Inhibition of the Nef regulatory protein of HIV-1 by a single-domain antibody

The Nef protein of HIV-1 is important for AIDS pathogenesis, but it is not targeted by current antiviral strategies. Here, we describe a single-domain antibody (sdAb) that binds to HIV-1 Nef with a high affinity (K(d) = 2 × 10(-9)M) and inhibited critical biologic activities of Nef both in vitro and in vivo. First, it interfered with the CD4 down-regulation activity of a broad panel of nef alleles through inhibition of the Nef effects on CD4 internalization from the cell surface. Second, it was able to interfere with the association of Nef with the cellular p21-activated kinase 2 as well as with the resulting inhibitory effect of Nef on actin remodeling. Third, it counteracted the Nef-dependent enhancement of virion infectivity and inhibited the positive effect of Nef on virus replication in peripheral blood mononuclear cells. Fourth, anti-Nef sdAb rescued Nef-mediated thymic CD4(+) T-cell maturation defects and peripheral CD4(+) T-cell activation in the CD4C/HIV-1(Nef) transgenic mouse model. Because all these Nef functions have been implicated in Nef effects on pathogenesis, this anti-Nef sdAb may represent an efficient tool to elucidate the molecular functions of Nef in the virus life cycle and could now help to develop new strategies for the control of AIDS.

Eukaryotic translation elongation factor 1A (eEF1A) domain I from S. cerevisiae is required but not sufficient for inter-species complementation

Ethanolamine phosphoglycerol (EPG) is a protein modification attached exclusively to eukaryotic elongation factor 1A (eEF1A). In mammals and plants, EPG is linked to conserved glutamate residues located in eEF1A domains II and III, whereas in the unicellular eukaryote Trypanosoma brucei, only domain III is modified by a single EPG. A biosynthetic precursor of EPG and structural requirements for EPG attachment to T. brucei eEF1A have been reported, but nothing is known about the EPG modifying enzyme(s). By expressing human eEF1A in T. brucei, we now show that EPG attachment to eEF1A is evolutionarily conserved between T. brucei and Homo sapiens. In contrast, S. cerevisiae eEF1A, which has been shown to lack EPG is not modified in T. brucei. Furthermore, we show that eEF1A cannot functionally complement across species when using T. brucei and S. cerevisiae as model organisms. However, functional complementation in yeast can be obtained using eEF1A chimera containing domains II or III from other species. In contrast, yeast domain I is strictly required for functional complementation in S. cerevisiae.

Proapoptotic BH3-only protein Bid is essential for death receptor-induced apoptosis of pancreatic beta-cells

OBJECTIVE: Apoptosis of pancreatic beta-cells is critical in both diabetes development and failure of islet transplantation. The role in these processes of pro- and antiapoptotic Bcl-2 family proteins, which regulate apoptosis by controlling mitochondrial integrity, remains poorly understood. We investigated the role of the BH3-only protein Bid and the multi-BH domain proapoptotic Bax and Bak, as well as prosurvival Bcl-2, in beta-cell apoptosis. RESEARCH DESIGN AND METHODS: We isolated islets from mice lacking Bid, Bax, or Bak and those overexpressing Bcl-2 and exposed them to Fas ligand, tumor necrosis factor (TNF)-alpha, and proinflammatory cytokines or cytotoxic stimuli that activate the mitochondrial apoptotic pathway (staurosporine, etoposide, gamma-radiation, tunicamycin, and thapsigargin). Nuclear fragmentation was measured by flow cytometry. RESULTS: Development and function of islets were not affected by loss of Bid, and Bid-deficient islets were as susceptible as wild-type islets to cytotoxic stimuli that cause apoptosis via the mitochondrial pathway. In contrast, Bid-deficient islets and those overexpressing antiapoptotic Bcl-2 were protected from Fas ligand-induced apoptosis. Bid-deficient islets were also resistant to apoptosis induced by TNF-alpha plus cycloheximide and were partially resistant to proinflammatory cytokine-induced death. Loss of the multi-BH domain proapoptotic Bax or Bak protected islets partially from death receptor-induced apoptosis. CONCLUSIONS: These results demonstrate that Bid is essential for death receptor-induced apoptosis of islets, similar to its demonstrated role in hepatocytes. This indicates that blocking Bid activity may be useful for protection of islets from immune-mediated attack and possibly also in other pathological states in which beta-cells are destroyed.

