Jennifer M. Kavran

Specificity and Promiscuity in Phosphoinositide Binding by Pleckstrin Homology Domains

Pleckstrin homology (PH) domains are small protein modules involved in recruitment of signaling molecules to cellular membranes, in some cases by binding specific phosphoinositides. We describe use of a convenient “dot-blot” approach to screen 10 different PH domains for those that recognize particular phosphoinositides. Each PH domain bound phosphoinositides in the assay, but only two (from phospholipase C-δ1and Grp1) showed clear specificity for a single species. Using soluble inositol phosphates, we show that the Grp1 PH domain (originally cloned on the basis of its phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) binding) binds specifically tod-myo-inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) (the PtdIns(3,4,5)P3headgroup) with K D = 27.3 nm, but bindsd-myo-inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) or d-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) over 80-fold more weakly. We show that this specificity allows localization of the Grp1 PH domain to the plasma membrane of mammalian cells only when phosphatidylinositol 3-kinase (PI 3-K) is activated. The presence of three adjacent equatorial phosphate groups was critical for inositol phosphate binding by the Grp1 PH domain. By contrast, another PH domain capable of PI 3-K-dependent membrane recruitment (encoded by EST684797) does not distinguish Ins(1,3,4)P3 from Ins(1,3,4,5)P3 (binding both with very high affinity), despite selecting strongly against Ins(1,4,5)P3. The remaining PH domains tested appear significantly less specific for particular phosphoinositides. Together with data presented in the literature, our results suggest that many PH domains bind similarly to multiple phosphoinositides (and in some cases phosphatidylserine), and are likely to be regulated in vivo by the most abundant species to which they bind. Thus, using the same simple approach to study several PH domains simultaneously, our studies suggest that highly specific phosphoinositide binding is a characteristic of relatively few cases.

Phosphatidylinositol-4,5-bisphosphate is required for endocytic coated vesicle formation

Receptor-mediated endocytosis via clathrin-coated vesicles has been extensively studied and, while many of the protein players have been identified, much remains unknown about the regulation of coat assembly and the mechanisms that drive vesicle formation [1]. Some components of the endocytic machinery interact with inositol polyphosphates and inositol lipids in vitro, implying a role for phosphatidylinositols in vivo [2] [3]. Specifically, the adaptor protein complex AP2 binds phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2), PtdIns(3)P, PtdIns(3,4,5)P3 and inositol phosphates. Phosphatidylinositol binding regulates AP2 self-assembly and the interactions of AP2 complexes with clathrin and with peptides containing endocytic motifs [4] [5]. The GTPase dynamin contains a pleckstrin homology (PH) domain that binds PtdIns(4,5)P2 and PtdIns(3,4,5)P3 to regulate GTPase activity in vitro [6] [7]. However, no direct evidence for the involvement of phosphatidylinositols in clathrin-mediated endocytosis exists to date. Using well-characterized PH domains as high affinity and high specificity probes in combination with a perforated cell assay that reconstitutes coated vesicle formation, we provide the first direct evidence that PtdIns(4,5)P2 is required for both early and late events in endocytic coated vesicle formation.

Transient Mammalian Cell Transfection with Polyethylenimine (PEI)

Standard protein expression systems, such as E. coli, often fail to produce folded, monodisperse, or functional eukaryotic proteins (see Small-scale Expression of Proteins in E. coli). The expression of these proteins is greatly benefited by using a eukaryotic system, such as mammalian cells, that contains the appropriate folding and posttranslational machinery. Here, we describe methods for both small- and large-scale transient expression in mammalian cells using polyethylenimine (PEI). We find this procedure to be more cost-effective and quicker than the more traditional route of generating stable cell lines. First, optimal transfection conditions are determined on a small-scale, using adherent cells. These conditions are then translated for use in large-scale suspension cultures. For further details on generating stable cell lines please (see Rapid creation of stable mammalian cell lines for regulated expression of proteins using the Gateway® Recombination Cloning Technology and Flp-In T-REx® lines or Generating mammalian stable cell lines by electroporation).

