The Streptomyces coelicolor Lipoate-protein Ligase Is a Circularly Permuted Version of the Escherichia coli Enzyme Composed of Discrete Interacting Domains

Lipoate-protein ligases are used to scavenge lipoic acid from the environment and attach the coenzyme to its cognate proteins, which are generally the E2 components of the 2-oxoacid dehydrogenases. The enzymes use ATP to activate lipoate to its adenylate, lipoyl-AMP, which remains tightly bound in the active site. This mixed anhydride is attacked by the ϵ-amino group of a specific lysine present on a highly conserved acceptor protein domain, resulting in the amide-linked coenzyme. The Streptomyces coelicolor genome encodes only a single putative lipoate ligase. However, this protein had only low sequence identity (<25%) to the lipoate ligases of demonstrated activity and appears to be a circularly permuted version of the known lipoate ligase proteins in that the canonical C-terminal domain seems to have been transposed to the N terminus. We tested the activity of this protein both by in vivo complementation of an Escherichia coli ligase-deficient strain and by in vitro assays. Moreover, when the domains were rearranged into a protein that mimicked the arrangement found in the canonical lipoate ligases, the enzyme retained complementation activity. Finally, when the two domains were separated into two proteins, both domain-containing proteins were required for complementation and catalysis of the overall ligase reaction in vitro. However, only the large domain-containing protein was required for transfer of lipoate from the lipoyl-AMP intermediate to the acceptor proteins, whereas both domain-containing proteins were required to form lipoyl-AMP.     

The Golgi-associated PDZ Domain Protein PIST/GOPC Stabilizes the β1-Adrenergic Receptor in Intracellular Compartments after Internalization

Many G-protein-coupled receptors carry C-terminal ligand motifs for PSD-95/discs large/ZO-1 (PDZ) domains; via interaction with PDZ domain-containing scaffold proteins, this allows for integration of receptors into signaling complexes. However, the presence of PDZ domain proteins attached to intracellular membranes suggests that PDZ-type interactions may also contribute to subcellular sorting of receptors. The protein interacting specifically with Tc10 (PIST; also known as GOPC) is a trans-Golgi-associated protein that interacts through its single PDZ domain with a variety of cell surface receptors. Here we show that PIST controls trafficking of the interacting β1-adrenergic receptor both in the anterograde, biosynthetic pathway and during postendocytic recycling. Overexpression and knockdown experiments show that PIST leads to retention of the receptor in the trans-Golgi network (TGN), to the effect that overexpressed PIST reduces activation of the MAPK pathway by β1-adrenergic receptor (β1AR) agonists. Receptors can be released from retention in the TGN by coexpression of the plasma membrane-associated scaffold PSD-95, which allows for transport of receptors to the plasma membrane. Stimulation of β1 receptors and activation of the cAMP pathway lead to relocation of PIST from the TGN to an endosome-like compartment. Here PIST colocalizes with SNX1 and the internalized β1AR and protects endocytosed receptors from lysosomal degradation. In agreement, β1AR levels are decreased in hippocampi of PIST-deficient mice. Our data suggest that PIST contributes to the fine-tuning of β1AR sorting both during biosynthetic and postendocytic trafficking.     

The Atomic Structure of the HIV-1 gp41 Transmembrane Domain and Its Connection to the Immunogenic Membrane-proximal External Region

The membrane-proximal external region (MPER) C-terminal segment and the transmembrane domain (TMD) of gp41 are involved in HIV-1 envelope glycoprotein-mediated fusion and modulation of immune responses during viral infection. However, the atomic structure of this functional region remains unsolved. Here, based on the high resolution NMR data obtained for peptides spanning the C-terminal segment of MPER and the TMD, we report two main findings: (i) the conformational variability of the TMD helix at a membrane-buried position; and (ii) the existence of an uninterrupted α-helix spanning MPER and the N-terminal region of the TMD. Thus, our structural data provide evidence for the bipartite organization of TMD predicted by previous molecular dynamics simulations and functional studies, but they do not support the breaking of the helix at Lys-683, as was suggested by some models to mark the initiation of the TMD anchor. Antibody binding energetics examined with isothermal titration calorimetry and humoral responses elicited in rabbits by peptide-based vaccines further support the relevance of a continuous MPER-TMD helix for immune recognition. We conclude that the transmembrane anchor of HIV-1 envelope is composed of two distinct subdomains: 1) an immunogenic helix at the N terminus also involved in promoting membrane fusion; and 2) an immunosuppressive helix at the C terminus, which might also contribute to the late stages of the fusion process. The unprecedented high resolution structural data reported here may guide future vaccine and inhibitor developments.     