PHYLOGENETIC ANALYSES, GENE DOSAGE EFFECT AND SELECTION IN MEMBERS OF THE LATERAL BOUNDARY DOMAIN (LBD) GENES TRANSCRIPTION FACTOR FAMILY

Wood formation is an economically and environmentally important process and has played a significant role in the evolution of terrestrial plants. Despite its significance, the molecular underpinnings of the process are still poorly understood. We have previously shown that four Lateral Boundary Domain (LBD) transcription factors have important roles in the regulation of wood formation with two (LBD1 and LBD4) involved in secondary phloem and ray cell development and two (LBD15 and LBD18) in secondary xylem formation. Here, we used comparative phylogenetic analyses to test potential roles of the four LBD genes in the evolution of woodiness. We studied the copy number and variation in DNA and amino acid sequences of the four LBDs in a wide range of woody and herbaceous plant taxa with fully sequenced and annotated genomes. LBD1 showed the highest gene copy number across the studied species, and LBD1 gene copy number was strongly and significantly correlated with the level of ray seriation. The lianas, cucumber and grape, with multiseriate ray cells showed the highest gene copy number (12 and 11, respectively). Because lianas’ growth habit requires significant twisting and bending, the less lignified ray parenchyma cells likely facilitate stem flexibility and maintenance of xylem conductivity. We further demonstrate conservation of amino acids in the LBD18 protein sequences that are specific to woody taxa. Neutrality tests showed evidence for strong purifying selection on these gene regions across various orders, indicating adaptive convergent evolution of LBD18. Structural modeling demonstrates that the conserved amino acids have a significant impact on the tertiary protein structure and thus are likely of significant functional importance.

Structural analysis of B-Box 2 from MuRF1: identification of a novel self-association pattern in a RING-like fold

The B-box motif is the defining feature of the TRIM family of proteins, characterized by a RING finger-B-box-coiled coil tripartite fold. We have elucidated the crystal structure of B-box 2 (B2) from MuRF1, a TRIM protein that supports a wide variety of protein interactions in the sarcomere and regulates the trophic state of striated muscle tissue. MuRF1 B2 coordinates two zinc ions through a cross-brace alpha/beta-topology typical of members of the RING finger superfamily. However, it self-associates into dimers with high affinity. The dimerization pattern is mediated by the helical component of this fold and is unique among RING-like folds. This B2 reveals a long shallow groove that encircles the C-terminal metal binding site ZnII and appears as the defining protein-protein interaction feature of this domain. A cluster of conserved hydrophobic residues in this groove and, in particular, a highly conserved aromatic residue (Y133 in MuRF1 B2) is likely to be central to this role. We expect these findings to aid the future exploration of the cellular function and therapeutic potential of MuRF1.

Biochemical characterization of PICK1, a PKC binding protein

Protein kinase C (PKC) is a family of serine-threonine kinases that are activated by a wide variety of hormones, neurotransmitters and growth factors. A single cell type contains multiple isoforms that are translocated to distinct and different subcellular sites upon mitogenic stimulus. Many different cellular responses are attributed to PKC activity though relatively few substrates or binding proteins have been definitively characterized. We used the hinge and catalytic domain of PKC$\alpha$ (PKC7) in a yeast two-hybrid screen to clone proteins that interact with C-kinase (PICKs). One protein which we have termed PICK1 may be involved in PKC$\alpha$-specific function at the level of the nuclear membrane after activation. Binding of PICK1 to PKC$\alpha$ has been shown to be isoform specific as it does not bind to PKC$\beta$II or PKC$\alpha$ in the yeast two-hybrid system. PICK1 mRNA expression level is highest in testis and brain with lower levels of expression in skeletal muscle, heart, kidney, lung and liver. PICK1 protein contains five PKC consensus phosphorylation sites and serves as an in vitro substrate for PKC. The PICK1 protein also contains a P-Loop motif that has been shown to bind ATP or GTP in the Ras family of oncoproteins as well as the G-Protein family. Proteins which bind ATP or GTP using this motif all have some sort of catalytic function although none has been identified for PICK1 as yet. PICK1 contains a DHR/GLGF motif at the N-terminus of the protein. The DHR/GLGF motif is contained in a number of recently described proteins and has been shown to mediate protein-protein interactions at the level of membranes and cytoskeleton. When both PKC$\alpha$ and PICK1 are co-expressed in Cos1 cells the two proteins co-localize to the perinucleus in immunoflouresence studies and co-immunoprecipitate. The binding site for PKC7 has been localized to amino acids 1-358 on PICK1 which contains the DHR/GLGF motif. Binding of PICK1 to PKC$\alpha$ requires the hinge and C-terminal domains of PKC$\alpha$. In vitro, PICK1 binds to PKC$\alpha$ and inhibits its activity as assayed by myelin basic protein phosphorylation. PICK1 also binds to TIS21, a primary response gene that is expressed in response to phorbol ester and growth factor treatment. The Caenorhabditis elegans homologue of PICK1 has been cloned and sequenced revealing a high degree of conservation in the DHR/GLGF motif. A more C-terminal region also shows a high degree of conservation, and the C. elegans PICK1 homologue binds to PKC7 suggesting a conservation of function. Taken together these results suggest that PICK1 may be involved in a PKC$\alpha$-specific function at the level of the nuclear membrane. ^

