Anchor Protein Research Articles

The imbalance of circulating T helper subsets and regulatory T cells in patients with LRBA deficiency: Correlation with disease severity.

Patients with lipopolysaccharides responsive beige-like anchor protein (LRBA) deficiency suffer from a variety of immunological abnormalities. In the current study, we investigated the role of T helper (Th) cell subsets and regulatory T (Treg) cellsand their related cytokines and transcription factors in the immune dysregulation of LRBA deficiency. The study population comprised of 13 LRBA-deficient patients and 13 age- andsex-matched healthy controls (HCs). Th subsets and Treg were examined by flow cytometry. The expression of determinant cytokines (interferon-γ [IFN-γ], interleukin [IL]-17, IL-22, and IL-10), and cell subset-specific transcription factors were evaluated before and after proliferation and activation stimuli. The frequencies of Th1, Th1-like Th17 and Th22 cells along with the expression of T-box transcription factor (TBET) and runt-related transcription factor 1 (RUNX1) were significantly increased in patients with LRBA. Moreover, IFN-γ and IL-22 production in LRBA-deficient CD4+ T cells were elevated after lymphocyte stimulation, particularly in patients with enteropathy. However, CD4+ CD25+ FoxP3+ CD127- cells were significantly decreased in LRBA-deficient patients compared with those of HCs, particularly in patients with autoimmunity. There was a negative correlation between the frequencies of CD4+ CD25+ FoxP3+ CD127- cells and Th1-like Th17 cells in LRBA-deficient patients, and an overlapping phenotype of autoimmunity and enteropathy were observed in ~70% of patients. The frequency of Th17 cells was lower in patients with enteropathy, while Th1-like Th17 cells were higher than in those without enteropathy. Our findings demonstrated an imbalance in Th subsets, mainly in Th1-like Th17 and Treg cells and their corresponding cytokines in LRBA deficiency, which might be important in the immunopathogenesis of autoimmunity and enteropathy.

Comparison of Immunogenicity and Protection of Two Pneumococcal Protein Vaccines Based on PsaA and PspA.

Streptococcus pneumoniae is a major cause of invasive pneumococcal disease, septicemia, and meningitis that can result in high morbidity rates in children under 5 years old. The current polysaccharide-based vaccines can provide type-specific immunity, but a broad-spectrum vaccine would provide greater coverage. Therefore, developing pneumococcal-protein-based vaccines that can extend to more serum types is highly important. In this study, we vaccinated mice via the subcutaneous (s.c.) route with a systemic vaccine that is a mixture of fusion protein PsaA-PspA23 and a single protein, PspA4, with aluminum hydroxide as an adjuvant. As a comparison, mice were immunized intranasally with a mucosal vaccine that is a mixture of PspA2-PA-BLP (where PA is protein anchor and BLP is bacterium-like particle) and PspA4-PA-BLP, via the intranasal (i.n.) route. The two immunization processes were followed by challenge with Streptococcus pneumoniae bacteria from two different PspA families. Specific IgG titers in the serum and specific IgA titers in the mucosa were determined following immunizations. Bacterial loads and survival rates after challenge were compared. Both the systemic vaccine and the mucosal vaccine induced a significant increase of IgG against PspAs. Only the mucosal vaccine also induced specific IgA in the mucosa. The two vaccines provided protection, but each vaccine showed an advantage. The systemic vaccine induced higher levels of serum antibodies, whereas the mucosal vaccine limited the bacterial load in the lung and blood. Therefore, coimmunizations with the two types of vaccines may be implemented in the future.

The DUF1669 domain of FAM83 family proteins anchor casein kinase 1 isoforms.

Members of the casein kinase 1 (CK1) family of serine-threonine protein kinases are implicated in the regulation of many cellular processes, including the cell cycle, circadian rhythms, and Wnt and Hedgehog signaling. Because these kinases exhibit constitutive activity in biochemical assays, it is likely that their activity in cells is controlled by subcellular localization, interactions with inhibitory proteins, targeted degradation, or combinations of these mechanisms. We identified members of the FAM83 family of proteins as partners of CK1 in cells. All eight members of the FAM83 family (FAM83A to FAM83H) interacted with the α and α-like isoforms of CK1; FAM83A, FAM83B, FAM83E, and FAM83H also interacted with the δ and ε isoforms of CK1. We detected no interaction between any FAM83 member and the related CK1γ1, CK1γ2, and CK1γ3 isoforms. Each FAM83 protein exhibited a distinct pattern of subcellular distribution and colocalized with the CK1 isoform(s) to which it bound. The interaction of FAM83 proteins with CK1 isoforms was mediated by the conserved domain of unknown function 1669 (DUF1669) that characterizes the FAM83 family. Mutations in FAM83 proteins that prevented them from binding to CK1 interfered with the proper subcellular localization and cellular functions of both the FAM83 proteins and their CK1 binding partners. On the basis of its function, we propose that DUF1669 be renamed the polypeptide anchor of CK1 domain.

