three VHH-TIE(XVIII) modules on a single polypeptide chain, we present an approach for the rational design of multispecific tandem trimerbodies with defined stoichiometry and controlled orientation. Using this technology we report here the construction and characterization of a tandem VHH-based trimerbody capable of simultaneously binding to three different antigens: carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR) and green fluorescence protein (GFP). Multispecific tandem VHH-based trimerbodies were well expressed in mammalian cells, had good biophysical properties and were capable of simultaneously binding their targeted antigens. Importantly, these antibodies were very effective in inhibiting the proliferation of human epidermoid carcinoma A431 cells. Multispecific VHH-based trimerbodies are therefore ideal candidates for future applications in various therapeutic areas.

Laminins are a major constituent of all basement membranes. The polymerisation of laminins at the cell surface is mediated by the three short arms of the cross-shaped laminin heterotrimer. The short arms contain repeats of laminin-type epidermal growth factor-like (LE) domains, interspersed with globular domains of unknown function. A single LF domain is inserted between LE5 and LE6 of the laminin β1 and β2 chains. We report the crystal structure at 1.85Å resolution of the laminin β2 LE5-LF-LE6 region. The LF domain consists of a β-sandwich related to bacterial family 35 carbohydrate binding modules, and more distantly to the L4 domains present in the short arms of laminin α and γ chains. An α-helical region mediates the extensive interaction of the LF domain with LE5. The relative arrangement of LE5 and LE6 is very similar to that of consecutive LE domains in uninterrupted LE tandems. Fitting atomic models to a low-resolution structure of the first eight domains of the laminin β1 chain determined by small-angle X-ray scattering suggests a deviation from the regular LE array at the LE4-LE5 junction. These results advance our understanding of laminin structure.

Adipose tissue inflammation is believed to play a pivotal role in the development obesity-related morbidities such as insulin resistance. However, it is not known how this (low-grade) inflammatory state develops. It has been proposed that the leakage of lipopolysaccharides (LPS), originating from the gut microbiota, through the gut epithelium could drive initiation of inflammation. To get a better understanding of which proteins and intracellular pathways are affected by LPS in adipocytes, we performed SILAC proteomic analysis and identified proteins that were altered in expression. Furthermore, we tested the anti-inflammatory compound resveratrol. A total of 927 proteins were quantified by the SILAC method and of these 57- and 64 were significantly up- and downregulated by LPS, respectively. Bioinformatic analysis (GO analysis) revealed that the upregulated proteins were especially involved in the pathways of respiratory electron transport chain and inflammation. The downregulated proteins were especially involved in protein glycosylation. One of the latter proteins, GALNT2, has previously been described to regulate the expression of liver lipases such as ANGPTL3 and apoC-III affecting lipid metabolism. Furthermore, LPS treatment reduced the protein levels of the insulin sensitizing adipokine, adiponectin, and proteins participating in the final steps of triglyceride- and cholesterol synthesis. Generally, resveratrol opposed the effect induced by LPS and, as such, functioning as an ameliorating factor in disease state. Using an unbiased proteomic approach, we present novel insight of how the proteome is altered in adipocytes in response to LPS as seen in obesity. We suggest that LPS partly exerts its detrimental effects by altering glycosylation processes of the cell, which is starting to emerge as important posttranscriptional regulators of protein expression. Furthermore, resveratrol could be a prime candidate in ameliorating dysfunctioning adipose tissue induced by inflammatory stimulation.

In the recently characterized Type IX Secretion System (T9SS), the conserved C-terminal domain (CTD) in secreted proteins functions as an outer membrane translocation signal for export of virulence factors to the cell surface in the Gram-negative Bacteroidetes phylum. In the periodontal pathogen Porphyromonas gingivalis, the CTD is cleaved off by PorU sortase in a sequence-independent manner, and anionic lipopolysaccharide (A-LPS) is attached to many translocated proteins, thus anchoring them to the bacterial surface. Here, we solved the atomic structure of the CTD of gingipain B (RgpB) from P. gingivalis, alone and together with a preceding immunoglobulin-superfamily domain (IgSF). The CTD was found to possess a typical Ig-like fold encompassing seven antiparallel β-strands organized in two β-sheets, packed into a β-sandwich structure that can spontaneously dimerise through C-terminal strand swapping. Small angle X-ray scattering (SAXS) revealed no fixed orientation of the CTD with respect to the IgSF. By introducing insertion or substitution of residues within the inter-domain linker in the native protein, we were able to show that despite the region being unstructured, it nevertheless is resistant to general proteolysis. These data suggest structural motifs located in the two adjacent Ig-like domains dictate the processing of CTDs by the T9SS secretion pathway.

