Expression of Cell-Adhesion Molecules in E. coli: A High Throughput Screening to Identify Paracellular Modulators.

Cell-adhesion molecules (CAMs) are responsible for cell-cell, cell-extracellular matrix, and cell-pathogen interactions. Claudins (CLDNs), occludin (OCLN), and junctional adhesion molecules (JAMs) are CAMs' components of the tight junction (TJ), the single protein structure tasked with safeguarding the paracellular space. The TJ is responsible for controlling paracellular permeability according to size and charge. Currently, there are no therapeutic solutions to modulate the TJ. Here, we describe the expression of CLDN proteins in the outer membrane of E. coli and report its consequences. When the expression is induced, the unicellular behavior of E. coli is replaced with multicellular aggregations that can be quantified using Flow Cytometry (FC). Our method, called iCLASP (inspection of cell-adhesion molecules aggregation through FC protocols), allows high-throughput screening (HTS) of small-molecules for interactions with CAMs. Here, we focused on using iCLASP to identify paracellular modulators for CLDN2. Furthermore, we validated those compounds in the mammalian cell line A549 as a proof-of-concept for the iCLASP method.

Molecular Characterization of the Extracellular Domain of Human Junctional Adhesion Proteins.

The junction adhesion molecule (JAM) family of proteins play central roles in the tight junction (TJ) structure and function. In contrast to claudins (CLDN) and occludin (OCLN), the other membrane proteins of the TJ, whose structure is that of a 4α-helix bundle, JAMs are members of the immunoglobulin superfamily. The JAM family is composed of four members: A, B, C and 4. The crystal structure of the extracellular domain of JAM-A continues to be used as a template to model the secondary and tertiary structure of the other members of the family. In this article, we have expressed the extracellular domains of JAMs fused with maltose-binding protein (MBP). This strategy enabled the work presented here, since JAM-B, JAM-C and JAM4 are more difficult targets due to their more hydrophobic nature. Our results indicate that each member of the JAM family has a unique tertiary structure in spite of having similar secondary structures. Surface plasmon resonance (SPR) revealed that heterotypic interactions among JAM family members can be greatly favored compared to homotypic interactions. We employ the well characterized epithelial cadherin (E-CAD) as a means to evaluate the adhesive properties of JAMs. We present strong evidence that suggests that homotypic or heterotypic interactions among JAMs are stronger than that of E-CADs.

Chimeric Claudins: A New Tool to Study Tight Junction Structure and Function.

The tight junction (TJ) is a structure composed of multiple proteins, both cytosolic and membranal, responsible for cell-cell adhesion in polarized endothelium and epithelium. The TJ is intimately connected to the cytoskeleton and plays a role in development and homeostasis. Among the TJ's membrane proteins, claudins (CLDNs) are key to establishing blood-tissue barriers that protect organismal physiology. Recently, several crystal structures have been reported for detergent extracted recombinant CLDNs. These structural advances lack direct evidence to support quaternary structure of CLDNs. In this article, we have employed protein-engineering principles to create detergent-independent chimeric CLDNs, a combination of a 4-helix bundle soluble monomeric protein (PDB ID: 2jua) and the apical-50% of human CLDN1, the extracellular domain that is responsible for cell-cell adhesion. Maltose-binding protein-fused chimeric CLDNs (MBP-CCs) used in this study are soluble proteins that retain structural and functional aspects of native CLDNs. Here, we report the biophysical characterization of the structure and function of MBP-CCs. MBP-fused epithelial cadherin (MBP-eCAD) is used as a control and point of comparison of a well-characterized cell-adhesion molecule. Our synthetic strategy may benefit other families of 4-α-helix membrane proteins, including tetraspanins, connexins, pannexins, innexins, and more.

A water-soluble DsbB variant that catalyzes disulfide-bond formation in vivo.

