LapB (YciM) orchestrates protein–protein interactions at the interface of lipopolysaccharide and phospholipid biosynthesis

The outer membrane (OM) of Gram-negative bacteria functions as an essential barrier and is characterized by an asymmetric bilayer with lipopolysaccharide (LPS) in the outer leaflet. The enzyme LpxC catalyzes the first committed step in LPS biosynthesis. It plays a critical role in maintaining the balance between LPS and phospholipids (PL), which are both derived from the same biosynthetic precursor. The essential inner membrane proteins YejM (PbgA, LapC), LapB (YciM), and the protease FtsH are known to account for optimal LpxC levels, but the mechanistic details are poorly understood. LapB is thought to be a bi-functional protein serving as an adaptor for FtsH-mediated turnover of LpxC and acting as a scaffold in the coordination of LPS biosynthesis. Here, we provide experimental evidence for the physical interaction of LapB with proteins at the biosynthetic node from where the LPS and PL biosynthesis pathways diverge. By a total of four in vivo and in vitro assays, we demonstrate protein–protein interactions between LapB and the LPS biosynthesis enzymes LpxA, LpxC, and LpxD, between LapB and YejM, the anti-adaptor protein regulating LapB activity, and between LapB and FabZ, the first PL biosynthesis enzyme. Moreover, we uncovered a new adaptor function of LapB in destabilizing not only LpxC but also LpxD. Overall, our study shows that LapB is a multi-functional protein that serves as a protein–protein interaction hub for key enzymes in LPS and PL biogenesis presumably by virtue of multiple tetratricopeptide repeat (TPR) motifs in its cytoplasmic C-terminal region.     

PRICE (PRotein Interface Conservation and Energetics): a server for the analysis of protein–protein interfaces

Residues in a protein–protein interface that are important for forming and stabilizing the interaction can usually be identified by looking at patterns of evolutionary conservation in groups of homologous proteins and also by the computational identification of binding hotspots. The PRICE (PRotein Interface Conservation and Energetics) server takes the coordinates of a protein–protein complex, dissects the interface into core and rim regions, and calculates (1) the degree of conservation (measured as the sequence entropy), as well as (2) the change in free energy of binding (∆∆G, due to alanine scanning mutagenesis) of interface residues. Results are displayed as color-coded plots and also made available for download. This enables the computational identification of binding hot spots, based on which further experiments can be designed. The method will aid in protein functional prediction by correct assignment of hot regions involved in binding. Consideration of sequence entropies for residues with large ∆∆G values may provide an indication of the biological relevance of the interface. Finally, the results obtained on a test set of alanine mutants has been compared to those obtained using other servers/methods. The PRICE server is a web application available at .     

Peroxisome biogenesis and protein import in plants,animals and yeasts: enigma and variations? (Review)

Peroxisomes are found in virtually all eukaryotic cells, where they play varied but essential metabolic roles, exemplified by the catastrophic effects of mutations that compromise peroxisome biogenesis and function. This review will aim to provide an accessible introduction to peroxisome biogenesis and protein import for the non-specialist, and draws together recent advances in peroxisome protein targeting and import in plants, animals and yeasts, seeking to define common themes and highlight variations. Despite much progress, many aspects of peroxisome biology remain an enigma, and current questions and controversies in the field are highlighted and discussed.     

Nitric oxide modulates sodium vitamin C transporter 2 (SVCT-2) protein expression via protein kinase G (PKG) and nuclear factor-κB (NF-κB)

Ascorbate is an important antioxidant, which also displays important functions in neuronal tissues, including the retina. The retina is responsible for the initial steps of visual processing, which is further refined in cerebral high-order centers. The retina is also a prototypical model for studying physiologic aspects of cells that comprise the nervous system. Of major importance also is the cellular messenger nitric oxide (NO). Previous studies have demonstrated the significance of NO for both survival and proliferation of cultured embryonic retinal cells. Cultured retinal cells express a high-affinity ascorbate transporter, and the release of ascorbate is delicately regulated by ionotropic glutamate receptors. Therefore, we proposed whether there is interplay between the ascorbate transport system and NO signaling pathway in retinal cells. Here we show compelling evidence that ascorbate uptake is tightly controlled by NO and its downstream signaling pathway in culture. NO also modulates the expression of SVCT-2, an effect mediated by cGMP and PKG. Kinetic studies suggest that NO increases the transport capacity for ascorbate, but not the affinity of SVCT-2 for its substrate. Interestingly, NO utilizes the NF-κB pathway, in a PKG-dependent manner, to modulate both SVCT-2 expression and ascorbate uptake. These results demonstrate that NO exerts a fine-tuned control of the availability of ascorbate to cultured retinal cells and strongly reinforces ascorbate as an important bioactive molecule in neuronal tissues.     