Progranulin (PGRN), a widely secreted growth factor, is involved in multiple biological functions, and mutations located within the PGRN gene (GRN) are a major cause of frontotemporal lobar degeneration with TDP-43-positive inclusions (FLTD-TDP). In light of recent reports suggesting PGRN functions as a protective neurotrophic factor and that sortilin (SORT1) is a neuronal receptor for PGRN, we used a Sort1-deficient (Sort1-/-) murine primary hippocampal neuron model to investigate whether PGRN's neurotrophic effects are dependent on SORT1. We sought to elucidate this relationship to determine what role SORT1, as a regulator of PGRN levels, plays in modulating PGRN's neurotrophic effects.

4-1BB (CD137) is an inducible costimulatory receptor that promotes expansion and survival of activated T cells; and IgG-based 4-1BB-agonistic monoclonal antibodies exhibited potent antitumor activity in clinical trials. However, the clinical development of those antibodies is restricted by major off-tumor toxicities associated with FcγR interactions. We have recently generated an EGFR-targeted 4-1BB-agonistic trimerbody that demonstrated strong antitumor activity and did not induce systemic inflammatory cytokine secretion and hepatotoxicity associated with first-generation 4-1BB agonists. Here, we generate a bispecific 4-1BB-agonistic trimerbody targeting the carcinoembryonic antigen (CEA) that is highly expressed in cancers of diverse origins. The CEA-targeted anti-4-1BB-agonistic trimerbody consists of three 4-1BB-specific single-chain fragment variable antibodies and three anti-CEA single-domain antibodies positioned around a murine collagen XVIII-derived homotrimerization domain. The trimerbody was produced as a homogenous, non-aggregating, soluble protein purifiable by standard affinity chromatographic methods. The purified trimerbody was found to be trimeric in solution, very efficient at recognizing 4-1BB and CEA, and potently costimulating T cells in vitro in the presence of CEA. Therefore, trimerbody-based tumor-targeted 4-1BB costimulation is a broadly applicable and clinically feasible approach to enhance the costimulatory environment of disseminated tumor lesions.

The slow-growing, anaerobic, coagulase-negative species Staphylococcus saccharolyticus is found on human skin and in clinical specimens but its pathogenic potential is unclear. Here, we investigated clinical isolates and sequenced the genomes of seven strains of S. saccharolyticus. Phylogenomic analyses showed that the closest relative of S. saccharolyticus is Staphylococcus capitis with an average nucleotide identity of 80%. Previously sequenced strains assigned to S. saccharolyticus are misclassified and belong to S. capitis. Based on single nucleotide polymorphisms of the core genome, the population of S. saccharolyticus can be divided into two clades that also differ in a few larger genomic islands as part of the flexible genome. An unexpected feature of S. saccharolyticus is extensive genome decay, with over 300 pseudogenes, indicating ongoing reductive evolution. Many genes of the core metabolism are not functional, rendering the species auxotrophic for several amino acids, which could explain its slow growth and need for fastidious growth conditions. Secreted proteins of S. saccharolyticus were determined; they include stress response proteins such as heat and oxidative stress-related factors, as well as immunodominant staphylococcal surface antigens and enzymes that can degrade host tissue components. The strains secrete lipases and a hyaluronic acid lyase. Hyaluronidase as well as urease activities were detected in biochemical assays, with clade-specific differences. Our study revealed that S. saccharolyticus has adapted its genome, possibly due to a recent change of habitat; moreover, the data imply that the species has tissue-invasive potential and might cause prosthetic joint infections.

The human epidermal growth factor receptor 2 (HER2) is an oncogene targeted by several kinase inhibitors and therapeutic antibodies. While the endosomal trafficking of many other receptor tyrosine kinases is known to regulate their oncogenic signalling, the prevailing view on HER2 is that this receptor is predominantly retained on the cell surface. Here, we find that sortilin-related receptor 1 (SORLA; SORL1) co-precipitates with HER2 in cancer cells and regulates HER2 subcellular distribution by promoting recycling of the endosomal receptor back to the plasma membrane. SORLA protein levels in cancer cell lines and bladder cancers correlates with HER2 levels. Depletion of SORLA triggers HER2 targeting to late endosomal/lysosomal compartments and impairs HER2-driven signalling and in vivo tumour growth. SORLA silencing also disrupts normal lysosome function and sensitizes anti-HER2 therapy sensitive and resistant cancer cells to lysosome-targeting cationic amphiphilic drugs. These findings reveal potentially important SORLA-dependent endosomal trafficking-linked vulnerabilities in HER2-driven cancers.