Escherichia coli DsbB is a transmembrane enzyme that catalyzes the reoxidation of the periplasmic oxidase DsbA by ubiquinone. Here, we sought to convert membrane-bound DsbB into a water-soluble biocatalyst by leveraging a previously described method for in vivo solubilization of integral membrane proteins (IMPs). When solubilized DsbB variants were coexpressed with an export-defective copy of DsbA in the cytoplasm of wild-type E. coli cells, artificial oxidation pathways were created that efficiently catalyzed de novo disulfide-bond formation in a range of substrate proteins, in a manner dependent on both DsbA and quinone. Hence, DsbB solubilization was achieved with preservation of both catalytic activity and substrate specificity. Moreover, given the generality of the solubilization technique, the results presented here should pave the way to unlocking the biocatalytic potential of other membrane-bound enzymes whose utility has been limited by poor stability of IMPs outside of their native lipid-bilayer context.

VDAC1 and the TSPO: Expression, Interactions, and Associated Functions in Health and Disease States.

The translocator protein (TSPO), located at the outer mitochondrial membrane (OMM), serves multiple functions and contributes to numerous processes, including cholesterol import, mitochondrial metabolism, apoptosis, cell proliferation, Ca2+ signaling, oxidative stress, and inflammation. TSPO forms a complex with the voltage-dependent anion channel (VDAC), a protein that mediates the flux of ions, including Ca2+, nucleotides, and metabolites across the OMM, controls metabolism and apoptosis and interacts with many proteins. This review focuses on the two OMM proteins TSPO and VDAC1, addressing their structural interaction and associated functions. TSPO appears to be involved in the generation of reactive oxygen species, proposed to represent the link between TSPO activation and VDAC, thus playing a role in apoptotic cell death. In addition, expression of the two proteins in healthy brains and diseased states is considered, as is the relationship between TSPO and VDAC1 expression. Both proteins are over-expressed in in brains from Alzheimer's disease patients. Finally, TSPO expression levels were proposed as a biomarker of some neuropathological settings, while TSPO-interacting ligands have been considered as a potential basis for drug development.

The Voltage-dependent Anion Channel 1 Mediates Amyloid ß Toxicity and Represents a Potential Target for Alzheimer Disease Therapy.

The voltage-dependent anion channel 1 (VDAC1), found in the mitochondrial outer membrane, forms the main interface between mitochondrial and cellular metabolisms, mediates the passage of a variety of molecules across the mitochondrial outer membrane, and is central to mitochondria-mediated apoptosis. VDAC1 is overexpressed in post-mortem brains of Alzheimer disease (AD) patients. The development and progress of AD are associated with mitochondrial dysfunction resulting from the cytotoxic effects of accumulated amyloid ß (Aß). In this study we demonstrate the involvement of VDAC1 and a VDAC1 N-terminal peptide (VDAC1-N-Ter) in Aß cell penetration and cell death induction. Aß directly interacted with VDAC1 and VDAC1-N-Ter, as monitored by VDAC1 channel conductance, surface plasmon resonance, and microscale thermophoresis. Preincubated Aß interacted with bilayer-reconstituted VDAC1 and increased its conductance ∼ 2-fold. Incubation of cells with Aß resulted in mitochondria-mediated apoptotic cell death. However, the presence of non-cell-penetrating VDAC1-N-Ter peptide prevented Aß cellular entry and Aß-induced mitochondria-mediated apoptosis. Likewise, silencing VDAC1 expression by specific siRNA prevented Aß entry into the cytosol as well as Aß-induced toxicity. Finally, the mode of Aß-mediated action involves detachment of mitochondria-bound hexokinase, induction of VDAC1 oligomerization, and cytochrome c release, a sequence of events leading to apoptosis. As such, we suggest that Aß-mediated toxicity involves mitochondrial and plasma membrane VDAC1, leading to mitochondrial dysfunction and apoptosis induction. The VDAC1-N-Ter peptide targeting Aß cytotoxicity is thus a potential new therapeutic strategy for AD treatment.

PSMB8 encoding the ß5i proteasome subunit is mutated in joint contractures, muscle atrophy, microcytic anemia, and panniculitis-induced lipodystrophy syndrome.