Intracellular membrane association of the N-terminal domain of classical swine fever virus NS4B determines viral genome replication and virulence.

Classical swine fever virus (CSFV) causes a highly contagious disease in pigs that can range from a severe haemorrhagic fever to a nearly unapparent disease, depending on the virulence of the virus strain. Little is known about the viral molecular determinants of CSFV virulence. The nonstructural protein NS4B is essential for viral replication. However, the roles of CSFV NS4B in viral genome replication and pathogenesis have not yet been elucidated. NS4B of the GPE-  vaccine strain and of the highly virulent Eystrup strain differ by a total of seven amino acid residues, two of which are located in the predicted trans-membrane domains of NS4B and were described previously to relate to virulence, and five residues clustering in the N-terminal part. In the present study, we examined the potential role of these five amino acids in modulating genome replication and determining pathogenicity in pigs. A chimeric low virulent GPE- -derived virus carrying the complete Eystrup NS4B showed enhanced pathogenicity in pigs. The in vitro replication efficiency of the NS4B chimeric GPE-  replicon was significantly higher than that of the replicon carrying only the two Eystrup-specific amino acids in NS4B. In silico and in vitro data suggest that the N-terminal part of NS4B forms an amphipathic α-helix structure. The N-terminal NS4B with these five amino acid residues is associated with the intracellular membranes. Taken together, this is the first gain-of-function study showing that the N-terminal domain of NS4B can determine CSFV genome replication in cell culture and viral pathogenicity in pigs.

Novel mechanisms for PKC family enzymes to regulate PI3K signaling pathway

Phosphatidylinositol 3-kinase (PI3K) generates membrane phospholipids that serve as second messengers to recruit signaling proteins to plasma membrane consequently regulating cell growth and survival. PI3K is a heterodimer consisting of a catalytic p110 subunit and a regulatory p85 subunit. Association of the p85 with other signal proteins is critical for induced PI3K activation. Activated PI3K, in turn, leads to signal flows through a variety of PI3K effectors including PDK1, AKT, GSK3, BAD, p70 S6K and NFκB. The PI3K pathway is under regulation by multiple signal proteins representing cross-talk between different signaling cascades. In this study, we have evaluated the role of protein kinase C family kinases on signaling through PI3K at multiple levels. Firstly, we observed that the action of PKC specific inhibitors like Ro-31-8220 and GF109203X was associated with an increased AKT phosphorylation and activity, suggesting that PKC kinases might play a negative role in the regulation of PI3K pathway. Then, we demonstrated the stimulation of AKT by PKC inhibition was dependent on functional PI3K enzyme and able to be transmitted to the AKT effector p70 S6K. Furthermore, we showed an inducible physical association between the PKCζ isotype and AKT, which was accompanied by an attenuated AKT activity. However, a kinase-dead form of PKC failed to affect AKT. In the second part of our research we revealed the ability of a different PKC family member, PKCδ to bind to the p85 subunit of PI3K in response to oxidative stress, a process requiring the activity of src tyrosine kinases. The interaction was demonstrated to be a direct and specific contact between the carboxyl terminal SH2 domain of p85 and tyrosine phosphorylated PKCδ. Several different types of agonists were capable to induce this association including tyrosine kinases and phorbol esters with PKCδ tyrosine phosphorylation being integral components. Finally, the PKCδ-PI3K complex was related to a reduction in the AKT phosphorylation induced by src. A kinase-deficient mutant of PKCδ was equally able to inhibit AKT signal as the wild type, indicative of a process independent of PKCδ catalytic activity. Altogether, our data illustrate different PKC isoforms regulating PI3K pathway at multiple levels, suggesting a mechanism to control signal flows through PI3K for normal cell activities. Although further investigation is required for full understanding of the regulatory mechanism, we propose that complex formation of signal proteins in PI3K pathway and specific PKC isoforms plays important role in their functional linkage. ^