How IGF-1 activates its receptor

The type I insulin-like growth factor receptor (IGF1R) is involved in growth and survival of normal and neoplastic cells. A ligand-dependent conformational change is thought to regulate IGF1R activity, but the nature of this change is unclear. We point out an underappreciated dimer in the crystal structure of the related Insulin Receptor (IR) with Insulin bound that allows direct comparison with unliganded IR and suggests a mechanism by which ligand regulates IR/IGF1R activity. We test this mechanism in a series of biochemical and biophysical assays and find the IGF1R ectodomain maintains an autoinhibited state in which the TMs are held apart. Ligand binding releases this constraint, allowing TM association and unleashing an intrinsic propensity of the intracellular regions to autophosphorylate. Enzymatic studies of full-length and kinase-containing fragments show phosphorylated IGF1R is fully active independent of ligand and the extracellular-TM regions. The key step triggered by ligand binding is thus autophosphorylation. DOI: http://dx.doi.org/10.7554/eLife.03772.001

All Mammalian Hedgehog Proteins Interact with Cell Adhesion Molecule, Down-regulated by Oncogenes (CDO) and Brother of CDO (BOC) in a Conserved Manner

Hedgehog (Hh) signaling proteins stimulate cell proliferation, differentiation, and tissue patterning at multiple points in animal development. A single Hh homolog is present in Drosophila, but three Hh homologs, Sonic Hh, Indian Hh, and Desert Hh, are present in mammals. Distribution, movement, and reception of Hh signals are tightly regulated, and abnormal Hh signaling is associated with developmental defects and cancer. In addition to the integral membrane proteins Patched and Smoothened, members of the Drosophila Ihog family of adhesion-like molecules have recently been shown to bind Hh proteins with micromolar affinity and positively regulate Hh signaling. Cell adhesion molecule-related, down-regulated by oncogenes (CDO) and Brother of CDO (BOC) are the closest mammalian relatives of Drosophila Ihog, and CDO binds Sonic Hh with micromolar affinity and positively regulates Hh signaling. Despite these similarities, structural and biochemical studies have shown that Ihog and CDO utilize nonorthologous domains and completely different binding modes to interact with cognate Hh proteins. We report here biochemical and x-ray structural studies of Sonic, Indian, and Desert Hh proteins both alone and complexed with active domains of CDO and BOC. These results show that all mammalian Hh proteins bind CDO and BOC in the same manner. We also show that interactions between Hh proteins and CDO are weakened at low pH. Formation of Hh-mediated Hh oligomers is thought to be an important feature of normal Hh signaling, but no conserved self-interaction between Hh proteins is apparent from inspection of 14 independent Hh-containing crystal lattices.

In vitro enzymatic characterization of near full length EGFR in activated and inhibited states.

The epidermal growth factor receptor (EGFR) is a single-pass transmembrane protein with an extracellular ligand-binding region and a cytoplasmic tyrosine kinase. Ligand binding activates the tyrosine kinase, which in turn initiates signaling cascades that influence cell proliferation and differentiation. EGFR activity is essential for normal development of many multicellular organisms, and inappropriate activation of EGFR is associated with multiple human cancers. Several drugs targeting EGFR activity are approved cancer therapies, and new EGFR-targeted therapies are being actively pursued. Much of what is known about EGFR structure and function is derived from studies of soluble receptor fragments. We report here an approach to producing an active, membrane-spanning form of EGFR of suitable purity, homogeneity, and quantity for structural and functional studies. We show that EGFR is capable of direct autophosphorylation of tyrosine 845, which is located on its kinase activation loop, and that the kinase activity of EGFR is approximately 500-fold higher in the presence of EGF vs the inhibitory anti-EGFR antibody cetuximab. The potencies of the small molecule EGFR kinase inhibitors erlotinib and lapatinib for various forms of EGFR were measured, and the therapeutic and mechanistic implications of these results considered.

46,XY Gonadal Dysgenesis due to a Homozygous Mutation in Desert Hedgehog (DHH) Identified by Exome Sequencing.