Both the N-terminal fragment and the protein–protein interaction domain (PDZ domain) are required for the pro-apoptotic activity of presenilin-associated protein PSAP

Presenilin-associated protein (PSAP) was originally identified as a PS1-associated, PDZ domain protein. In a subsequent study, PSAP was found to be a mitochondrial apoptotic molecule. In this study, we cloned the PSAP gene and found that it is composed of 12 exons and localizes on chromosome 6. To better understand the structure and function of PSAP, we have generated a series of antibodies that recognize different regions of PSAP. Using these antibodies, we found that PSAP is expressed in four isoforms as a result of differential splicing of exon 8 in addition to the use of either the first or the second ATG codon as the start codon. We also found that all these isoforms are localized in the mitochondria and are pro-apoptotic. Furthermore, our data revealed that the PDZ domain and N-terminal fragment are required for the pro-apoptotic activity of PSAP.     

Chk1 modulates the interaction between myosin phosphatase targeting protein 1 (MYPT1) and protein phosphatase 1cβ (PP1cβ)

Polo-like kinase 1 (Plk1) is an instrumental kinase that modulates many aspects of the cell cycle. Previous investigations have indicated that Plk1 is a target of the DNA damage response, and Plk1 inhibition is dependent on ATM/ATR and Chk1. But the exact mechanism remains elusive. In a proteomic screen to identify Chk1-interacting proteins, we found that myosin phosphatase targeting protein 1 (MYPT1) was present in the immunocomplex. MYPT1 is phosphorylated by CDK1, thus recruiting protein phosphatase 1β (PP1cβ) to dephosphorylate and inactivate Plk1. Here we identified that Chk1 directly interacts with MYPT1 and preferentially phosphorylates MYPT1 at Ser20, which is essential for MYPT1-PP1cβ interaction and subsequent Plk1 dephosphorylation. Phosphorylation of Ser20 is abolished during mitotic damage when Chk1 is inhibited. The degradation of MYPT1 is also regulated by Chk1 phosphorylation. Our results thus unveil the underlying machinery that attenuates Plk1 activity during mitotic damage through Chk1-induced phosphorylation of MYPT1.     

Hydrogen sulfide and L-cysteine increase phosphatidylinositol 3,4,5-trisphosphate (PIP3) and glucose utilization by inhibiting phosphatase and tensin homolog (PTEN) protein and activating phosphoinositide 3-kinase (PI3K)/serine/threonine protein kinase (AKT)/protein kinase Cζ/λ (PKCζ/λ) in 3T3l1 adipocytes

This work examined the novel hypothesis that reduced levels of H(2)S or L-cysteine (LC) play a role in the impaired glucose metabolism seen in diabetes. 3T3L1 adipocytes were treated with high glucose (HG, 25 mM) in the presence or absence of LC or H(2)S. Both LC and H(2)S treatments caused an increase in phosphatidylinositol-3,4,5 trisphosphate (PIP3), AKT phosphorylation, and glucose utilization in HG-treated cells. The effect of LC on PIP3 and glucose utilization was prevented by propargylglycine, an inhibitor of cystathionine γ-lyase that catalyzes H(2)S formation from LC. This demonstrates that H(2)S mediates the effect of LC on increased PIP3 and glucose utilization. H(2)S and LC caused phosphatidylinositol 3-kinase activation and PTEN inhibition. Treatment with LC, H(2)S, or PIP3 increased the phosphorylation of IRS1, AKT, and PKCζ/λ as well as GLUT4 activation and glucose utilization in HG-treated cells. This provides evidence that PIP3 is involved in the increased glucose utilization observed in cells supplemented with LC or H(2)S. Comparative signal silencing studies with siAKT2 or siPKCζ revealed that PKCζ phosphorylation is more effective for the GLUT4 activation and glucose utilization in LC-, H(2)S-, or PIP3-treated cells exposed to HG. This is the first report to demonstrate that H(2)S or LC can increase cellular levels of PIP3, a positive regulator of glucose metabolism. The PIP3 increase is mediated by PI3K activation and inhibition of PTEN but not of SHIP2. This study provides evidence for a molecular mechanism by which H(2)S or LC can up-regulate the insulin-signaling pathways essential for maintenance of glucose metabolism.     

