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.     

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

Special issue dedicated to Dr. Paul Greengard     

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.     

Solvent accessibility, protein surfaces, and protein folding.

Studies of the native structures of proteins, together with measurements of the thermodynamic properties of the transition between unfolded and native states, have defined the major components of the forces that stabilize native protein structures. However, the nature of the intermediates in the folding process remains largely hypothetical. It is a fairly widespread and not implausible assumption that the intermediates in the folding of a monomeric protein contain the same kinds of secondary and tertiary structures that appear in the native conformation, and that, although unstable, their lifetimes are prolonged by forces similar to those that stabilize the native structure. We wished to examine what happens if, during the folding of a monomeric protein, regions of secondary structure come together to form an intermediate of reduced instability. We applied calculations of accessible surface area (a measure of hydrophobic stabilization) and parameterized nonbonded energy calculations (measuring the strengths of van der Waals forces) to identify the kinds of stabilizing interactions that might be available to such an intermediate. First, we analyzed the total buried surface area of two types of proteins into contributions from formation of secondary structure alone, interaction of pairs of secondary-structural elements, the formation of the structure alone, interaction of pairs of secondary-structural elements, the formation of the complete secondary structure without the turns, and the complete native structure. The formation of secondary structure alone, without tertiary-structural interactions, buries roughly half the surface that the complete structure does. We then analyzed in more detail the approach of two alpha-helices to form a complex, as an illustrative example of the nature of the interaction between compact structural units which remain fairly rigid during their interaction. Many features of the results are not limited to the interaction of alpha-helices. (The results therefore neither confirm nor refute the hypothesis that alpha-helices are intermediates in the folding proteins). We find that the first forces to be felt upon approach arise from solvent conditions on the relative position and orientation of the two helices as does the close packing which optimizes the van der Waals interactions at shorter distances apart. Therefore there appears to be a range of distances in which hydrophobic interactions could create a nonspecific complex between two helices in which the side chains might have sufficient time to seek the proper interdigitation observed in the native structure, where the two helices are in intimate contact. Indeed, we find that only in the final stages of approach is the native geometry the most stable; in the region in which solvent-exclusion forces predominate, the conformation with helix axes parallel is more stable than the native conformation, in the cases we examined...     

Protein interactions among Fe65, the low-density lipoprotein receptor-related protein, and the amyloid precursor protein

The adapter protein Fe65 has been proposed to be the link between the intracellular domains of the amyloid precursor protein, APP (AICD), and the low-density lipoprotein receptor-related protein (LRP-CT). Functional linkage between these two proteins has been established, and mutations within LRP-CT affect the amount of Aβ produced from APP. Previous work showed that AICD binds to protein interaction domain 2 (PID2) of Fe65. Although the structure of PID1 was determined recently, all attempts to demonstrate LRP-CT binding to this domain failed. We used biophysical experiments and binding studies to investigate the binding among these three proteins. Full-length Fe65 bound more weakly to AICD than did N-terminally truncated forms; however, the intramolecular domain-domain interactions that had been proposed to inhibit binding could not be observed using amide H-D exchange. Surprisingly, when LRP-CT is phosphorylated at Tyr4507, it bound to Fe65 PID1 despite the fact that this domain belongs to the Dab-like subclass of PIDs that are not supposed to be phosphorylation-dependent. Mutation of a critical arginine abolished binding, providing further proof of the phosphorylation dependence. Fe65 PID1 thus provides a link between the Dab-like class and the IRS-like class of PIDs and is the first Dab-like family member to show phosphorylation-dependent binding.     

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.     

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.     

The interface of protein structure, protein biophysics, and molecular evolution

Abstract The interface of protein structural biology, protein biophysics, molecular evolution, and molecular population genetics forms the foundations for a mechanistic understanding of many aspects of protein biochemistry. Current efforts in interdisciplinary protein modeling are in their infancy and the state-of-the art of such models is described. Beyond the relationship between amino acid substitution and static protein structure, protein function, and corresponding organismal fitness, other considerations are also discussed. More complex mutational processes such as insertion and deletion and domain rearrangements and even circular permutations should be evaluated. The role of intrinsically disordered proteins is still controversial, but may be increasingly important to consider. Protein geometry and protein dynamics as a deviation from static considerations of protein structure are also important. Protein expression level is known to be a major determinant of evolutionary rate and several considerations including selection at the mRNA level and the role of interaction specificity are discussed. Lastly, the relationship between modeling and needed high-throughput experimental data as well as experimental examination of protein evolution using ancestral sequence resurrection and in vitro biochemistry are presented, towards an aim of ultimately generating better models for biological inference and prediction.     

Functional significance of the interaction between the mRNA-binding protein, Nab2, and the nuclear pore-associated protein, Mlp1, in mRNA export