We performed homozygosity mapping in two recently reported pedigrees from Portugal and Mexico with an autosomal-recessive autoinflammatory syndrome characterized by joint contractures, muscle atrophy, microcytic anemia, and panniculitis-induced lipodystrophy (JMP). This revealed only one homozygous region spanning 2.4 Mb (5818 SNPs) on chromosome 6p21 shared by all three affected individuals from both families. We directly sequenced genes involved in immune response located in this critical region, excluding the HLA complex genes. We found a homozygous missense mutation c.224C>T (p.Thr75Met) in the proteasome subunit, beta-type, 8 (PSMB8) gene in affected patients from both pedigrees. The mutation segregated in an autosomal-recessive fashion and was not detected in 275 unrelated ethnically matched healthy subjects. PSMB8 encodes a catalytic subunit of the 20S immunoproteasomes called ß5i. Immunoproteasome-mediated proteolysis generates immunogenic epitopes presented by major histocompatibility complex (MHC) class I molecules. Threonine at position 75 is highly conserved and its substitution with methionine disrupts the tertiary structure of PSMB8. As compared to normal lymphoblasts, those from an affected patient showed significantly reduced chymotrypsin-like proteolytic activity mediated by immunoproteasomes. We conclude that mutations in PSMB8 cause JMP syndrome, most probably by affecting MHC class I antigen processing.

Insulin receptor-inspired soluble insulin binder.

The insulin receptor (IR) is a 320 kDa membrane receptor tyrosine kinase mediating the pleiotropic actions of insulin, leading to phosphorylation of several intracellular substrates including serine/threonine-protein kinase (AKT1), and IR autophosphorylation. Structural details of the IR have been recently revealed. A high-binding insulin site, L1 (Kd =2 nM), consists of two distant domains in the primary sequence of the IR. Our design simplified the L1 binding site and transformed it into a soluble insulin binder (sIB). The sIB, a 17 kDa protein, binds insulin with 38 nM affinity. The sIB competes with IR for insulin and reduces by more than 50% phosphorylation of AKT1 in HEK 293 T cells, with similar effects on IR autophosphorylation. The sIB represents a new tool for research of insulin binding and signaling properties.

Human 1-acylglycerol-3-phosphate O-acyltransferase isoforms 1 and 2: biochemical characterization and inability to rescue hepatic steatosis in Agpat2(-/-) gene lipodystrophic mice.

Loss-of-function mutations in 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) 2 in humans and mice result in loss of both the white and brown adipose tissues from birth. AGPAT2 generates precursors for the synthesis of glycerophospholipids and triacylglycerols. Loss of adipose tissue, or lipodystrophy, results in hyperinsulinemia, diabetes mellitus, and severe hepatic steatosis. Here, we analyzed biochemical properties of human AGPAT2 and its close homolog, AGPAT1, and we studied their role in liver by transducing their expression via recombinant adenoviruses in Agpat2(-/-) mice. The in vitro substrate specificities of AGPAT1 and AGPAT2 are quite similar for lysophosphatidic acid and acyl-CoA. Protein homology modeling of both the AGPATs with glycerol-3-phosphate acyltransferase 1 (GPAT1) revealed that they have similar tertiary protein structure, which is consistent with their similar substrate specificities. When co-expressed, both isoforms co-localize to the endoplasmic reticulum. Despite such similarities, restoring AGPAT activity in liver by overexpression of either AGPAT1 or AGPAT2 in Agpat2(-/-) mice failed to ameliorate the hepatic steatosis. From these studies, we suggest that the role of AGPAT1 or AGPAT2 in liver lipogenesis is minimal and that accumulation of liver fat is primarily a consequence of insulin resistance and loss of adipose tissue in Agpat2(-/-) mice.

Using the Power of Junctional Adhesion Molecules Combined with the Target of CAR-T to Inhibit Cancer Proliferation, Metastasis and Eradicate Tumors.