BACKGROUND 46,XY disorders of sex development (DSD) comprise a heterogeneous group of congenital conditions. Mutations in a variety of genes can affect gonadal development or androgen biosynthesis/action and thereby influence the development of the internal and external genital organs. OBJECTIVE The objective of the study was to identify the genetic cause in two 46,XY sisters of a consanguineous family with DSD and gonadal tumor formation. METHODS We used a next-generation sequencing approach by exome sequencing. Electrophysiological and high-resolution ultrasound examination of peripheral nerves as well as histopathological examination of the gonads were performed. RESULTS We identified a novel homozygous R124Q mutation in the desert hedgehog gene (DHH), which alters a conserved residue among the three mammalian Hedgehog ligands sonic hedgehog, Indian hedgehog, and desert hedgehog. No other relevant mutations in DSD-related genes were encountered. The gonads of one patient showed partial gonadal dysgenesis with loss of Leydig cells in tubular areas with seminoma in situ and a hyperplasia of Leydig cell-like cells expressing CYP17A1 in more dysgenetic parts of the gonad. In addition, both patients suffer from a polyneuropathy. High-resolution ultrasound revealed a structural change of peripheral nerve structure that fits well to a minifascicle formation of peripheral nerves. CONCLUSION Mutations in DHH play a role in 46,XY gonadal dysgenesis and are associated with seminoma formation and a neuropathy with minifascicle formation. Gonadal dysgenesis in these cases may be due to impairment of Sertoli cell-Leydig cell interaction during gonadal development.

Regulation of S-Adenosylhomocysteine Hydrolase by Lysine Acetylation

Background: S-Adenosylhomocysteine hydrolase (SAHH) regulates methyltransferase reactions and is acetylated on two lysines. Results: We prepared acetylated semisynthetic SAHH and determined the impact of acetylation on structure and activity. Conclusion: Lys acetylation of SAHH changes hydrogen bonding patterns in its NAD+ binding regions and reduces its catalytic activity. Significance: Acetylation of SAHH may serve to regulate global alterations in cellular methylation. S-Adenosylhomocysteine hydrolase (SAHH) is an NAD+-dependent tetrameric enzyme that catalyzes the breakdown of S-adenosylhomocysteine to adenosine and homocysteine and is important in cell growth and the regulation of gene expression. Loss of SAHH function can result in global inhibition of cellular methyltransferase enzymes because of high levels of S-adenosylhomocysteine. Prior proteomics studies have identified two SAHH acetylation sites at Lys401 and Lys408 but the impact of these post-translational modifications has not yet been determined. Here we use expressed protein ligation to produce semisynthetic SAHH acetylated at Lys401 and Lys408 and show that modification of either position negatively impacts the catalytic activity of SAHH. X-ray crystal structures of 408-acetylated SAHH and dually acetylated SAHH have been determined and reveal perturbations in the C-terminal hydrogen bonding patterns, a region of the protein important for NAD+ binding. These crystal structures along with mutagenesis data suggest that such hydrogen bond perturbations are responsible for SAHH catalytic inhibition by acetylation. These results suggest how increased acetylation of SAHH may globally influence cellular methylation patterns.

A naturally occurring repeat protein with high internal sequence identity defines a new class of TPR-like proteins

Linear repeat proteins often have high structural similarity and low (∼25%) pairwise sequence identities (PSI) among modules. We identified a unique P. anserina (Pa) sequence with tetratricopeptide repeat (TPR) homology, which contains longer (42 residue) repeats (42PRs) with an average PSI >91%. We determined the crystal structure of five tandem Pa 42PRs to 1.6 Å, and examined the stability and solution properties of constructs containing three to six Pa 42PRs. Compared with 34-residue TPRs (34PRs), Pa 42PRs have a one-turn extension of each helix, and bury more surface area. Unfolding transitions shift to higher denaturant concentration and become sharper as repeats are added. Fitted Ising models show Pa 42PRs to be more cooperative than consensus 34PRs, with increased magnitudes of intrinsic and interfacial free energies. These results demonstrate the tolerance of the TPR motif to length variation, and provide a basis to understand the effects of helix length on intrinsic/interfacial stability.

Single cell cloning of a stable mammalian cell line.

Isolate cell lines with improved stability or expression properties from a parental cell line.