Chaperone-like features of bovine serum albumin: a comparison with α-crystallin

The chaperone behaviour of bovine serum albumin was compared with that of α-crystallin. The chaperone activity was assessed by measuring: (i) the ability to antagonize protein aggregation induced by heat; (ii) the capability to protect the activity of thermally stressed enzymes and (iii) the effectiveness in assisting the functional recovery of chemically denatured sorbitol dehydrogenase. Despite the lack of structural analogies, both proteins show several functional similarities in preventing inactivation of thermally stressed enzymes and in reactivating chemically denatured sorbitol dehydrogenase. As with α-crystallin, the chaperone action of bovine serum albumin appears to be ATP independent. Bovine serum albumin appears significantly less effective than α-crystallin only in preventing thermally induced protein aggregation. A possible relationship between chaperone function and structural organization is proposed. Together, our results indicate that bovine serum albumin acts as a molecular chaperone and that, for its particular distribution, can be included in the extracellular chaperone family. Received 29 August 2005; received after revision 23 September 2005; accepted 12 October 2005     

Structure and function of small heat shock/alpha-crystallin proteins: established concepts and emerging ideas

Small heat shock/alpha-crystallin proteins are defined by conserved sequence of approximately 90 amino acid residues, termed the alpha-crystallin domain, which is bounded by variable amino- and carboxy-terminal extensions. These proteins form oligomers, most of uncertain quaternary structure, and oligomerization is prerequisite to their function as molecular chaperones. Sequence modelling and physical analyses show that the secondary structure of small heat shock/alpha-crystallin proteins is predominately beta-pleated sheet. Crystallography, site-directed spin-labelling and yeast two-hybrid selection demonstrate regions of secondary structure within the alpha-crystallin domain that interact during oligomer assembly, a process also dependent on the amino terminus. Oligomers are dynamic, exhibiting subunit exchange and organizational plasticity, perhaps leading to functional diversity. Exposure of hydrophobic residues by structural modification facilitates chaperoning where denaturing proteins in the molten globule state associate with oligomers. The flexible carboxy-terminal extension contributes to chaperone activity by enhancing the solubility of small heat shock/alpha-crystallin proteins. Site-directed mutagenesis has yielded proteins where the effect of the change on structure and function depends upon the residue modified, the organism under study and the analytical techniques used. Most revealing, substitution of a conserved arginine residue within the alpha-crystallin domain has a major impact on quaternary structure and chaperone action probably through realignment of beta-sheets. These mutations are linked to inherited diseases. Oligomer size is regulated by a stress-responsive cascade including MAPKAP kinase 2/3 and p38. Phosphorylation of small heat shock/alpha-crystallin proteins has important consequences within stressed cells, especially for microfilaments.     

Signal regulation by family conspiracy

The signal regulating proteins (SIRPs) are a family of ubiquitously expressed transmembrane glycoproteins composed of two subgroups: SIRPα and SIRPβ, containing more than ten members. SIRPα has been shown to inhibit signalling through a variety of receptors including receptor tyrosine kinases and cytokine receptors. This function involves protein tyrosine kinases and is dependent on immunoreceptor tyrosine-based inhibition motifs which recruit key protein tyrosine phosphatases to the membrane. Negative regulation by SIRPα may also involve its ligand, CD47, in a bi-directional signalling mechanism. The SIRPβ subtype has no cytoplasmic domain but instead associates with at least one other transmembrane protein (DAP-12, or KARAP). DAP-12 possesses immunoreceptor tyrosine-based activation motifs within its cytoplasmic domain that are thought to link SIRPβ to activating machinery. SIRPα and SIRPβ thus have complementary roles in signal regulation and may conspire to tune the response to a stimulus. Received 6 July 2000; revised 2 August 2000; accepted 5 August 2000     