Interdomain Hydrophobic Interactions Modulate the Thermostability of Microbial Esterases from the Hormone-Sensitive Lipase Family

Microbial hormone-sensitive lipases (HSLs) contain a CAP domain and a catalytic domain. However, it remains unclear how the CAP domain interacts with the catalytic domain to maintain the stability of microbial HSLs. Here, we isolated an HSL esterase, E40, from a marine sedimental metagenomic library. E40 exhibited the maximal activity at 45 °C and was quite thermolabile, with a half-life of only 2 min at 40 °C, which may be an adaptation of E40 to the permanently cold sediment environment. The structure of E40 was solved to study its thermolability. Structural analysis showed that E40 lacks the interdomain hydrophobic interactions between loop 1 of the CAP domain and α7 of the catalytic domain compared with its thermostable homologs. Mutational analysis showed that the introduction of hydrophobic residues Trp²⁰² and Phe²⁰³ in α7 significantly improved E40 stability and that a further introduction of hydrophobic residues in loop 1 made E40 more thermostable because of the formation of interdomain hydrophobic interactions. Altogether, the results indicate that the absence of interdomain hydrophobic interactions between loop 1 and α7 leads to the thermolability of E40. In addition, a comparative analysis of the structures of E40 and other thermolabile and thermostable HSLs suggests that the interdomain hydrophobic interactions between loop 1 and α7 are a key element for the thermostability of microbial HSLs. Therefore, this study not only illustrates the structural element leading to the thermolability of E40 but also reveals a structural determinant for HSL thermostability.     

The Cytoplasmic Domain of A Disintegrin and Metalloproteinase 10 (ADAM10) Regulates Its Constitutive Activity but Is Dispensable for Stimulated ADAM10-dependent Shedding

The membrane-anchored metalloproteinase a disintegrin and metalloprotease 10 (ADAM10) is required for shedding of membrane proteins such as EGF, betacellulin, the amyloid precursor protein, and CD23 from cells. ADAM10 is constitutively active and can be rapidly and post-translationally enhanced by several stimuli, yet little is known about the underlying mechanism. Here, we use ADAM10-deficient cells transfected with wild type or mutant ADAM10 to address the role of its cytoplasmic and transmembrane domain in regulating ADAM10-dependent protein ectodomain shedding. We report that the cytoplasmic domain of ADAM10 negatively regulates its constitutive activity through an ER retention motif but is dispensable for its stimulated activity. However, chimeras with the extracellular domain of ADAM10 and the transmembrane domain of ADAM17 with or without the cytoplasmic domain of ADAM17 show reduced stimulated shedding of the ADAM10 substrate betacellulin, whereas the ionomycin-stimulated shedding of the ADAM17 substrates CD62-L and TGFα is not affected. Moreover, we show that influx of extracellular calcium activates ADAM10 but is not essential for its activation by APMA and BzATP. Finally, the rapid stimulation of ADAM10 is not significantly affected by incubation with proprotein convertase inhibitors for up to 8 h, arguing against a major role of increased prodomain removal in the rapid stimulation of ADAM10. Thus, the cytoplasmic domain of ADAM10 negatively influences constitutive shedding through an ER retention motif, whereas the cytoplasmic domain and prodomain processing are not required for the rapid activation of ADAM10-dependent shedding events.     