Contribution of transmembrane and cytoplasmic domains to membrane protein association

Membrane proteins are critical to every aspect of cell physiology, with their association mediating important biological functions. The transmembrane and cytoplasmic domains are known to be important for their association. In order to characterize their role in detail, we have applied different biophysical techniques in detergent micelles to two model systems. The first study involves FcRγ, a single transmembrane domain protein existing as a disulfide linked homodimer. We investigated the role of a conserved transmembrane polar residue and the cytoplasmic tail in FcRγ homo-interactions. Our results by various biophysical techniques including SDS-PAGE, circular dichroism and sedimentation equilibrium in detergent micelles indicate importance of both the transmembrane polar residue and cytoplasmic tail in maintaining proper conformation for FcRγ homo-interactions. A contrasting second study was on L-selectin, another single transmembrane domain protein with a large extracellular domain and a short cytoplasmic tail. Previous cross-linking experiments indicate its possible dimerization. However, the purified fragment of L-selectin and corresponding mutants did not dimerize when analyzed by TOXCAT assay, sedimentation equilibrium and fluorescence resonance energy transfer. It was likely that the presence of juxtamembrane positively charged residues led to decreased migrational rates in SDS PAGE. In conclusion, complementary biophysical techniques should be used with care when studying membrane protein association in detergent micelles. As an extension to our study on L-selectin, we also investigated its interaction with Calmodulin (CaM) in detergent micelles. CaM was found to interact with different detergents. We applied fluorescence and NMR spectroscopy to characterize the interaction of both the apo and Ca 2+ bound form of CaM, with commonly used detergents, below and above their respective critical micelle concentrations. The interaction of apo-CaM with detergents was found to vary with the nature of the detergent head group, whereas Ca2+-CaM interacted with individual detergent molecules irrespective of the nature of their head group. NMR titration experiments of CaM with detergents indicated involvement of specific residues from the N-lobe, linker and C-lobe of CaM. ^

Identification by mass spectrometry of the phosphorylated residue responsible for activation of the catalytic domain of myosin I heavy chain kinase, a member of the PAK/STE20 family

Myosin I heavy chain kinase from Acanthamoeba castellanii is activated in vitro by autophosphorylation (8–10 mol of P per mol). The catalytically active C-terminal domain produced by trypsin cleavage of the phosphorylated kinase contains 2–3 mol of P per mol. However, the catalytic domain expressed in a baculovirus–insect cell system is fully active as isolated without autophosphorylation in vitro. We now show that the expressed catalytic domain is inactivated by incubation with acid phosphatase and regains activity upon autophosphorylation. The state of phosphorylation of all of the hydroxyamino acids in the catalytic domain were determined by mass spectrometry of unfractionated protease digests. Ser-627 was phosphorylated in the active, expressed catalytic domain, lost its phosphate when the protein was incubated with phosphatase, and was rephosphorylated when the dephosphorylated protein was incubated with ATP. No other residue was significantly phosphorylated in any of the three samples. Thus, phosphorylation of Ser-627, which is in the same position as the Ser and Thr residues that are phosphorylated in many other kinases, is necessary and sufficient for full activity of the catalytic domain. Ser-627 is also phosphorylated when full-length, native kinase is activated by autophosphorylation.

The C-type lectin domains of lecticans, a family of aggregating chondroitin sulfate proteoglycans, bind tenascin-R by protein– protein interactions independent of carbohydrate moiety

The lecticans are a family of chondroitin sulfate proteoglycans including aggrecan, versican, neurocan, and brevican. The C-terminal globular domains of lecticans are structurally related to selectins, consisting of a C-type lectin domain flanked by epidermal growth factor and complement regulatory protein domains. The C-type lectin domain of versican has been shown to bind tenascin-R, an extracellular matrix protein specifically expressed in the nervous system, and the interaction was presumed to be mediated by a carbohydrate–protein interaction. In this paper, we show that the C-type lectin domain of brevican, another lectican that is specifically expressed in the nervous system, also binds tenascin-R. Surprisingly, this interaction is mediated by a protein–protein interaction through the fibronectin type III domains 3–5 of tenascin-R, independent of any carbohydrates or sulfated amino acids. The lectin domains of versican and other lecticans also bind the same domain of tenascin-R by protein–protein interactions. Surface plasmon resonance analysis revealed that brevican lectin has at least a 10-fold higher affinity than the other lectican lectins. Tenascin-R is coprecipitated with brevican from adult rat brain extracts, suggesting that tenascin-R and brevican form complexes in vivo. These results demonstrate that the C-type lectin domain can interact with fibronectin type III domains through protein–protein interactions, and suggest that brevican is a physiological tenascin-R ligand in the adult brain.