Decades of evidence suggest that alterations in the adhesion properties of neoplastic cells endow them with an invasive and migratory phenotype. Tight junctions (TJs) are present in endothelial and epithelial cells. Tumors arise from such tissues, thus, the role of TJ proteins in the tumor microenvironment is highly relevant. In the TJ, junctional adhesion molecules (JAM) play a key role in assembly of the TJ and control of cell-cell adhesion. Reprogramming of immune cells using chimeric antigen receptors (CAR) to allow for target recognition and eradication of tumors is an FDA approved therapy. The best-studied CAR-T cells recognize CD19, a B-cell surface molecule. CD19 is not a unique marker for tumors, liquid or solid. To address this limitation, we developed a biologic containing three domains: (1) pH-low-insertion peptide (pHLIP), which recognizes the low pH of the cancer cells, leading to the insertion of the peptide into the plasma membrane. (2) An extracellular domain of JAM proteins that fosters cell-cell interactions. (3) CD19 to be targeted by CAR-T cells. Our modular design only targets cancer cells and when coupled with anti-CD19 CAR-T cells, it decreases proliferation and metastasis in at least two cancer cell lines.

Calcium regulates the interplay between the tight junction and epithelial adherens junction at the plasma membrane.

The apical junctional complex (AJC) is a membrane protein ultrastructure that regulates cell adhesion and homeostasis. The tight junction (TJ) and the adherens junction (AJ) are substructures of the AJC. The interplay between TJ and AJ membrane proteins to assemble the AJC remains unclear. We employed synthetic biology strategies to express the basic membrane elements of a simple AJC-the adhesive extracellular domains of junctional adhesion molecule A (JAM-A), epithelial cadherin, claudin 1, and occludin-to study their interactions. Our results suggest that calcium concentration fluctuations and JAM-A, acting as an interface molecule between the TJ and AJ, orchestrate their interplay. Calcium affects the secondary structure, oligomerization, and binding affinity of homotypic and heterotypic interactions of TJ and AJ components, thus acting as a molecular switch influencing AJC dynamics.

A universal glycoenzyme biosynthesis pipeline that enables efficient cell-free remodeling of glycans.

The ability to reconstitute natural glycosylation pathways or prototype entirely new ones from scratch is hampered by the limited availability of functional glycoenzymes, many of which are membrane proteins that fail to express in heterologous hosts. Here, we describe a strategy for topologically converting membrane-bound glycosyltransferases (GTs) into water soluble biocatalysts, which are expressed at high levels in the cytoplasm of living cells with retention of biological activity. We demonstrate the universality of the approach through facile production of 98 difficult-to-express GTs, predominantly of human origin, across several commonly used expression platforms. Using a subset of these water-soluble enzymes, we perform structural remodeling of both free and protein-linked glycans including those found on the monoclonal antibody therapeutic trastuzumab. Overall, our strategy for rationally redesigning GTs provides an effective and versatile biosynthetic route to large quantities of diverse, enzymatically active GTs, which should find use in structure-function studies as well as in biochemical and biomedical applications involving complex glycomolecules.

Why human cytochrome P450c21 is a progesterone 21-hydroxylase.

Human cytochrome P450c21 (steroid 21-hydroxylase, CYP21A2) catalyzes the 21-hydroxylation of progesterone (P4) and its preferred substrate 17α-hydroxyprogestrone (17OHP4). CYP21A2 activities, which are required for cortisol and aldosterone biosynthesis, involve the formation of energetically disfavored primary carbon radicals. Therefore, we hypothesized that the binding of P4 and 17OHP4 to CYP21A2 restricts access of the reactive heme-oxygen complex to the C-21 hydrogen atoms, suppressing oxygenation at kinetically more favorable sites such as C-17 and C-16, which are both hydroxylated by cytochrome P450c17 (CYP17A1). We reasoned that expansion of the CYP21A2 substrate-binding pocket would increase substrate mobility and might yield additional hydroxylation activities. We built a computer model of CYP21A2 based principally on the crystal structure of CYP2C5, which also 21-hydroxylates P4. Molecular dynamics simulations indicate that binding of the steroid nucleus perpendicular to the plane of the CYP21A2 heme ring limits access of the heme oxygen to the C-21 hydrogen atoms. Residues L107, L109, V470, I471, and V359 were found to contribute to the CYP21A2 substate-binding pocket. Mutation of V470 and I471 to alanine or glycine preserved P4 21-hydroxylase activity, and mutations of L107 or L109 were inactive. Mutations V359A and V359G, in contrast, acquired 16α-hydroxylase activity, accounting for 40% and 90% of the P4 metabolites, respectively. We conclude that P4 binds to CYP21A2 in a fundamentally different orientation than to CYP17A1 and that expansion of the CYP21A2 substrate-binding pocket allows additional substrate trajectories and metabolic switching.