Respiratory manifestations in LPS-responsive beige-like anchor (LRBA) protein-deficient patients.

Pulmonary manifestations are common in LRBA deficiency. Respiratory characteristics in LRBA-deficient patients should be investigated, monitored, and treated from the time of diagnosis. What is Known: • Lipopolysaccharide-responsive beige-like anchor (LRBA) deficiency is a syndrome of primary immune deficiency and immune dysregulation. • Studies concerning the pulmonary characteristics of LRBA-deficient patients are lacking. What is New: • Respiratory manifestations include infections, bronchiectasis, interstitial lung disease, thoracic lymphadenopathy, and clubbing. • Awareness to pulmonary morbidity in LRBA-deficient patients and involvement of a pulmonologist in the workup and clinical decision-making is important. • Respiratory characteristics in LRBA-deficient patients should be investigated, monitored, and treated from a young age.

Comparative Genomics Reveals the Core Gene Toolbox for the Fungus-Insect Symbiosis.

ABSTRACTModern genomics has shed light on many entomopathogenic fungi and expanded our knowledge widely; however, little is known about the genomic features of the insect-commensal fungi. Harpellales are obligate commensals living in the digestive tracts of disease-bearing insects (black flies, midges, and mosquitoes). In this study, we produced and annotated whole-genome sequences of nine Harpellales taxa and conducted the first comparative analyses to infer the genomic diversity within the members of the Harpellales. The genomes of the insect gut fungi feature low (26% to 37%) GC content and large genome size variations (25 to 102 Mb). Further comparisons with insect-pathogenic fungi (from both Ascomycota and Zoopagomycota), as well as with free-living relatives (as negative controls), helped to identify a gene toolbox that is essential to the fungus-insect symbiosis. The results not only narrow the genomic scope of fungus-insect interactions from several thousands to eight core players but also distinguish host invasion strategies employed by insect pathogens and commensals. The genomic content suggests that insect commensal fungi rely mostly on adhesion protein anchors that target digestive system, while entomopathogenic fungi have higher numbers of transmembrane helices, signal peptides, and pathogen-host interaction (PHI) genes across the whole genome and enrich genes as well as functional domains to inactivate the host inflammation system and suppress the host defense. Phylogenomic analyses have revealed that genome sizes of Harpellales fungi vary among lineages with an integer-multiple pattern, which implies that ancient genome duplications may have occurred within the gut of insects.

Design and synthesis of PEGylated amphiphilic block oligomers as membrane anchors for stable binding to lipid bilayer membranes

Cell surface engineering is a potentially powerful method for manipulating living cells by decorating the cell membrane with specific molecules. Possible applications include cell therapy, drug delivery systems, bio-imaging, and tissue engineering. The stable binding of synthetic molecules to serve as artificial membrane protein anchors is a promising approach for appending functional molecules to the cell surface. However, such synthetic molecules have previously shown limitations, including cytotoxicity and low cell surface affinity. We synthesized amphiphilic block oligomers, using ruthenium-catalyzed living radical polymerization, as novel membrane anchors for stable binding to lipid bilayer membranes. AB and ABA-type amphiphilic block oligomers were synthesized with poly(ethylene glycol) methacrylate (PEGMA) and varying butyl methacrylate (BMA) contents (PEGMA/BMA ratios of 25/5–25/50). These PEGylated oligomers showed high binding efficiencies (up to 92%) for liposomes, which served as model cell membranes, and low cytotoxicity in K562 cells. Both the BMA content and the block segment sequence in the copolymers strongly affected their binding efficiencies. Oligomers with an ABA-type block structure were much more effective than AB-type block oligomers, random oligomers, or PEGMA homo-oligomers for stable membrane binding. Thus, precise control of the primary structures of the amphiphilic oligomers enabled tuning of their binding efficiencies. These amphiphilic block oligomers hold promise as novel membrane anchors in many biomedical applications. A series of amphiphilic block oligomers were designed and synthesized using ruthenium-catalyzed living radical polymerization with poly(ethylene glycol) and butyl methacrylates (BMA). These PEGylated oligomers showed high binding efficiencies for liposomal preparations as model cell membranes, and also had low cytotoxicity. BMA contents and monomer sequences in the copolymers strongly affected their binding efficiencies. Current method enabled precise control of the primary structures of amphiphilic oligomers, allowing tuning of their binding efficiencies. These amphiphilic block oligomers have promise as novel membrane anchors for many biomedical applications.

