PACT, a protein activator of the interferon-induced protein kinase, PKR.

PKR, a latent protein kinase, mediates the antiviral actions of interferon. It is also involved in cellular signal transduction, apoptosis, growth regulation and differentiation. Although in virus-infected cells, viral double-stranded (ds) RNA can serve as a PKR activator, cellular activators have remained obscure. Here, we report the cloning of PACT, a cellular protein activator of PKR. PACT heterodimerized with PKR and activated it in vitro in the absence of dsRNA. In mammalian cells, overexpression of PACT caused PKR activation and, in yeast, co-expression of PACT enhanced the anti-growth effect of PKR. Thus, PACT has the hallmarks of a direct activator of PKR.     

Oxidative stress,neurodegeneration, and the balance of protein degradation and protein synthesis

Oxidative stress occurs in a variety of disease settings and is strongly linked to the development of neuron death and neuronal dysfunction. Cells are equipped with numerous pathways to prevent the genesis, as well as the consequences, of oxidative stress in the brain. In this review we discuss the various forms and sources of oxidative stress in the brain and briefly discuss some of the complexities in detecting the presence of oxidative stress. We then focus the review on the interplay between the diverse cellular proteolytic pathways and their roles in regulating oxidative stress in the brain. Additionally, we discuss the involvement of protein synthesis in regulating the downstream effects of oxidative stress. Together, these components of the review demonstrate that the removal of damaged proteins by effective proteolysis and the synthesis of new and protective proteins are vital in the preservation of brain homeostasis during periods of increased levels of reactive oxygen species. Last, studies from our laboratory and others have demonstrated that protein synthesis is intricately linked to the rates of protein degradation, with impairment of protein degradation sufficient to decrease the rates of protein synthesis, which has important implications for successfully responding to periods of oxidative stress. Specific neurodegenerative diseases, including Alzheimer disease, Parkinson disease, amyotrophic lateral sclerosis, and stroke, are discussed in this context. Taken together, these findings add to our understanding of how oxidative stress is effectively managed in the healthy brain and help elucidate how impairments in proteolysis and/or protein synthesis contribute to the development of neurodegeneration and neuronal dysfunction in a variety of clinical settings.     

YajL, the prokaryotic homolog of the Parkinsonism-associated protein DJ-1, protects cells against protein sulfenylation

YajL is the closest Escherichia coli homolog of the Parkinsonism-associated protein DJ-1, a multifunctional oxidative stress response protein whose biochemical function remains unclear. We recently described the oxidative-stress-dependent aggregation of proteins in yajL mutants and the oxidative-stress-dependent formation of mixed disulfides between YajL and members of the thiol proteome. We report here that yajL mutants display increased protein sulfenic acids levels and that formation of mixed disulfides between YajL and its protein substrates in vivo is inhibited by the sulfenic acid reactant dimedone, suggesting that YajL preferentially forms disulfides with sulfenylated proteins. YajL (but not YajL(C106A)) also forms mixed disulfides in vitro with the sulfenylated form of bovine serum albumin. The YajL-serum albumin disulfides can be subsequently reduced by glutathione or dihydrolipoic acid. We also show that DJ-1 can form mixed disulfides with sulfenylated E. coli proteins and with sulfenylated serum albumin. These results suggest that YajL and possibly DJ-1 function as covalent chaperones involved in the detection of sulfenylated proteins by forming mixed disulfides with them and that these disulfides are subsequently reduced by low-molecular-weight thiols.     

ProtoMap: automatic classification of protein sequences, a hierarchy of protein families, and local maps of the protein space

We investigate the space of all protein sequences in search of clusters of related proteins. Our aim is to automatically detect these sets, and thus obtain a classification of all protein sequences. Our analysis, which uses standard measures of sequence similarity as applied to an all-vs.-all comparison of SWISSPROT, gives a very conservative initial classification based on the highest scoring pairs. The many classes in this classification correspond to protein subfamilies. Subsequently we merge the subclasses using the weaker pairs in a two-phase clustering algorithm. The algorithm makes use of transitivity to identify homologous proteins; however, transitivity is applied restrictively in an attempt to prevent unrelated proteins from clustering together. This process is repeated at varying levels of statistical significance. Consequently, a hierarchical organization of all proteins is obtained. The resulting classification splits the protein space into well-defined groups of proteins, which are closely correlated with natural biological families and superfamilies. Different indices of validity were applied to assess the quality of our classification and compare it with the protein families in the PROSITE and Pfam databases. Our classification agrees with these domain-based classifications for between 64.8% and 88.5% of the proteins. It also finds many new clusters of protein sequences which were not classified by these databases. The hierarchical organization suggested by our analysis reveals finer subfamilies in families of known proteins as well as many novel relations between protein families.     