The parvins

The parvins are a family of proteins involved in linking integrins and associated proteins with intracellular pathways that regulate actin cytoskeletal dynamics and cell survival. Both α-parvin (PARVA) and β-parvin (PARVB) localize to focal adhesions and function in cell adhesion, spreading, motility and survival through interactions with partners, such as integrin-linked kinase (ILK), paxillin, α-actinin and testicular kinase 1. A complex of PARVA with ILK and the LIM protein PINCH-1 is critical for cell survival in a variety of cells, including certain cancer cells, kidney podocytes and cardiac myocytes. While PARVA inhibits the activities of Rac1 and testicular kinase 1 and cell spreading, PARVB binds αPIX and α-actinin, and can promote cell spreading. In contrast to PARVA, PARVB inhibits ILK activity and reverses some of its oncogenic effects in cancer cells. This review focuses on the structure and function of the parvins and some possible roles in human diseases. Received 5 August 2005; received after revision 5 September 2005; accepted 22 September 2005     

��-Glucosidases

β-Glucosidases (3.2.1.21) are found in all domains of living organisms, where they play essential roles in the removal of nonreducing terminal glucosyl residues from saccharides and glycosides. β-Glucosidases function in glycolipid and exogenous glycoside metabolism in animals, defense, cell wall lignification, cell wall β-glucan turnover, phytohormone activation, and release of aromatic compounds in plants, and biomass conversion in microorganisms. These functions lead to many agricultural and industrial applications. β-Glucosidases have been classified into glycoside hydrolase (GH) families GH1, GH3, GH5, GH9, and GH30, based on their amino acid sequences, while other β-glucosidases remain to be classified. The GH1, GH5, and GH30 β-glucosidases fall in GH Clan A, which consists of proteins with (β/α)₈-barrel structures. In contrast, the active site of GH3 enzymes comprises two domains, while GH9 enzymes have (α/α)₆ barrel structures. The mechanism by which GH1 enzymes recognize and hydrolyze substrates with different specificities remains an area of intense study.     

Protein kinase CK2 and new binding partners during spermatogenesis

Protein kinase CK2 is an ubiquitously expressed enzyme that is absolutely necessary for the survival of cells. Besides the holoenzyme consisting of the regulatory β-subunit and the catalytic α- or α′-subunit, the subunits exist in separate forms. The subunits bind to a number of other cellular proteins. We show the expression of individual subunits as well as interaction with the transitional nuclear protein TNP1 and with the motor neuron protein KIF5C during spermatogenesis. TNP1 is a newly identified binding partner of the α-subunit of CK2. CK2α and KIF5C were found in late spermatogenesis, whereas CK2β and TNP1 were found in early spermatogenesis. CK2α, CK2α′, TNP1, and KIF5C were detected in the acrosome of spermatozoa, while CK2β was detectable in the mid-piece. Combinations of CK2 subunits might determine interactions with other proteins during spermatogenesis. KIF5C as a kinesin motor neuron protein is probably involved in the redistribution of proteins during spermatogenesis.     

Fractal symmetry of protein interior: what have we learned?