The current understanding of the biological identity that nanoparticles may acquire in a given biological milieu is mostly inferred from the hard component of the protein corona (HC). The composition of soft corona (SC) proteins and their biological relevance have remained elusive due to the lack of analytical separation methods. Here, we identify a set of specific corona proteins with weak interactions at silica and polystyrene nanoparticles by using an in situ click-chemistry reaction. We show that these SC proteins are present also in the HC, but are specifically enriched after the capture, suggesting that the main distinction between HC and SC is the differential binding strength of the same proteins. Interestingly, the weakly interacting proteins are revealed as modulators of nanoparticle-cell association mainly through their dynamic nature. We therefore highlight that weak interactions of proteins at nanoparticles should be considered when evaluating nano-bio interfaces.

SORL1 is strongly associated with both sporadic and familial forms of Alzheimer's disease (AD), but a lack of information about alternatively spliced transcripts currently limits our understanding of the role of SORL1 in AD. Here, we describe a SORL1 transcript (SORL1-38b) characterized by inclusion of a novel exon (E38b) that encodes a truncated protein. We identified E38b-containing transcripts in several brain regions, with the highest expression in the cerebellum and showed that SORL1-38b is largely located in neuronal dendrites, which is in contrast to the somatic distribution of transcripts encoding the full-length SORLA protein (SORL1-fl). SORL1-38b transcript levels were significantly reduced in AD cerebellum in three independent cohorts of postmortem brains, whereas no changes were observed for SORL1-fl. A trend of lower 38b transcript level in cerebellum was found for individuals carrying the risk variant at rs2282649 (known as SNP24), although not reaching statistical significance. These findings suggest synaptic functions for SORL1-38b in the brain, uncovering novel aspects of SORL1 that can be further explored in AD research.

Mirolysin is a secretory protease of Tannerella forsythia, a member of the dysbiotic oral microbiota responsible for periodontitis. In this study, we show that mirolysin latency is achieved by a "cysteine-switch" mechanism exerted by Cys23 in the N-terminal profragment. Mutation of Cys23 shortened the time needed for activation of the zymogen from several days to 5 min. The mutation also decreased the thermal stability and autoproteolysis resistance of promirolysin. Mature mirolysin is a thermophilic enzyme and shows optimal activity at 65 °C. Through NMR-based fragment screening, we identified a small molecule (compound (cpd) 9) that blocks promirolysin maturation and functions as a competitive inhibitor (Ki = 3.2 µM), binding to the S1' subsite of the substrate-binding pocket. Cpd 9 shows superior specificity and does not interact with other T. forsythia proteases or Lys/Arg-specific proteases.

The established causal genes in Alzheimer's disease (AD), APP, PSEN1, and PSEN2, are functionally characterized using biomarkers, capturing an in vivo profile reflecting the disease's initial preclinical phase. Mutations in SORL1, encoding the endosome recycling receptor SORLA, are found in 2%-3% of individuals with early-onset AD, and SORL1 haploinsufficiency appears to be causal for AD. To test whether SORL1 can function as an AD causal gene, we use CRISPR-Cas9-based gene editing to develop a model of SORL1 haploinsufficiency in Göttingen minipigs, taking advantage of porcine models for biomarker investigations. SORL1 haploinsufficiency in young adult minipigs is found to phenocopy the preclinical in vivo profile of AD observed with APP, PSEN1, and PSEN2, resulting in elevated levels of β-amyloid (Aβ) and tau preceding amyloid plaque formation and neurodegeneration, as observed in humans. Our study provides functional support for the theory that SORL1 haploinsufficiency leads to endosome cytopathology with biofluid hallmarks of autosomal dominant AD.

Whether and how the pathogenic disruptions in endosomal trafficking observed in Alzheimer's disease (AD) are linked to its anatomical vulnerability remain unknown. Here, we began addressing these questions by showing that neurons are enriched with a second retromer core, organized around VPS26b, differentially dedicated to endosomal recycling. Next, by imaging mouse models, we show that the trans-entorhinal cortex, a region most vulnerable to AD, is most susceptible to VPS26b depletion-a finding validated by electrophysiology, immunocytochemistry, and behavior. VPS26b was then found enriched in the trans-entorhinal cortex of human brains, where both VPS26b and the retromer-related receptor SORL1 were found deficient in AD. Finally, by regulating glutamate receptor and SORL1 recycling, we show that VPS26b can mediate regionally selective synaptic dysfunction and SORL1 deficiency. Together with the trans-entorhinal's unique network properties, hypothesized to impose a heavy demand on endosomal recycling, these results suggest a general mechanism that can explain AD's regional vulnerability.