The heterotrimeric G protein subunits Gα(q) and Gβ(1) have lysophospholipase D activity

In a previous study we purified a novel lysoPLD (lysophospholipase D) which converts LPC (lysophosphatidylcholine) into a bioactive phospholipid, LPA (lysophosphatidic acid), from the rat brain. In the present study, we identified the purified 42 and 35 kDa proteins as the heterotrimeric G protein subunits Gα(q) and Gβ(1) respectively. When FLAG-tagged Gα(q) or Gβ(1) was expressed in cells and purified, significant lysoPLD activity was observed in the microsomal fractions. Levels of the hydrolysed product choline increased over time, and the Mg(2+) dependency and substrate specificity of Gα(q) were similar to those of lysoPLD purified from the rat brain. Mutation of Gα(q) at amino acids Lys(52), Thr(186) or Asp(205), residues that are predicted to interact with nucleotide phosphates or catalytic Mg(2+), dramatically reduced lysoPLD activity. GTP does not compete with LPC for the lysoPLD activity, indicating that these substrate-binding sites are not identical. Whereas the enzyme activity of highly purified FLAG-tagged Gα(q) overexpressed in COS-7 cells was ~4 nmol/min per mg, the activity from Neuro2A cells was 137.4 nmol/min per mg. The calculated K(m) and V(max) values for lysoPAF (1-O-hexadecyl-sn-glycero-3-phosphocholine) obtained from Neuro2A cells were 21 μM and 0.16 μmol/min per mg respectively, similar to the enzyme purified from the rat brain. These results reveal a new function for Gα(q) and Gβ(1) as an enzyme with lysoPLD activity. Tag-purified Gα(11) also exhibited a high lysoPLD activity, but Gα(i) and Gα(s) did not. The lysoPLD activity of the Gα subunit is strictly dependent on its subfamily and might be important for cellular responses. However, treatment of Hepa-1 cells with Gα(q) and Gα(11) siRNAs (small interfering RNAs) did not change lysoPLD activity in the microsomal fraction. Clarification of the physiological relevance of lysoPLD activity of these proteins will need further studies.     

Two distinct aspects of coupling between Gα(i) protein and G protein-activated K+ channel (GIRK) revealed by fluorescently labeled Gα(i3) protein subunits

G protein-activated K(+) channels (Kir3 or GIRK) are activated by direct interaction with Gβγ. Gα is essential for specific signaling and regulates basal activity of GIRK (I(basal)) and kinetics of the response elicited by activation by G protein-coupled receptors (I(evoked)). These regulations are believed to occur within a GIRK-Gα-Gβγ signaling complex. Fluorescent energy resonance transfer (FRET) studies showed strong GIRK-Gβγ interactions but yielded controversial results regarding the GIRK-Gα(i/o) interaction. We investigated the mechanisms of regulation of GIRK by Gα(i/o) using wild-type Gα(i3) (Gα(i3)WT) and Gα(i3) labeled at three different positions with fluorescent proteins, CFP or YFP (xFP). Gα(i3)xFP proteins bound the cytosolic domain of GIRK1 and interacted with Gβγ in a guanine nucleotide-dependent manner. However, only an N-terminally labeled, myristoylated Gα(i3)xFP (Gα(i3)NT) closely mimicked all aspects of Gα(i3)WT regulation except for a weaker regulation of I(basal). Gα(i3) labeled with YFP within the Gα helical domain preserved regulation of I(basal) but failed to restore fast I(evoked). Titrated expression of Gα(i3)NT and Gα(i3)WT confirmed that regulation of I(basal) and of the kinetics of I(evoked) of GIRK1/2 are independent functions of Gα(i). FRET and direct biochemical measurements indicated much stronger interaction between GIRK1 and Gβγ than between GIRK1 and Gα(i3). Thus, Gα(i/o)βγ heterotrimer may be attached to GIRK primarily via Gβγ within the signaling complex. Our findings support the notion that Gα(i/o) actively regulates GIRK. Although regulation of I(basal) is a function of Gα(i)(GDP), our new findings indicate that regulation of kinetics of I(evoked) is mediated by Gα(i)(GTP).     