Androgens, estrogens, and hydroxysteroid dehydrogenases.

The task of regulating both production and activity of potent androgens and estrogens in human physiology is largely relegated to the hydroxysteroid dehydrogenases (HSDs). Although over two dozen enzymes with HSD activities have been described, we only understand the physiology of a small number, and for only one enzyme has the function been unequivocally determined by the study of human mutations. The physiology of the HSDs derive from their enzymatic activities, which in turn derive from their respective structures. In general, pairs of enzymes that drive steroid flux in opposite directions are found, and we have been studying the biochemical principles which enable dichotomous enzymes to perform their specific functions. In general, these directional preferences in intact cells are governed by relative affinities for nicotinamide adenine dinucleotide (phosphate) cofactors [NAD(P)(H)] and concentration gradients of these cofactors in subcellular compartments. For the reductive HSDs human 17betaHSD type 1 and rat AKR1C9, we can attenuate or reverse the directional preference in intact cells by site-directed mutagenesis in the cofactor-binding domain or by glucose deprivation, but the magnitude of such changes vary with the different enzymes.

An intracellular loop (IL2) residue confers different basal constitutive activities to the human lutropin receptor and human thyrotropin receptor through structural communication between IL2 and helix 6, via helix 3.

The human lutropin receptor (hLHR) and human TSH receptor (hTSHR) are G protein-coupled receptors that play key roles in reproductive and thyroid physiology, respectively. We show using a quantitative assessment of cAMP production as a function of cell surface receptor expression that the hTSHR possesses greater basal constitutive activity than the hLHR. Further studies were undertaken to test the hypothesis that different potential Gs-coupling motifs identified in IL2 of the hTSHR and hLHR contribute to their different basal constitutive activities. Although mutating the receptors to interchange their potential Gs-coupling motifs reversed their relative activities, we show this to be due to the swapping of one IL2 residue (Q476 in the hLHR; R531 in the hTSHR). Molecular dynamics simulations show that the effect of the hLHR(Q476R) mutation, switching the structural features of the hLHR toward those of the hTSHR, is greater than the switching effect of the hTSHR(R531Q) mutant toward the hLHR. The structural model of the hLHR(Q476R) mutant can be considered as a hybrid of wild-type (wt) hTSHR and constitutively active mutant hLHR forms. In this hLHR(Q476R) mutant, IL2 adopts a structure similar to IL2 of the wt hTSHR, but it shares with the hLHR constitutively active mutant the solvent exposure and the reciprocal arrangement of helices 3, 5, and 6, including the weakening of the wt native R3.50-D6.30 interaction. Our results suggest a H3-mediated structural connection between IL2 and the cytosolic extension of H6. Thus, IL2 contributes significantly to the inactive and active state ensembles of these G protein-coupled receptors.

Chimeras of the rat and human FSH receptors (FSHRs) identify residues that permit or suppress transmembrane 6 mutation-induced constitutive activation of the FSHR via rearrangements of hydrophobic interactions between helices 6 and 7.