Nuclear export of mRNA requires several key mRNA-binding proteins that recognize and remodel the mRNA and target it for export via interactions with the nuclear pore complex. In Saccharomyces cerevisiae, the shuttling heterogeneous nuclear ribonucleoprotein, Nab2, which is essential for mRNA export, specifically recognizes poly(A) RNA and binds to the nuclear pore-associated protein, myosin-like protein 1 (Mlp1), which functions in mRNA export and quality control. Specifically, the N-terminal domain of Nab2 (Nab2-N; residues 1-97) interacts directly with the C-terminal globular domain of Mlp1 (CT-Mlp1: residues 1490-1875). Recent structural and binding studies focused on Nab2-N have shown that Nab2-N contains a hydrophobic patch centered on Phe(73) that is critical for interaction with Mlp1. Engineered amino acid changes within this patch disrupt the Nab2/Mlp1 interaction in vitro. Given the importance of Nab2 and Mlp1 to mRNA export, we have examined the Nab2/Mlp1 interaction in greater detail and analyzed the functional consequences of disrupting the interaction in vivo. We find that the Nab2-binding domain of Mlp1 (Mlp1-NBD) maps to a 183-residue region (residues 1586-1768) within CT-Mlp1, binds directly to Nab2 with micromolar affinity, and confers nuclear accumulation of poly(A) RNA. Furthermore, we show that cells expressing a Nab2 F73D mutant that cannot interact with Mlp1 exhibit nuclear accumulation of poly(A) RNA and that this nab2 F73D mutant genetically interacts with alleles of two essential mRNA export genes, MEX67 and YRA1. These data provide in vivo evidence for a model of mRNA export in which Nab2 is important for targeting mRNAs to the nuclear pore for export.     

Geometry, thermodynamics, and protein

We derive a new continuous free energy formula for protein folding. We obtain the formula first by adding hydrophobic effect to a classical free energy formula for cavities in water. We then obtain the same formula by geometrically pursuing the structure that fits best the well-known global geometric features of native structures of globular proteins: 1. high density; 2. small surface area; 3. hydrophobic core; 4. forming domains for long polypeptide chains. Conformations of a protein are presented as an all atom CPK model where each atom is a ball B(x_i,r_i). All conformations satisfy generally defined steric conditions. For each conformation P of a globular protein, there is a closed thermodynamic system Ω_P⊃P bounded by the molecular surface M_P. Both methods derive the same free energy aV(P)+bA(P)+cW(P), where a,b,c>0, V(P), A(P), and W(P) are volume of Ω_P, area of M_P, and area of the hydrophobic surface W_P⊂M_P, which quantifies hydrophobic effect.Minimizing W(P) is sufficient to produce statistically significant native like secondary structures and hydrogen bonds in the proteins we simulated.     

Structure, function and interactions of the PufX protein

The PufX protein is an important component of the reaction centre-light-harvesting 1 (RC-LH1) complex of Rhodobacter species of purple photosynthetic bacteria. Early studies showed that removal of the PufX protein causes changes in the structure of the RC-LH1 complex that result in a loss of the capacity for photosynthetic growth, and that this loss can be overcome though further mutations that change the structure of the LH1 antenna. More recent studies have examined interactions of the PufX protein with other components of the RC-LH1 complex. This review considers our current understanding of the structure and function of the PufX protein, how this protein interacts with other components of the photosynthetic membrane, and its influence on the oligomeric state of the RC-LH1 complex and the larger-scale architecture of the photosynthetic membrane.     

Metal affinity protein precipitation: effects of mixing, protein concentration, and modifiers on protein fractionation

Previous work by us and others has shown that mixing impacts apparent protein solubility in single protein precipitations. In this work, we probe the effects of contacting conditions on fractional precipitation behavior at the bench scale. We have chosen metal affinity precipitation as our model system; the kinetics of this mode of precipitation are very rapid and largely irreversible and, consequently, mixing conditions govern the extent of fractionation and purity of the product in such a process. Our experimental strategy involved a three-pronged approach to control the effects contacting conditions on precipitate yield, purity, and particle size distribution. First, we studied the impact of process variables that control precipitant concentrations in the reactor including impeller speed and precipitant addition rate. Second, we controlled the rate of precipitation by changing the initial protein concentration to alter the protein-protein collision rate. Third, we examined the role of the molecular-level kinetics of affinity precipitation by using modifiers that compete with surface moieties to bind the metal ion, thereby reducing its availability. Our model process and protein system consisted of zinc precipitations of mixtures of bovine serum albumin and bovine gamma-globulins, carried out at a nominal 1-L scale; glycine was examined as a modifier. Faster impeller speeds and lower precipitant addition rates increased the desired protein yields, decreased purities, and reduced average precipitate particle size. Higher initial protein concentrations were found to produce precipitates with higher yields, lower purities and diminished particle size. Experiments with glycine indicated that modifiers in the precipitant solution serve to increase product purity, decrease yield, and increase the average particle size in bench-scale precipitations. (c) 1995 John Wiley & Sons, Inc.     

Identification and characterization of a novel peritrophic matrix protein, Ae-Aper50, and the microvillar membrane protein, AEG12, from the mosquito, Aedes aegypti

Immuno-screening of an adult Aedes aegypti midgut cDNA expression library with anti-peritrophic matrix antibodies identified cDNAs encoding a novel peritrophic matrix protein, termed Ae. aegypti Adult Peritrophin 50 (Ae-Aper50), and the epithelial cell-surface membrane protein, AEG12. Both genes are expressed exclusively in the midguts of adult female mosquitoes and their expression is strongly induced by blood feeding. Ae-Aper50 has a predicted secretory signal peptide and five chitin-binding domains with intervening mucin-like domains. Localization of Ae-Aper50 to the peritrophic matrix was demonstrated by immuno-electron microscopy. Recombinant Ae-Aper50 expressed in baculovirus-infected insect cells binds chitin in vitro. Site-directed mutagenesis was used to study the role that cysteine residues from a single chitin-binding domain play in the binding to a chitin substrate. Most of the cysteine residues proved to be critical for binding. AEG12 has a putative secretory signal peptide at the amino-terminus and a putative glycosyl-phosphatidylinositol (GPI) anchor signal at its carboxyl-terminus and the protein was localized by immuno-electron microscopy to the midgut epithelial cell microvilli.