Secretion of surfactant protein C, an integral membrane protein, requires the N-terminal propeptide

Proteolytic processing of surfactant protein C (SP-C) proprotein in multivesicular bodies of alveolar type II cells results in a 35-residue mature peptide, consisting of a transmembrane domain and a 10-residue extramembrane domain. SP-C mature peptide is stored in lamellar bodies (a lysosomal-like organelle) and secreted with surfactant phospholipids into the alveolar space. This study was designed to identify the peptide domain of SP-C required for sorting and secretion of this integral membrane peptide. Deletion analyses in transiently transfected PC12 cells and isolated mouse type II cells suggested the extramembrane domain of mature SP-C was cytosolic and sufficient for sorting to the regulated secretory pathway. Intratracheal injection of adenovirus encoding SP-C mature peptide resulted in secretion into the alveolar space of wild type mice but not SP-C (-/-) mice. SP-C secretion in null mice was restored by the addition of the N-terminal propeptide. The cytosolic domain, consisting of the N- terminal propeptide and extramembrane domain of mature SP-C peptide, supported secretion of the transmembrane domain of platelet-derived growth factor receptor. Collectively, these studies indicate that the N-terminal propeptide of SP-C is required for intracellular sorting and secretion of SP-C.     

Role of the deinhibitor protein in the interconversion of the ATP,Mg-dependent protein phosphatase

The small molecular weight (± 9,000) heat stable deinhibitor protein, isolated from dog liver, not only protects the multisubstrate protein phosphatase from inhibition by inhibitor-1 and the modulator protein. It prevents the conversion of the active enzyme to the ATP,Mg-dependent enzyme form brought about by the modulator protein, and also affects the activation of the ATP,Mg-dependent protein phosphatase, probably by stabilizing the enzyme in its active conformation during the reversible activation by protein kinase F_A. Therefore the deinhibitor protein could be an important factor in the process of glycogen synthesis, which requires glycogen synthase and phosphorylase as dephosphorylated enzymes.     

Self-association of the Lentivirus protein, Nef

Background

The HIV-1 pathogenic factor, Nef, is a multifunctional protein present in the cytosol and on membranes of infected cells. It has been proposed that a spatial and temporal regulation of the conformation of Nef sequentially matches Nef's multiple functions to the process of virion production. Further, it has been suggested that dimerization is required for multiple Nef activities. A dimerization interface has been proposed based on intermolecular contacts between Nefs within hexagonal Nef/FynSH3 crystals. The proposed dimerization interface consists of the hydrophobic B-helix and flanking salt bridges between R105 and D123. Here, we test whether Nef self-association is mediated by this interface and address the overall significance of oligomerization.

Results

By co-immunoprecipitation assays, we demonstrated that HIV-1Nef exists as monomers and oligomers with about half of the Nef protomers oligomerized. Nef oligomers were found to be present in the cytosol and on membranes. Removal of the myristate did not enhance the oligomerization of soluble Nef. Also, SIVNef oligomerizes despite lacking a dimerization interface functionally homologous to that proposed for HIV-1Nef. Moreover, HIV-1Nef and SIVNef form hetero-oligomers demonstrating the existence of homologous oligomerization interfaces that are distinct from that previously proposed (R105-D123). Intracellular cross-linking by formaldehyde confirmed that SF2Nef dimers are present in intact cells, but surprisingly self-association was dependent on R105, but not D123. SIV_MAC239Nef can be cross-linked at its only cysteine, C55, and SF2Nef is also cross-linked, but at C206 instead of C55, suggesting that Nefs exhibit multiple dimeric structures. ClusPro dimerization analysis of HIV-1Nef homodimers and HIV-1Nef/SIVNef heterodimers identified a new potential dimerization interface, including a dibasic motif at R105-R106 and a six amino acid hydrophobic surface.

Conclusions

We have demonstrated significant levels of intracellular Nef oligomers by immunoprecipitation from cellular extracts. However, our results are contrary to the identification of salt bridges between R105 and D123 as necessary for self-association. Importantly, binding between HIV-1Nef and SIVNef demonstrates evolutionary conservation and therefore significant function(s) for oligomerization. Based on modeling studies of Nef self-association, we propose a new dimerization interface. Finally, our findings support a stochastic model of Nef function with a dispersed intracellular distribution of Nef oligomers.     

Direct interaction of the trans-Golgi network membrane protein, TGN38, with the F-actin binding protein, neurabin.