Aldosterone Impairs Mitochondrial Function in Human Cardiac Fibroblasts via A-Kinase Anchor Protein 12

Aldosterone (Aldo) contributes to mitochondrial dysfunction and cardiac oxidative stress. Using a proteomic approach, A-kinase anchor protein (AKAP)-12 has been identified as a down-regulated protein by Aldo in human cardiac fibroblasts. We aim to characterize whether AKAP-12 down-regulation could be a deleterious mechanism which induces mitochondrial dysfunction and oxidative stress in cardiac cells. Aldo down-regulated AKAP-12 via its mineralocorticoid receptor, increased oxidative stress and induced mitochondrial dysfunction characterized by decreased mitochondrial-DNA and Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) expressions in human cardiac fibroblasts. CRISPR/Cas9-mediated knock-down of AKAP-12 produced similar deleterious effects in human cardiac fibroblasts. CRISPR/Cas9-mediated activation of AKAP-12 blunted Aldo effects on mitochondrial dysfunction and oxidative stress in human cardiac fibroblasts. In Aldo-salt-treated rats, cardiac AKAP-12, mitochondrial-DNA and PGC-1α expressions were decreased and paralleled increased oxidative stress. In myocardial biopsies from patients with aortic stenosis (AS, n = 26), AKAP-12, mitochondrial-DNA and PGC-1α expressions were decreased as compared to Controls (n = 13). Circulating Aldo levels inversely correlated with cardiac AKAP-12. PGC-1α positively associated with AKAP-12 and with mitochondrial-DNA. Aldo decreased AKAP-12 expression, impairing mitochondrial biogenesis and increasing cardiac oxidative stress. AKAP-12 down-regulation triggered by Aldo may represent an important event in the development of mitochondrial dysfunction and cardiac oxidative stress.

ITRAQ-based quantitative proteomic analysis of conidia and mycelium in the filamentous fungus Metarhizium robertsii

Metarhizium robertsii is widely applied in biological control via conidia application. To clarify the proteomic differences between conidia and mycelia and explore the underlying mechanisms of conidia as a unit responsible for dispersal and environmental stress, we carried out an iTRAQ (isobaric tags for relative and absolute quantitation)-based quantitative proteomic analysis for two developmental stages from M. robertsii. A total of 2052 proteins were detected, and 90 showed differential protein abundance between the conidia and mycelia. These 90 proteins were primarily associated with stress resistance, amino acid and protein metabolism, and energy metabolism. Further bioinformatics analysis showed that these proteins could be mapped to 52 pathways, five of which were significantly enriched after mapping to KEGG pathways. Interestingly, many proteins involved in the significantly enriched pathway of peroxisome, biosynthesis of secondary metabolites and glyoxylate and dicarboxylate metabolism, including catalase, peroxisomal membrane anchor protein, formate dehydrogenase and isocitrate lyase, were identified with higher abundance in conidia. The results deepened our understanding of the conidia proteome in M. robertsii and provide a basis for further exploration for improving the efficiency of the fungi as biocontrol agents.