The application of fractal dimension-based constructs to probe the protein interior dates back to the development of the concept of fractal dimension itself. Numerous approaches have been tried and tested over a course of (almost) 30 years with the aim of elucidating the various facets of symmetry of self-similarity prevalent in the protein interior. In the last 5 years especially, there has been a startling upsurge of research that innovatively stretches the limits of fractal-based studies to present an array of unexpected results on the biophysical properties of protein interior. In this article, we introduce readers to the fundamentals of fractals, reviewing the commonality (and the lack of it) between these approaches before exploring the patterns in the results that they produced. Clustering the approaches in major schools of protein self-similarity studies, we describe the evolution of fractal dimension-based methodologies. The genealogy of approaches (and results) presented here portrays a clear picture of the contemporary state of fractal-based studies in the context of the protein interior. To underline the utility of fractal dimension-based measures further, we have performed a correlation dimension analysis on all of the available non-redundant protein structures, both at the level of an individual protein and at the level of structural domains. In this investigation, we were able to separately quantify the self-similar symmetries in spatial correlation patterns amongst peptide–dipole units, charged amino acids, residues with the π-electron cloud and hydrophobic amino acids. The results revealed that electrostatic environments in the interiors of proteins belonging to ‘α/α toroid’ (all-α class) and ‘PLP-dependent transferase-like’ domains (α/β class) are highly conducive. In contrast, the interiors of ‘zinc finger design’ (‘designed proteins’) and ‘knottins’ (‘small proteins’) were identified as folds with the least conducive electrostatic environments. The fold ‘conotoxins’ (peptides) could be unambiguously identified as one type with the least stability. The same analyses revealed that peptide–dipoles in the α/β class of proteins, in general, are more correlated to each other than are the peptide–dipoles in proteins belonging to the all-α class. Highly favorable electrostatic milieu in the interiors of TIM-barrel, α/β-hydrolase structures could explain their remarkably conserved (evolutionary) stability from a new light. Finally, we point out certain inherent limitations of fractal constructs before attempting to identify the areas and problems where the implementation of fractal dimension-based constructs can be of paramount help to unearth latent information on protein structural properties.     

Kinetic and structural evidence of the alkenal/one reductase specificity of human ζ-crystallin

Human ζ-crystallin is a Zn²⁺-lacking medium-chain dehydrogenase/reductase (MDR) included in the quinone oxidoreductase (QOR) family because of its activity with quinones. In the present work a novel enzymatic activity was characterized: the double bond α,β-hydrogenation of medium-chain 2-alkenals and 3-alkenones. The enzyme is especially active with lipid peroxidation products such as 4-hydroxyhexenal, and a role in their detoxification is discussed. This specificity is novel in the QOR family, and it is similar to that described in the distantly related alkenal/one reductase family. Moreover, we report the X-ray structure of ζ-crystallin, which represents the first structure solved for a tetrameric Zn²⁺-lacking MDR, and which allowed the identification of the active-site lining residues. Docking simulations suggest a role for Tyr53 and Tyr59 in catalysis. The kinetics of Tyr53Phe and Tyr59Phe mutants support the implication of Tyr53 in binding/catalysis of alkenal/one substrates, while Tyr59 is involved in the recognition of 4-OH-alkenals.     

Cytolytic and K+ channel blocking activities of β-KTx and scorpine-like peptides purified from scorpion venoms

Among the scorpion venom components whose function are poorly known or even show contrasting pharmacological results are those called “orphan peptides”. The most widely distributed are named β-KTx or scorpine-like peptides. They contain three disulfide bridges with two recognizable domains: a freely moving N-terminal amino acid sequence and a tightly folded C-terminal region with a cysteine-stabilized α/β (CS-αβ) motif. Four such peptides and three cloned genes are reported here. They were assayed for their cytolytic, antimicrobial and K ⁺ channel-blocking activities. Two main characteristics were found: the existence of an unusual structural and functional diversity, whereby the full-length peptide can lyse cells or kill microorganisms, and a C-terminal domain containing the CS-αβ motif that can block K ⁺ channels. Furthermore, sequence analyses and phylogenetic reconstructions are used to discuss the evolution of this type of peptide and to highlight the versatility of the CS-αβ structures. Received 13 August 2007; received after revision 30 October 2007; accepted 2 November 2007     

Biased binding of class IA phosphatidyl inositol 3-kinase subunits to inducible costimulator (CD278)