TGN38 is a type I integral membrane protein that constitutively cycles between the trans-Golgi network (TGN) and plasma membrane. The cytosolic domain of TGN38 interacts with AP2 clathrin adaptor complexes via the tyrosine-containing motif (-SDYQRL-) to direct internalization from the plasma membrane. This motif has previously been shown to direct both internalization and subsequent TGN targeting of TGN38. We have used the cytosolic domain of TGN38 in a two-hybrid screen, and we have identified the brain-specific F-actin binding protein neurabin-I as a TGN38-binding protein. We demonstrate a direct interaction between TGN38 and the ubiquitous homologue of neurabin-I, neurabin-II (also called spinophilin). We have used a combination of yeast two-hybrid and in vitro protein interaction assays to show that this interaction is dependent on the serine (but not tyrosine) residue of the known TGN38 trafficking motif. We show that TGN38 interacts with the coiled coil region of neurabin in vitro and binds preferentially with the dimeric form of neurabin. TGN38 and neurabin also interact in vivo as demonstrated by coimmunoprecipitation from stably transfected PC12 cells. These data suggest that neurabin provides a direct physical link between TGN38-containing membranes and the actin cytoskeleton.     

Interaction of the heart-specific LIM domain protein, FHL2, with DNA-binding nuclear protein, hNP220

Using a yeast two-hybrid library screen, we have identified that the heart specific FHL2 protein, four-and-a-half LIM protein 2, interacted with human DNA-binding nuclear protein, hNP220. Domain studies by the yeast two-hybrid interaction assay revealed that the second LIM domain together with the third and the fourth LIM domains of FHL2 were responsible to the binding with hNP220. Using green fluorescent protein (GFP)-FHL2 and blue fluorescent protein (BFP)-hNP220 fusion proteins co-expressed in the same cell, we demonstrated a direct interaction between FHL2 and hNP220 in individual nucleus by two-fusion Fluorescence Resonance Energy Transfer (FRET) assay. Besides, Western blot analysis using affinity-purified anti-FHL2 antipeptide antibodies confirmed a 32-kDa protein of FHL2 in heart only. Virtually no expression of FHL2 protein was detected in brain, liver, lung, kidney, testis, skeletal muscle, and spleen. Moreover, the expression of FHL2 protein was also detectable in the human diseased heart tissues. Our results imply that FHL2 protein can shuttle between cytoplasm and nucleus and may act as a molecular adapter to form a multicomplex with hNP220 in the nucleus, thus we speculate that FHL2 may be particularly important for heart muscle differentiation and the maintenance of the heart phenotype.     

The viral death protein Apoptin interacts with Hippi,the protein interactor of Huntingtin-interacting protein 1

Apoptin, a chicken anemia virus-encoded protein, induces apoptosis in human tumor cells but not in normal cells. The tumor-specific activity of Apoptin is correlated with its nuclear localization in tumor cells. In an attempt to elucidate the molecular mechanism of Apoptin-induced apoptosis, we identified human Hippi, the protein interactor and apoptosis co-mediator of Huntingtin interacting protein 1, as one of the Apoptin-associated proteins by yeast two-hybrid screen. We also demonstrated that Hippi could interact with Apoptin both in vitro and in human cells. Furthermore, subcellular localization studies showed that Hippi and Apoptin perfectly colocalized in the cytoplasm of normal human HEL cells, whereas in cancerous HeLa cells most Apoptin and Hippi were located separately in the nucleus and cytoplasm and, thus, showed only a modest colocalization. Mapping studies indicate that Hippi binds within the self-multimerization domain of Apoptin, and Apoptin binds to the C-terminal half of Hippi, including its death effector domain-like motif. Our results suggest that the Apoptin-Hippi interaction may play a role in the suppression of apoptosis in normal cells.     

Overproduction, isolation and determination of the amino-terminal sequence of the SecY protein, a membrane protein involved in protein export in Escherichia coli

The gene product of secY (prlA) is an integral membrane protein with an essential role in protein export in Escherichia coli. When the protein was overproduced, using a plasmid, it was degraded rapidly in the cell. The lon or the htpR mutation did not slow down this degradation, but low-temperature growth conditions (30 degrees C) did so appreciably. On the other hand, the copy number of the pUC8-based plasmid was higher at higher temperatures. Thus, the plasmid was first amplified at 42 degrees C and the protein was then accumulated at 30 degrees C. The SecY protein was isolated in sodium dodecyl sulfate (SDS)-denatured form from the membranes of the overproducing cells, using SDS-SDS two-dimensional gel electrophoresis. Its NH2-terminal sequence confirmed the secY reading frame and the translation initiation site assigned previously. The SecY protein does not undergo NH2-terminal processing except for the removal of the initiator methionine.     