SorCS1 variants and amyloid precursor protein (APP) are co‐transported in neurons but only SorCS1c modulates anterograde APP transport

Processing of amyloid precursor protein (APP) into amyloid‐β peptide (Aβ) is crucial for the development of Alzheimer's disease (AD). Because this processing is highly dependent on its intracellular itinerary, altered subcellular targeting of APP is thought to directly affect the degree to which Aβ is generated. The sorting receptor SorCS1 has been genetically linked to AD, but the underlying molecular mechanisms are poorly understood. We analyze two SorCS1 variants; one, SorCS1c, conveys internalization of surface‐bound ligands whereas the other, SorCS1b, does not. In agreement with previous studies, we demonstrate co‐immunoprecipitation and co‐localization of both SorCS1 variants with APP. Our results suggest that SorCS1c and APP are internalized independently, although they mostly share a common post‐endocytic pathway. We introduce functional Venus‐tagged constructs to study SorCS1b and SorCS1c in living cells. Both variants are transported by fast anterograde axonal transport machinery and about 30% of anterograde APP‐positive transport vesicles contain SorCS1. Co‐expression of SorCS1b caused no change of APP transport kinetics, but SorCS1c reduced the anterograde transport rate of APP and increased the number of APP‐positive stationary vesicles. These data suggest that SorCS1 and APP share trafficking pathways and that SorCS1c can retain APP from insertion into anterograde transport vesicles.

     

Identification and engineering of the cytochalasin gene cluster from Aspergillus clavatus NRRL 1

Cytochalasins are a group of fungal secondary metabolites with diverse structures and bioactivities, including cytochalasin E produced by Aspergillus clavatus, which is a potent anti-angiogenic agent. Here, we report the identification and characterization of the cytochalasin gene cluster from A. clavatus NRRL 1. As a producer of cytochalasin E and K, the genome of A. clavatus was analyzed and the ∼30 kb ccs gene cluster was identified based on the presence of a polyketide synthase–nonribosomal peptide synthetases (PKS–NRPS) and a putative Baeyer–Villiger monooxygenase (BVMO). Deletion of the central PKS–NRPS gene, ccsA, abolished the production of cytochalasin E and K, confirming the association between the natural products and the gene cluster. Based on bioinformatic analysis, a putative biosynthetic pathway is proposed. Furthermore, overexpression of the pathway specific regulator ccsR elevated the titer of cytochalasin E from 25 mg/L to 175 mg/L. Our results not only shed light on the biosynthesis of cytochalasins, but also provided genetic tools for increasing and engineering the production.     

Shift in chicken intestinal gene association networks after infection with Salmonella

A primary infection of Salmonella enteritidis causes a spatial-temporal dependent change in the gene expression patterns in the intestine of chickens (Gallus gallus). This is the result of a dynamic intestinal response to adapt to the altered environment and to optimize its ‘health’ and functionality under the new circumstances. By inferring gene association networks (GANs), the complexities of and changes in biological networks can be uncovered. Within such GANs highly interacting (hub) genes can be identified, which are supposed to be high-level regulators connected to multiple processes. By exploring the intestinal expression of genes differing between control and Salmonella infected chicken in a time-dependent manner differences in GANs were found. In control chickens more developmental processes were observed, whereas in infected chickens relatively more processes were associated to ‘defense/pathogen response’. Moreover the conserved protein domains of the identified hub genes in controls were nuclear-associated, whereas hub genes in infected chickens were involved in ‘cellular communication’. The shift in topology and functionality of the intestinal GANs in control and Salmonella infected animals and the identification of GAN-specific hubs is a first step to understand the complexity of biological networks and processes regulating intestinal health and functionality under normal and disturbed conditions.     

Thermal inactivation of the reductase domain of cytochrome P450 BM3

Although the reductase domain of cytochrome P450 BM3 (BMR) catalyzes the reduction of cytochrome c and 2,6-dichlorophenolindophenol, we observed a catalytically independent loss of activity. By varying the incubation time for the enzyme prior to reaction initiation, we measured an inactivation rate of 0.22 min(-1). We hypothesized that either an active BMR dimer dissociates to an inactive monomer or BMR undergoes denaturation. We were not able to trap or destabilize a dimer, and BMR inactivation proved to be irreversible. Addition of excess FMN only slightly decreased the rate of inactivation from 0.22 to 0.13 min(-1), indicating inactivation likely does not reflect loss of flavin. When inactivation rates as a function of temperature were fit to the Arrhenius equation, the energy required to inactivate BMR was 9.9 kcal mol(-1)--equivalent to a few hydrogen bonds. The potential instability of BMR under certain conditions raises concerns for the use of BMR as a model or surrogate P450 reductase in other systems.