High-light-inducible proteins HliA and HliB: pigment binding and protein–protein interactions

High-light-inducible proteins (Hlips) are single-helix transmembrane proteins that are essential for the survival of cyanobacteria under stress conditions. The model cyanobacterium Synechocystis sp. PCC 6803 contains four Hlip isoforms (HliA-D) that associate with Photosystem II (PSII) during its assembly. HliC and HliD are known to form pigmented (hetero)dimers that associate with the newly synthesized PSII reaction center protein D1 in a configuration that allows thermal dissipation of excitation energy. Thus, it is expected that they photoprotect the early steps of PSII biogenesis. HliA and HliB, on the other hand, bind the PSII inner antenna protein CP47, but the mode of interaction and pigment binding have not been resolved. Here, we isolated His-tagged HliA and HliB from Synechocystis and show that these two very similar Hlips do not interact with each other as anticipated, rather they form HliAC and HliBC heterodimers. Both dimers bind Chl and β-carotene in a quenching conformation and associate with the CP47 assembly module as well as later PSII assembly intermediates containing CP47. In the absence of HliC, the cellular levels of HliA and HliB were reduced, and both bound atypically to HliD. We postulate a model in which HliAC-, HliBC-, and HliDC-dimers are the functional Hlip units in Synechocystis. The smallest Hlip, HliC, acts as a ‘generalist’ that prevents unspecific dimerization of PSII assembly intermediates, while the N-termini of ‘specialists’ (HliA, B or D) dictate interactions with proteins other than Hlips.

     

N(α)-Acetylation of yeast ribosomal proteins and its effect on protein synthesis

N(α)-Acetyltransferases (NATs) cause the N(α)-acetylation of the majority of eukaryotic proteins during their translation, although the functions of this modification have been largely unexplored. In yeast (Saccharomyces cerevisiae), four NATs have been identified: NatA, NatB, NatC, and NatD. In this study, the N(α)-acetylation status of ribosomal protein was analyzed using NAT mutants combined with two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS). A total of 60 ribosomal proteins were identified, of which 17 were N(α)-acetylated by NatA, and two by NatB. The N(α)-acetylation of two of these, S17 and L23, by NatA was not previously observed. Furthermore, we tested the effect of ribosomal protein N(α)-acetylation on protein synthesis using the purified ribosomes from each NAT mutant. It was found that the protein synthesis activities of ribosomes from NatA and NatB mutants were decreased by 27% and 23%, respectively, as compared to that of the normal strain. Furthermore, we have shown that ribosomal protein N(α)-acetylation by NatA influences translational fidelity in the presence of paromomycin. These results suggest that ribosomal protein N(α)-acetylation is necessary to maintain the ribosome's protein synthesis function.     

Assessment and significance of protein–protein interactions during development of protein biopharmaceuticals

Early development of protein biotherapeutics using recombinant DNA technology involved progress in the areas of cloning, screening, expression and recovery/purification. As the biotechnology industry matured, resulting in marketed products, a greater emphasis was placed on development of formulations and delivery systems requiring a better understanding of the chemical and physical properties of newly developed protein drugs. Biophysical techniques such as analytical ultracentrifugation, dynamic and static light scattering, and circular dichroism were used to study protein–protein interactions during various stages of development of protein therapeutics. These studies included investigation of protein self-association in many of the early development projects including analysis of highly glycosylated proteins expressed in mammalian CHO cell cultures. Assessment of protein–protein interactions during development of an IgG1 monoclonal antibody that binds to IgE were important in understanding the pharmacokinetics and dosing for this important biotherapeutic used to treat severe allergic IgE-mediated asthma. These studies were extended to the investigation of monoclonal antibody–antigen interactions in human serum using the fluorescent detection system of the analytical ultracentrifuge. Analysis by sedimentation velocity analytical ultracentrifugation was also used to investigate competitive binding to monoclonal antibody targets. Recent development of high concentration protein formulations for subcutaneous administration of therapeutics posed challenges, which resulted in the use of dynamic and static light scattering, and preparative analytical ultracentrifugation to understand the self-association and rheological properties of concentrated monoclonal antibody solutions.     