Structure and Interaction Analysis of Human R7‐RGS/Gβ5/R7BP complexes

The R7‐RGS/Gβ5 protein duplex functions as a GTPase‐activating protein (GAP) to facilitate the Gαi/o intrinsic GTPase activity. The R7‐RGS subfamily of RGS (Regulator of G protein Signaling) proteins comprises RGS6, RGS7, RGS9 and RGS11. The R7 family‐binding protein (R7BP) anchors the R7‐RGS/Gβ5 duplex to the cell membrane to enhance its GAP activity. Recently it was shown that knock‐out of R7BP in mice led to decreased acute and chronic itch sensation, indicating that R7BP has a central role in itch response. We are developing interfering peptides to block the R7BP and R7‐RGS/Gβ5 interactions as potential inhibitors of chronic itch. We demonstrate here the use of cross‐linking mass spectrometry (XL‐MS) to obtain structural clues about human R7‐RGS/Gβ5/R7BP triplexes. To this end, we first transfected plasmids encoding human RGS7, Gβ5 and R7BP into Expi293 cells and purified the protein triplex to 95% purity. The triplex was treated with the cleavable cross‐linking reagent DSSO (disuccinimidyl sulfoxide) at different protein:DSSO molar ratios and analyzed by mass spectrometry. The experiment was repeated using the human RGS9/Gβ5/R7BP triplex. Both experiments yielded peptides involved in the RGS7/9‐R7BP interaction, with one R7BP peptide common to both. We believe this peptide region to be an essential interaction surface on R7BP and an ideal target for interfering peptide design to block the R7‐RGS/R7BP interaction. Taken together, we demonstrate that XL‐MS is an effective way to shed light on structural features of the R7‐RGS/Gβ5/R7BP triplex to facilitate the development of novel anti‐itch drugs.Support or Funding InformationNational Institute of Diabetes and Digestive and Kidney Diseases Intramural Funding, NIHThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of Variants in RET and IHH Pathway Members in a Large Family With History of Hirschsprung Disease

Hirschsprung disease (HSCR) is an inherited congenital disorder characterized by absence of enteric ganglia in the distal part of the gut. Variants in ret proto-oncogene (RET) have been associated with up to 50% of familial and 35% of sporadic cases. We searched for variants that affect disease risk in a large, multigenerational family with history of HSCR in a linkage region previously associated with the disease (4q31.3-q32.3) and exome wide. We performed exome sequencing analyses of a family in the Netherlands with 5 members diagnosed with HSCR and 2 members diagnosed with functional constipation. We initially focused on variants in genes located in 4q31.3-q32.3; however, we also performed an exome-wide analysis in which known HSCR or HSCR-associated gene variants predicted to be deleterious were prioritized for further analysis. Candidate genes were expressed in HEK293, COS-7, and Neuro-2a cells and analyzed by luciferase and immunoblot assays. Morpholinos were designed to target exons of candidate genes and injected into 1-cell stage zebrafish embryos. Embryos were allowed to develop and stained for enteric neurons. Within the linkage region, we identified 1 putative splice variant in the lipopolysaccharide responsive beige-like anchor protein gene (LRBA). Functional assays could not confirm its predicted effect on messenger RNA splicing or on expression of the mab-21 like 2 gene (MAB21L2), which is embedded in LRBA. Zebrafish that developed following injection of the lrba morpholino had a shortened body axis and subtle gut morphological defects, but no significant reduction in number of enteric neurons compared with controls. Outside the linkage region, members of 1 branch of the family carried a previously unidentified RET variant or an in-frame deletion in the glial cell line derived neurotrophic factor gene (GDNF), which encodes a ligand of RET. This deletion was located 6 base pairs before the last codon. We also found variants in the Indian hedgehog gene (IHH) and its mediator, the transcription factor GLI family zinc finger 3 (GLI3). When expressed in cells, the RET-P399L variant disrupted protein glycosylation and had altered phosphorylation following activation by GDNF. The deletion in GDNF prevented secretion of its gene product, reducing RET activation, and the IHH-Q51K variant reduced expression of the transcription factor GLI1. Injection of morpholinos that target ihhreduced the number of enteric neurons to 13% ± 1.4% of control zebrafish. In a study of a large family with history of HSCR, we identified variants in LRBA, RET, the gene encoding the RET ligand (GDNF), IHH, and a gene encoding a mediator of IHH signaling (GLI3). These variants altered functions of the gene products when expressed in cells and knockout of ihh reduced the number of enteric neurons in the zebrafish gut.