D-AKAP2, a novel protein kinase A anchoring protein with a putative RGS domain

Subcellular localization directed by specific A kinase anchoring proteins (AKAPs) is a mechanism for compartmentalization of cAMP-dependent protein kinase (PKA). Using a two-hybrid screen, a novel AKAP was isolated. Because it interacts with both the type I and type II regulatory subunits, it was defined as a dual specific AKAP or D-AKAP1. Here we report the cloning and characterization of another novel cDNA isolated from that screen. This new member of the D-AKAP family, D-AKAP2, also binds both types of regulatory subunits. A message of 5 kb pairs was detected for D-AKAP2 in all embryonic stages and in all adult tissues tested. In brain, skeletal muscle, kidney, and testis, a 10-kb mRNA was identified. In testis, several small mRNAs were observed. Therefore, D-AKAP2 represents a novel family of proteins. cDNA cloning from a mouse testis library identified the full length D-AKAP2. It is composed of 372 amino acids which includes the R binding fragment, residues 333–372, at its C-terminus. Based on coprecipitation assays, the R binding domain interacts with the N-terminal dimerization domain of RIα and RIIα. A putative RGS domain was identified near the N-terminal region of D-AKAP2. The presence of this domain raises the intriguing possibility that D-AKAP2 may interact with a Gα protein thus providing a link between the signaling machinery at the plasma membrane and the downstream kinase.

Genetic definition of a protein-splicing domain: Functional mini-inteins support structure predictions and a model for intein evolution

Inteins are protein-splicing elements, most of which contain conserved sequence blocks that define a family of homing endonucleases. Like group I introns that encode such endonucleases, inteins are mobile genetic elements. Recent crystallography and computer modeling studies suggest that inteins consist of two structural domains that correspond to the endonuclease and the protein-splicing elements. To determine whether the bipartite structure of inteins is mirrored by the functional independence of the protein-splicing domain, the entire endonuclease component was deleted from the Mycobacterium tuberculosis recA intein. Guided by computer modeling studies, and taking advantage of genetic systems designed to monitor intein function, the 440-aa Mtu recA intein was reduced to a functional mini-intein of 137 aa. The accuracy of splicing of several mini-inteins was verified. This work not only substantiates structure predictions for intein function but also supports the hypothesis that, like group I introns, mobile inteins arose by an endonuclease gene invading a sequence encoding a small, functional splicing element.

Three distinct domains of SSI-1/SOCS-1/JAB protein are required for its suppression of interleukin 6 signaling

Cytokine-inducible protein SSI-1 [signal transducers and activators of transcription (STAT)-induced STAT inhibitor 1, also referred to as SOCS-1 (suppressor of cytokine signaling 1) or JAB (Janus kinase-binding protein)] negatively regulates cytokine receptor signaling by inhibition of JAK kinases. The SSI family of proteins includes eight members that are structurally characterized by an SH2 domain and a C-terminal conserved region that we have called the SC-motif. In this study, we investigated the roles of these domains in the function of SSI-1. Results of reporter assays demonstrated that the pre-SH2 domain (24 aa in front of the SH2 domain) and the SH2 domain of SSI-1 were required for the suppression by SSI-1 of interleukin 6 signaling. Coexpression studies of COS7 cells revealed that these domains also were required for inhibition of three JAKs (JAK1, JAK2, and TYK2). Furthermore, deletion of the SH2 domain, but not the pre-SH2 domain, resulted in loss of association of SSI-1 with TYK2. Thus, SSI-1 associates with JAK family kinase via its SH2 domain, and the pre-SH2 domain is required for the function of SSI-1. Deletion of the SC-motif markedly reduced expression of SSI-1 protein in M1 cells, and this reduction was reversed by treatment with proteasome inhibitors, suggesting that this motif is required to protect the SSI-1 molecule from proteolytic degradation. Based on these findings, we concluded that three distinct domains of SSI-1 (the pre-SH2 domain, the SH2 domain, and the SC-motif) cooperate in the suppression of interleukin 6 signaling.