Carbohydrate tolerance,serum albumin and protein values of normal and “waster” marmosets (Callithrix jacchus)

The major objective of this study was to establish standard glucose and lactose tolerance curves for the common marmoset (Callithrix jacchus). These data were utilized to establish criteria for detection of abnormal glucose tolerance and characterization of some aspects of the “marmoset wasting syndrome” which has been observed in this species. Glucose and lactose tolerance tests were performed on healthy animals and typical “marmoset wasters.” Eighteen normal animals were 18 to 36 months old and weighed 194–280 g. Six “wasters” were in the age range of 24 to 84 months and weighed 163–253 g. Seven experiments were carried out for each glucose tolerance test. In each trial it was observed that the serum glucose concentration (SGC) of the healthy animal after 90 min was two times higher than the pre-administration concentrations. The SGC returned to the pre-administration concentration within 150–300 min in animals administered glucose at dosages of 2 g/kg and 1 g/kg of body weight. However, at the dosage level of 5 g/kg body weight, the SGC of the animals tripled after 30 min and required 300 min to return to the pre-administration level. The 2 g/kg dosage level was chosen as typical. When similar experiments were conducted with animals identified as “chronic wasters,” all of the animals except one were observed to be inefficient in the absorption of glucose. When lactose was administered at a level of 4 g/kg, similar results were obtained. Normal and “waster” marmosets were also subjected to serum total protein, albumin and electrophoresis determinations in an effort to establish additional criteria that may be utilized in the identification of the “marmoset wasting” syndrome. Serum albumin was significantly higher in the “waster” marmosets 30 min following an oral administration of glucose than was observed in normal animals. Total protein values were not significantly lower in the “wasters” when subjected to the same tolerance test. The albumin level in normal animals was not affected by similar glucose tolerance tests. The electrophoretic patterns of serum protein for normal animals exhibited more bands than was observed in patterns of serum protein for “waster” marmosets. From these data, it seems logical that these diagnostic tests may be useful in developing a profile for the early detection of the “wasting” syndrome in marmosets.     

Peptides and peptidomimetics as regulators of protein–protein interactions

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Acyl coenzyme A-binding protein (ACBP) is phosphorylated and secreted by retinal Müller astrocytes following protein kinase C activation

Horizontal optokinetic stimulation of rabbit retina in vivo evokes increased expression of acyl coenzyme A-binding protein (ACBP), also known as 'diazepam binding inhibitor,' from retinal Müller cells. If the expressed ACBP were also secreted by Müller cells, then stimulus-evoked secretion of ACBP could influence the activity of GABA_A receptor-expressing retinal neurons. In this study, we examine in vitro whether ACBP is secreted by Müller glial cells and Müller-like QNR/K2 cells following stimulation with elevated levels of KCl and phorbol myristic acetate (PMA). KCl and PMA stimulation evoked secretion of threonine-phosphorylated ACBP. A sequence analysis of ACBP shows that it has five potential phosphorylation sites: Two threonine sites fit a protein kinase C phosphorylation pattern. Two threonine sites fit a casein kinase II (CK2) pattern. One serine site fits a CK2 pattern. As CK2 is not expressed in QNR/K2 cells, it is probable that protein kinase C accounts for the phosphorylation of ACBP in these cells and for the PMA-evoked secretion of ACBP. Serine phosphorylation was constitutive. Horizontal optokinetic stimulation increased threonine-phosphorylated ACBP in rabbit retina. Phosphorylation of ACBP may influence its target affinity. We used a proteolytic fragment of ACBP, octadecaneuropeptide (ODN), to investigate how threonine phosphorylation influences its affinity for GABA_A receptors. Threonine-phosphorylated ODN had a stronger affinity for GABA_A receptors than did unphosphorylated ODN or unphosphorylated ACBP. We conclude that stimulus-induced Müller cell secretion of phosphorylated ACBP could influence the GABAergic transmission in neighboring retinal neurons.