Although a large number of naturally occurring activating mutations of the human LH receptor (hLHR) and human TSH receptor (hTSHR) have been identified, only one activating mutation of the human FSH receptor (hFSHR) has been found. Furthermore, mutations of several residues within the i3/transmembrane domain (TM) 6 region of the hFSHR that were done based upon known constitutively activating mutations of the human LHR were found to have no effect on hFSHR signaling. One of the hFSHR mutations examined in this context was the substitution of a highly conserved aspartate (D581) in TM6 with glycine. We show herein that although the basal activity of the rat FSHR (rFSHR) is similar to the hFSHR, mutation of the comparable residue (D580) in the rFSHR causes marked constitutive activation. Taking advantage of the high degree of amino acid identity between the rat and human FSHRs, we have used chimeras and point substitutions to determine the precise residues that suppress or permit constitutive activity by the D580/581G mutation. Thus, the simultaneous substitution of M576 in TM6 and H615 in TM7 of the hFSHR with the cognate rFSHR residues (threonine and tyrosine, respectively) now renders the hFSHR(D581G) mutant constitutively active. Conversely, the substitution of Y614 of the rFSHR with the cognate hFSHR residue (histidine) fully suppresses the constitutive activity of the rFSHR (D580G) mutant. Computer models of the human and rat FSHRs and mutants thereof were created based upon the crystal structure of rhodopsin. These models suggest that differences in hydrophobic interactions between TMs 6 and 7 of the rat and human FSHRs may account for the ability of TM6 of the rat, but not human, FSHR to adopt an active conformation as a result of the D580/581G mutation.

Beyond the cytoplasm of Escherichia coli: localizing recombinant proteins where you want them.

Recombinant protein expression in Escherichia coli represents a cornerstone of the biotechnology enterprise. While cytoplasmic expression in this host has received the most attention, achieving substantial yields of correctly folded proteins in this compartment can sometimes be met with difficulties. These issues can often be overcome by targeting protein expression to extracytoplasmic compartments (e.g., membrane, periplasm) or to the culture medium. This chapter discusses various strategies for exporting proteins out of the cytoplasm as well as tools for monitoring and optimizing these different export mechanisms.

Evolutionary comparisons predict that dimerization of human cytochrome P450 aromatase increases its enzymatic activity and efficiency.

Estrogen is an essential vertebrate hormone synthesized from androgens involving multiple hydroxylations, catalyzed by cytochrome P450 aromatase (P450arom or CYP19) enzymes. Despite their importance, very few comparative studies have been conducted on vertebrate and/or mammalian P450arom enzymes, either structurally or functionally. Here we directly compared the human (h-) and porcine gonadal (p(g)-) P450arom, as p(g)-P450arom has very low catalytic efficiency, with a ten-fold higher affinity (Km) for a substrate (androstenedione) and ten-fold reduction in turnover (Vmax). We recombinantly expressed these proteins and compared their interactions on a membrane using a quartz crystal microbalance (QCM) and also with the electron donor protein cytochrome P450 oxidoreductase (CPR). Changes in frequency and dissipation in the QCM supported the h-P450arom forming a homodimer that agreed with the FRET data, but not p(g)-P450arom. Analysis of the X-ray crystal structure of the h-P450arom suggested a likely site of homo-dimerization and found that certain key interacting residues were not conserved in pg-P450arom. Molecular dynamics simulations provide support for the importance of these residues in homo-dimerization. Here we propose that the lower affinity and higher activity with reduced release of intermediate metabolites by the h-P450arom is as a consequence of its ability to form homodimers. The functional implications of dimerization provide an important mechanistic step in the requirement for efficient aromatization.

Making water-soluble integral membrane proteins in vivo using an amphipathic protein fusion strategy.

Integral membrane proteins (IMPs) play crucial roles in all cells and represent attractive pharmacological targets. However, functional and structural studies of IMPs are hindered by their hydrophobic nature and the fact that they are generally unstable following extraction from their native membrane environment using detergents. Here we devise a general strategy for in vivo solubilization of IMPs in structurally relevant conformations without the need for detergents or mutations to the IMP itself, as an alternative to extraction and in vitro solubilization. This technique, called SIMPLEx (solubilization of IMPs with high levels of expression), allows the direct expression of soluble products in living cells by simply fusing an IMP target with truncated apolipoprotein A-I, which serves as an amphipathic proteic 'shield' that sequesters the IMP from water and promotes its solubilization.