Blockage of AKAP12 accelerates angiotensin II (Ang II)-induced cardiac injury in mice by regulating the transforming growth factor β1 (TGF-β1) pathway

Hypertension is a multifactorial chronic inflammatory disease that leads to cardiac remodeling. A-kinase anchor protein 12 (AKAP12) is a scaffolding protein that has multiple functions in various biological events, including the regulation of vessel integrity and differentiation of neural barriers in blood. However, the role of AKAP12 in angiotensin II (Ang II)-induced cardiac injury remains unclear. In the present study, Ang II infusion reduced AKAP12 expressions in the hearts of wild-type (WT) mice, and AKAP12 knockout (KO) enhanced the infiltration of inflammatory cells. In addition, AKAP12 deletion accelerated Ang II-induced cardiac histologic alterations and dysfunction. Further, AKAP12−/− aggravated heart failure by promoting the inflammation, oxidative stress, cellular apoptosis, and autophagy induced by Ang II. Furthermore, AKAP12 KO elevated Ang II-induced cardiac fibrosis, as indicated by the following: (1) Masson trichrome staining showed that Ang II infusion markedly increased fibrotic areas of the WT mouse heart, which was greatly accelerated in AKAP12−/− mice; (2) immunohistochemistry analysis showed increased expression of transforming growth factor β1 (TGF-β1) and α-smooth muscle actin (α-SMA) in the AKAP12−/− mouse heart; (3) reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) analysis showed increased expression of fibrosis-related molecules in the AKAP12-deficient mouse heart; and (4) Western blot analysis indicated significantly higher upregulation of p-SMAD2/3 in the AKAP12−/− mouse heart. In vitro, AKAP12 knockdown in HL-1 cells was responsible for TGF-β1-induced inflammation, the generation of reactive oxygen species (ROS), apoptosis, autophagy, and fibrosis. Furthermore, overexpression of AKAP12 reduced fibrosis triggered by TGF-β1 in cells. Overall, our study suggests that fibrosis induced by Ang II may be alleviated by AKAP12 expression through inactivation of the TGF-β1 pathway.

A Novel PspA Protein Vaccine Intranasal Delivered by Bacterium-Like Particles Provides Broad Protection Against Pneumococcal Pneumonia in Mice

ABSTRACTBackground: Streptococcus pneumoniae is a major pathogen accounting for a large number of pneumococcal disease in worldwide. Due to the mucosal immune pathway induces both systemic and mucosal immune responses, the potential strategy to prevent pneumococcal disease may be to develop a mucosal vaccine.Method: In this study, we developed an intranasal pneumococcal protein vaccine based on a bacterium-like particle (BLP) delivery system. PspA is expressed and exposed on the surface of all pneumococcal strains, which confers the potential to induce immune responses to protect against pneumococcal infection. We fused one of the pneumococcal surface proteins (PspA, family2 clade4) with the protein anchor (PA) protein in order to display PspA on the surface of BLPs.Result: The current results showed that intranasal immunization with BLPs/PspA-PA efficiently induced both PspA-specific IgG in the serum and PspA-specific IgA in mucosal washes. And intranasal immunization of BLPs/PspA-PA could provide complete protection in a mouse challenge model with pneumococci of different two clades of both homologous and heterologous PspA families.Discussion and conclusion: Thus, targeted delivery of multiple bacterial antigens via BLPs may prevent pneumococcal disease by inducing both systemic and mucosal immune responses.

The C-terminal helix of ribosomal P stalk recognizes a hydrophobic groove of elongation factor 2 in a novel fashion.

Archaea and eukaryotes have ribosomal P stalks composed of anchor protein P0 and aP1 homodimers (archaea) or P1•P2 heterodimers (eukaryotes). These P stalks recruit translational GTPases to the GTPase-associated center in ribosomes to provide energy during translation. The C-terminus of the P stalk is known to selectively recognize GTPases. Here we investigated the interaction between the P stalk and elongation factor 2 by determining the structures of Pyrococcus horikoshii EF-2 (PhoEF-2) in the Apo-form, GDP-form, GMPPCP-form (GTP-form), and GMPPCP-form bound with 11 C-terminal residues of P1 (P1C11). Helical structured P1C11 binds to a hydrophobic groove between domain G and subdomain G′ of PhoEF-2, where is completely different from that of aEF-1α in terms of both position and sequence, implying that such interaction characteristic may be requested by how GTPases perform their functions on the ribosome. Combining PhoEF-2 P1-binding assays with a structural comparison of current PhoEF-2 structures and molecular dynamics model of a P1C11-bound GDP form, the conformational changes of the P1C11-binding groove in each form suggest that in response to the translation process, the groove has three states: closed, open, and release for recruiting and releasing GTPases.

The chondrocyte channelome: A narrative review.