Involvement of Tetrahymena intermediate filament protein, a 49K protein, in the oral morphogenesis

To study the biological function of Tetrahymena intermediate-type filament protein (a 49K protein), we examined the immunofluorescence localization of 49K protein within Tetrahymena cells. The results showed that the immunofluorescence was localized in the oral apparatus, mitochondria and mucocysts. Among them, the fluorescence in the oral apparatus was of high interest in its unique region and vicissitude in the cell cycle: a tau-shaped region of the oral apparatus intensely fluoresced during interphase, but the fluorescence completely disappeared during dividing phase. The tau-shaped region corresponded to 'posterior connectives' and the root part of 'deep fiber', to the conjunction parts of microtubule bundles. In the those parts, there was electron-dense material in the microtubule bundles. Hence, it is conceivable that 49K protein corresponds to the dense material and has a function of microtubule bundle conjunction. On the other hand, disappearance of immunofluorescence from the old oral apparatus of most dividing cells reflected the oral apparatus regression and remodelling which have been known as necessary sequential events in the cell cycle. We observed that oral fluorescence disappeared concurrently with the onset of oral regression and of constriction of division furrow, whereas at a late dividing stage immunofluorescence began to appear simultaneously in both new and old oral apparatus. Thus, the 49K protein may play a crucial role(s) not only in the morphogenesis of oral primordia but also in the transient morphogenesis in the old oral system.     

The cGMP-dependent protein kinase-gene,protein, and function

Special issue dedicated to Dr. Paul Greengard     

Seminalplasmin,the major basic protein of bull seminal plasma,is a secretory protein of the seminal vesicles

From the experimental results of three independent methods: (1) indirect immunofluorescence employing monospecific anti-seminalplasmin-IgGs, (2) cell-free translation of poly(A)⁺ RNA from seminal vesicle and testicular tissue, as well as (3) Northern analysis of poly(A)⁺ RNA of the latter tissues with a synthetic seminalplasmin-specific antisense DNA probe, it is concluded that the biosynthesis of seminalplasmin occurs in seminal vesicles but not in testis.     

The effect of the 540-kilodalton actin cross-linking protein, actin-binding protein, on the mechanical properties of F-actin

This study describes the effect of actin-binding protein derived from rabbit lung macrophages on the mechanical properties of F-actin. The dynamic storage modulus, G'(omega), and loss modulus, G"(omega) of F-actin, at concentrations from 1 to 4 mg/ml, in the absence or presence of actin-binding protein at molar ratios to actin of 1:1000 to 1:125, were measured at frequencies ranging from 3 X 10(-3) to 0.5 Hz. Actin-binding protein increased the dynamic moduli of F-actin, but this increase was much greater as either the actin-binding protein/actin ratio or the total protein concentration increased. Moreover, there was a convergence of the values of G' and G" at high frequencies for F-actin which became more prominent upon the addition of actin-binding protein. The value of the modulus obtained by an extrapolation of these data to actin concentrations similar to that found in the cell cortex was close to values which have been obtained by direct measurements. The addition of actin-binding protein to an F-actin solution enabled it to reach an equilibrium strain following the application of a stress, in contrast to pure F-actin. These data allow a more rigorous definition of the "sol" to "gel" transition and suggest that the cross-linking of actin filaments by actin-binding protein leads to the formation of a network structure whose underlying mechanism of mechanical behavior is short range intrafilament bending in contrast to the classical rubber network.     

Role of PTB-like protein,a neuronal RNA-binding protein,during the differentiation of PC12 cells

PTB-like protein (PTBLP) is a new homologue of pyrimidine tract binding protein (PTB), and has been cloned as a possible autoantigen in cancer-associated retinopathy. PTBLP has two functional domains, the nuclear localization signal and the RNA recognition motifs (RRMs). Full-length PTBLP (PTBLP-L) has four RRMs, and its alternative splicing product (PTBLP-S) lacks the third and fourth RRMs. Although PTBLPs are expressed in neuronal tissues, the function of PTBLPs has not been determined. We have studed whether PTBLP plays a role in neuronal differentiation using PC12 cells. During the process of nerve growth factor-induced neuronal differentiation of PC12 cells, PTBLP-L was down-regulated whereas PTBLP-S was up-regulated. Transfection of PTBLP-L into PC12 cells led to the suppression of neuronal differentiation. In PTBLP-S transfected cells, however, this suppression was not evident. When both PTBLP-L and PTBLP-S were co-transfected, the suppressive effect of PTBLP-L decreased. In differentiated cells, PTBLP-S localized in the nucleus and PTBLP-L was found dispersed throughout the cytoplasm and neuronal growth cone. These findings suggest that PTBLP-L acts as a negative regulator of neuronal differentiation and PTBLP-S acts as a competitor of PTBLP-L.