To better understand T lymphocyte costimulation by inducible costimulator (ICOS; H4; CD278), we analyzed proteins binding to ICOS peptides phosphorylated at the Y₁₉₁MFM motif. Phosphorylated ICOS binds class IA phosphatidyl inositol 3-kinase (PI3-K) p85α, p50-55α and p85β regulatory subunits and p110α, p110δ and p110β catalytic subunits. Intriguingly, T cells expressed high levels of both p110α or p110δ catalytic subunits, yet ICOS peptides, cell surface ICOS or PI3-kinase class IA regulatory subunits preferentially coprecipitated p110α catalytic subunits. Silencing p110α or p110δ partially inhibited Akt/PKB activation induced by anti-CD3 plus anti-ICOS antibodies. However, silencing p110α enhanced and silencing p110δ inhibited Erk activation. Both p110α- and p110δ-specific inhibitors blocked cytokine secretion induced by TCR/CD3 activation with or without ICOS costimulus, but only p110α inhibitors blocked ICOS-induced cell elongation. Thus, p110α and p110δ are essential to optimal T cell activation, but their abundance and activity differentially tune up distinct ICOS signaling pathways.     

Glycoprotein IIb/IIIa antagonists - from bench to practice

The central role played by the α_IIbβ₃ receptor in platelet aggregation, and hence in platelet thrombosis, has led to the development of a number of parenteral and oral glycoprotein (GP) IIb/IIIa inhibitors for use in cardiovascular disease states, such as acute coronary syndromes and stroke. The predominant effect of these agents is to inhibit platelet aggregation, although studies of α_IIbβ₃ receptor function and various GP IIb/IIIa inhibitors have demonstrated the potential for these agents to produce effects on other aspects of platelet function, in addition to non-platelet effects. Overall, clinical studies have demonstrated an impressive beneficial effect for parenteral agents in reducing ischemic complications following percutaneous intervention, and a more modest beneficial effect in the treatment of patients with acute coronary syndromes. Trials with oral GP IIb/IIIa inhibitors in similar patient populations have demonstrated toxicity, manifested by an increased mortality in treated patients. Increased understanding of molecular aspects of both α_IIbβ₃ receptor function and the effects of GP IIb/IIIa inhibition may help explain some of the inconsistency in recently reported clinical studies with parenteral agents, and the frank toxicity of oral agents. Such studies may also hold the key to the development of newer agents with enhanced therapeutic benefit. Received 10 September 2001; received after revision 22 October 2001; accepted 2 November 2001     

Liver X receptors in cardiovascular and metabolic disease

Liver X receptors (LXRs) α and β are nuclear oxysterol receptors and metabolic sensors initially found to regulate cholesterol metabolism and lipid biosynthesis. Recent studies have elucidated the importance of LXR in the development of cardiovascular diseases and metabolic disorders. LXR agonists prevent development of atherosclerosis by modulation of metabolic as well as inflammatory gene expression in rodent models. Moreover, LXR activation inhibits hepatic gluconeogenesis and lowers serum glucose levels, indicating possible application of LXR activation in the treatment of diabetes mellitus. However, first-generation LXR agonists elevate hepatic and serum trigylceride levels, making subtype-specific agonists and selective LXR modulators rather than unselective LXR agonists a potential pharmacological strategy. This review summarizes the multiple physiological and pathophysiological implications of LXRs and observations that identify LXRs as potential targets for therapeutic interventions in human cardiovascular and metabolic disease. Received 30 August 2005; received after revision 10 October 2005; accepted 4 November 2005     

Crystallin proteins and amyloid fibrils

Improper protein folding (misfolding) can lead to the formation of disordered (amorphous) or ordered (amyloid fibril) aggregates. The major lens protein, α-crystallin, is a member of the small heat-shock protein (sHsp) family of intracellular molecular chaperone proteins that prevent protein aggregation. Whilst the chaperone activity of sHsps against amorphously aggregating proteins has been well studied, its action against fibril-forming proteins has received less attention despite the presence of sHsps in deposits found in fibril-associated diseases (e.g. Alzheimer’s and Parkinson’s). In this review, the literature on the interaction of αB-crystallin and other sHsps with fibril-forming proteins is summarized. In particular, the ability of sHsps to prevent fibril formation, their mechanisms of action and the possible in vivo consequences of such associations are discussed. Finally, the fibril-forming propensity of the crystallin proteins and its implications for cataract formation are described along with the potential use of fibrillar crystallin proteins as bionanomaterials. Received 13 June 2008; received after revision 29 July 2008; accepted 05 August 2008     