Chondrocytes are the main cells in the extracellular matrix (ECM) of articular cartilage and possess a highly differentiated phenotype that is the hallmark of the unique physiological functions of this specialised load-bearing connective tissue. The plasma membrane of articular chondrocytes contains a rich and diverse complement of membrane proteins, known as the membranome, which defines the cell surface phenotype of the cells. The membranome is a key target of pharmacological agents and is important for chondrocyte function. It includes channels, transporters, enzymes, receptors, and anchors for intracellular, cytoskeletal and ECM proteins and other macromolecular complexes. The chondrocyte channelome is a sub-compartment of the membranome and includes a complete set of ion channels and porins expressed in these cells. Many of these are multi-functional proteins with "moonlighting" roles, serving as channels, receptors and signalling components of larger molecular assemblies. The aim of this review is to summarise our current knowledge of the fundamental aspects of the chondrocyte channelome, discuss its relevance to cartilage biology and highlight its possible role in the pathogenesis of osteoarthritis (OA). Excessive and inappropriate mechanical loads, an inflammatory micro-environment, alternative splicing of channel components or accumulation of basic calcium phosphate crystals can result in an altered chondrocyte channelome impairing its function. Alterations in Ca2+ signalling may lead to defective synthesis of ECM macromolecules and aggravated catabolic responses in chondrocytes, which is an important and relatively unexplored aspect of the complex and poorly understood mechanism of OA development.

Yeast arming systems: pros and cons of different protein anchors and other elements required for display.

Yeast display is a powerful strategy that consists in exposing peptides or proteins of interest on the cell surface of this microorganism. Ever since initial experiments with this methodology were carried out, its scope has extended and many applications have been successfully developed in different science and technology fields. Several yeast display systems have been designed, which all involve introducting into yeast cells the gene fusions that contain the coding regions of a signal peptide, an anchor protein, to properly attach the target to the cell surface, and the protein of interest to be exposed, all of which are controlled by a strong promoter. In this work, we report the description of such elements for the alternative systems introduced by focusing particularly on anchor proteins. The comparisons made between them are included whenever possible, and the main advantages and inconveniences of each one are discussed. Despite the huge number of publications on yeast surface display and the revisions published to date, this topic has not yet been widely considered. Finally, given the growing interest in developing systems for non-Saccharomyces yeasts, the main strategies reported for some are also summarized.

Kinome and phosphoproteome of high-grade meningiomas reveal AKAP12 as a central regulator of aggressiveness and its possible role in progression

There is a need to better understand meningioma oncogenesis for biomarker discovery and development of targeted therapies. Histological or genetic criteria do not accurately predict aggressiveness. Post-translational studies in meningioma progression are lacking. In the present work, we introduce a combination of mass spectrometry-based phosphoproteomics and peptide array kinomics to profile atypical and anaplastic (high-grade) meningiomas. In the discovery set of fresh-frozen tissue specimens (14), the A-kinase anchor protein 12 (AKAP12) protein was found downregulated across the grades. AKAP12 knockdown in benign meningioma cells SF4433 increases proliferation, cell cycle, migration, invasion, and confers an anaplastic profile. Differentially regulated pathways were characteristic of high-grade meningiomas. Low AKAP12 expression in a larger cohort of patients (75) characterized tumor invasiveness, recurrence, and progression, indicating its potential as a prognostic biomarker. These results demonstrate AKAP12 as a central regulator of meningioma aggressiveness with a possible role in progression.

Structural and functional characterization of shaft, anchor, and tip proteins of the Mfa1 fimbria from the periodontal pathogen Porphyromonas gingivalis

Very little is known about how fimbriae of Bacteroidetes bacteria are assembled. To shed more light on this process, we solved the crystal structures of the shaft protein Mfa1, the regulatory protein Mfa2, and the tip protein Mfa3 from the periodontal pathogen Porphyromonas gingivalis. Together these build up part of the Mfa1 fimbria and represent three of the five proteins, Mfa1-5, encoded by the mfa1 gene cluster. Mfa1, Mfa2 and Mfa3 have the same overall fold i.e., two β-sandwich domains. Upon polymerization, the first β-strand of the shaft or tip protein is removed by indigenous proteases. Although the resulting void is expected to be filled by a donor-strand from another fimbrial protein, the mechanism by which it does so is still not established. In contrast, the first β-strand in Mfa2, the anchoring protein, is firmly attached by a disulphide bond and is not cleaved. Based on the structural information, we created multiple mutations in P. gingivalis and analysed their effect on fimbrial polymerization and assembly in vivo. Collectively, these data suggest an important role for the C-terminal tail of Mfa1, but not of Mfa3, affecting both polymerization and maturation of downstream fimbrial proteins.