Retargeting of T lymphocytes toward cancer cells by bispecific antibodies has demonstrated its therapeutic potential, with one such antibody approved for the treatment of acute lymphoblastic leukemia (blinatumomab) and several other in clinical trials. However, improvement of their efficacy and selectivity for solid tumors is still required. Here, we describe a novel tandem T-cell recruiting trispecific antibody for the treatment of colorectal cancer (CRC). This construct, termed trispecific T-cell engager (TriTE), consists of a CD3-specific single-chain Fv (scFv) flanked by anti-epidermal growth factor receptor (EGFR) and anti-epithelial cell adhesion molecule (EpCAM) single-domain VHH antibodies. The TriTE was well expressed in mammalian and yeast cells, bound the cognate antigens of the three parental antibodies, and enabled the specific cytolysis of EGFR- and/or EpCAM-expressing cancer cells, without inducing T cell activation and cytoxicity against double-negative (EGFR-EpCAM-) cancer cells. Bivalent bispecific targeting of double-positive HCT116 cells by TriTE improved in vitro potency up to 100-fold compared to single-positive cells and significantly prolonged survival in vivo. In addition, it was less efficient at killing single-positive target cells than the corresponding bispecific controls, leading to potentially enhanced tumor specificity. Moreover, dual targeting of two tumor-associated antigens may contribute toward preventing the tumor escape by antigen loss caused by selective pressures from conventional single-targeting T-cell engagers, and may help to overcome antigenic heterogeneity.

Cytosine arabinoside (AraC) is one of the main therapeutic treatments for several types of cancer, including acute myeloid leukaemia. However, after a high-dose AraC chemotherapy regime, patients develop severe neurotoxicity and cell death in the central nervous system leading to cerebellar ataxia, dysarthria, nystagmus, somnolence and drowsiness. AraC induces apoptosis in dividing cells. However, the mechanism by which it leads to neurite degeneration and cell death in mature neurons remains unclear. We hypothesise that the upregulation of the death receptor p75NTR is responsible for AraC-mediated neurodegeneration and cell death in leukaemia patients undergoing AraC treatment. To determine the role of AraC-p75NTR signalling in the cell death of mature neurons, we used mature cerebellar granule neurons' primary cultures from p75NTR knockout and p75NTRCys259 mice. Evaluation of neurite degeneration, cell death and p75NTR signalling was done by immunohistochemistry and immunoblotting. To assess the interaction between AraC and p75NTR, we performed cellular thermal shift and AraTM assays as well as Homo-FRET anisotropy imaging. We show that AraC induces neurite degeneration and programmed cell death of mature cerebellar granule neurons in a p75NTR-dependent manner. Mechanistically, Proline 252 and Cysteine 256 residues facilitate AraC interaction with the transmembrane domain of p75NTR resulting in uncoupling of p75NTR from the NFκB survival pathway. This, in turn, exacerbates the activation of the cell death/JNK pathway by recruitment of TRAF6 to p75NTR. Our findings identify p75NTR as a novel molecular target to develop treatments for counteract AraC-mediated cell death of mature neurons.

The SORL1 gene has recently emerged as a strong Alzheimer's Disease (AD) risk gene. Over 500 different variants have been identified in the gene and the contribution of individual variants to AD development and progression is still largely unknown. Here, we describe a family consisting of 2 parents and 5 offspring. Both parents were affected with dementia and one had confirmed AD pathology with an age of onset >75 years. All offspring were affected with AD with ages at onset ranging from 53yrs-74yrs. DNA was available from the parent with confirmed AD and 5 offspring. We identified a coding variant, p.(Arg953Cys), in SORL1 in 5 of 6 individuals affected by AD. Notably, variant carriers had severe AD pathology, and the SORL1 variant segregated with TDP-43 pathology (LATE-NC). We further characterized this variant and show that this Arginine substitution occurs at a critical position in the YWTD-domain of the SORL1 translation product, SORL1. Functional studies further show that the p.R953C variant leads to retention of the SORL1 protein in the endoplasmic reticulum which leads to decreased maturation and shedding of the receptor and prevents its normal endosomal trafficking. Together, our analysis suggests that p.R953C is a pathogenic variant of SORL1 and sheds light on mechanisms of how missense SORL1 variants may lead to AD.

Fuchs' endothelial corneal dystrophy (FECD) is a major corneal disorder affecting the innermost part of the cornea, leading to visual impairment. As the morphological changes in FECD are mainly observed in the extracellular matrix of the Descemet's membrane/endothelial layer, we determined the protein profiles of diseased and control tissues using two relative quantitation MS methods. The first quantitation method, based on the areas of the extracted ion chromatograms, quantified the 51 and 48 most abundant proteins of the Descemet's membrane/endothelial layer in patient and control tissues, respectively, of which 10 were significantly regulated. The results indicated that the level of type VIII collagen was unaltered even though the protein previously has been shown to be implicated in familial early-onset forms of the disease. Using the second relative quantitation method, iTRAQ, we identified 22 differentially regulated proteins, many of which are extracellular proteins known to be involved in proper assembly of the basement membrane in other tissues. In total, 26 differentially regulated proteins were identified, of which 6 proteins were regulated in both methods. These results support that the morphological changes observed in FECD are caused in part by an aberrant assembly of the extracellular matrix within the Descemet's membrane/endothelial layer.