A-Kinase Anchor Protein 12 Is Required for Oligodendrocyte Differentiation in Adult White Matter.

Oligodendrocyte precursor cells (OPCs) give rise to oligodendrocytes in cerebral white matter. However, the underlying mechanisms that regulate this process remain to be fully defined, especially in adult brains. Recently, it has been suggested that signaling via A-kinase anchor protein 12 (AKAP12), a scaffolding protein that associates with intracellular molecules such as protein kinase A, may be involved in Schwann cell homeostasis and peripheral myelination. Here, we asked whether AKAP12 also regulates the mechanisms of myelination in the CNS. AKAP12 knockout mice were compared against wild-type (WT) mice in a series of neurochemical and behavioral assays. Compared with WTs, 2-months old AKAP12 knockout mice exhibited loss of myelin in white matter of the corpus callosum, along with perturbations in working memory as measured by a standard Y-maze test. Unexpectedly, very few OPCs expressed AKAP12 in the corpus callosum region. Instead, pericytes appeared to be one of the major AKAP12-expressing cells. In a cell culture model system, conditioned culture media from normal pericytes promoted in-vitro OPC maturation. However, conditioned media from AKAP12-deficient pericytes did not support the OPC function. These findings suggest that AKAP12 signaling in pericytes may be required for OPC-to-oligodendrocyte renewal to maintain the white matter homeostasis in adult brain. Stem Cells 2018;36:751-760.

Sporobeads: The Utilization of the Bacillus subtilis Endospore Crust as a Protein Display Platform.

Upon starvation, the soil bacterium Bacillus subtilis forms an intracellular, metabolically inactive endospore. Its core contains the DNA, encased by three protein layers protecting it against a multitude of environmental threats. The outermost layer, the crust, harbors great potential as a protein-displaying platform: a gene of interest can be translationally fused to a crust protein gene, resulting in endospores displaying the desired protein on their surface. To unlock this potential in a standardized fashion, we designed a suite of 12 vectors (Sporovectors), based on the BioBrick cloning standard. With these vectors, proteins can easily be fused N- or C-terminally to the six crust proteins CotV, CotW, CotX, CotY, CotZ, and CgeA under the control of the strongest crust gene promoter PcotYZ. All Sporovectors were evaluated with GFP and two different laccases. On the basis of our data, CotY and CotZ represent the best anchor proteins. But there are significant differences in activity and functional stability between the two tested laccases. Our vector suite is a powerful tool to generate and evaluate a vast variety of functionalized endospores. It allows quickly identifying the best anchor and fusion site for the protein of interest. Our findings demonstrate that the crust of B. subtilis endospores is an inexpensive and easy platform for displaying different proteins of interest.

Membrane Anchoring of α-Helical Proteins: Role of Tryptophan.

The function of membrane proteins relies on a defined orientation of protein relative to lipid. In apparent correlation to protein anchoring, tryptophan residues are enriched in the lipid headgroup region. To characterize the thermodynamic and structural basis of this relationship in α-helical membrane proteins, we examined the role of three conserved tryptophans in the folding of the heterodimeric integrin αIIbβ3 transmembrane (TM) complex in phospholipid bicelles and mammalian membranes. In the homogenous lipid environment of bicelles, tryptophan was replaceable by residues of distinct polarities. The appropriate polarity was guided by the electrostatic potential of the tryptophan surrounding, suggesting that tryptophan can complement diverse environments by adjusting the orientation of its anisotropic side chain to achieve site-specific anchoring. As a sole membrane anchor, tryptophan made a contribution of 0.4 kcal/mol to TM complex stability in bicelles. In membranes, it proved more difficult to replace tryptophan even by tyrosine, indicating a superior capacity to interact with heterogeneous lipids of biological membranes. Interestingly, at intracellular TM helix ends, where integrin activation is initiated, sequence motifs that interact with lipids via opposing polarity patterns were found to restrict TM helix orientations beyond tryptophan anchoring. In contrast to bicelles, phenylalanine became the least accepted substitute in membranes, demonstrating an increased role of the hydrophobic effect. Altogether, our study implicates a wide amphiphilic range of tryptophan, membrane complexity, and the hydrophobic effect to be important factors in tryptophan membrane anchoring.