Tauroursodeoxycholic acid prevents E22Q Alzheimer’s Aβ toxicity in human cerebral endothelial cells

The vasculotropic E22Q mutant of the amyloid-β (Aβ) peptide is associated with hereditary cerebral hemorrhage with amyloidosis Dutch type. The cellular mechanism(s) of toxicity and nature of the AβE22Q toxic assemblies are not completely understood. Comparative assessment of structural parameters and cell death mechanisms elicited in primary human cerebral endothelial cells by AβE22Q and wild-type Aβ revealed that only AβE22Q triggered the Bax mitochondrial pathway of apoptosis. AβE22Q neither matched the fast oligomerization kinetics of Aβ42 nor reached its predominant β-sheet structure, achieving a modest degree of oligomerization with a secondary structure that remained a mixture of β and random conformations. The endogenous molecule tauroursodeoxycholic acid (TUDCA) was a strong modulator of AβE22Q-triggered apoptosis but did not significantly change the secondary structures and fibrillogenic propensities of Aβ peptides. These data dissociate the pro-apoptotic properties of Aβ peptides from their distinct mechanisms of aggregation/fibrillization in vitro, providing new perspectives for modulation of amyloid toxicity. Received 20 November 2008; received after revision 12 December 2008; accepted 12 January 2009     

Multiple catalytically active thioredoxin folds: a winning strategy for many functions

The Thioredoxin (Trx) fold is a versatile protein scaffold consisting of a four-stranded β-sheet surrounded by three α-helices. Various insertions are possible on this structural theme originating different proteins, which show a variety of functions and specificities. During evolution, the assembly of different Trx fold domains has been used many times to build new multi-domain proteins able to perform a large number of catalytic functions. To clarify the interaction mode of the different Trx domains within a multi-domain structure and how their combination can affect catalytic performances, in this review, we report on a structural and functional analysis of the most representative proteins containing more than one catalytically active Trx domain: the eukaryotic protein disulfide isomerases (PDIs), the thermophilic protein disulfide oxidoreductases (PDOs) and the hybrid peroxiredoxins (Prxs).     

β-Lactam resistance in Staphylococcus aureus: the adaptive resistance of a plastic genome

Staphylococci have two mechanisms for resistance to β-lactam antibiotics. One is the production of β-lactamases, enzymes that hydrolytically destroy β-lactams. The other is the expression of penicillin-binding protein 2a (PBP 2a), which is not susceptible to inhibition by β-lactam antibiotics. Strains of S. aureus exhibiting either β-lactamase or PBP 2a-directed resistance (or both) have established a considerable ecological niche among human pathogens. The emergence and subsequent spread of bacterial strains designated as methicillin-resistant S. aureus (MRSA), from the 1960s to the present, has created clinical difficulties for nosocomial treatment on a global scale. The recent variants of MRSA that are resistant to glycopeptide antibiotics (such as vancomycin) have ushered in a new and disconcerting chapter in the evolution of this organism. Received 2 April 2005; received after revision 15 July 2005; accepted 25 July 2005     

KIF5C: A new binding partner for protein kinase CK2 with a preference for the CK2α’ subunit

Protein kinase CK2 is a highly conserved serine/threonine kinase that is ubiquitously expressed in eukaryotic cells. CK2 is a constitutively active tetrameric enzyme composed of two catalytic α and/or α’-subunits and two regulatory β-subunits. There is increasing evidence that the individual subunits may have independent functions and that they are asymmetrically distributed inside the cell. To gain a better understanding of the functions of the individual subunits, we employed a yeast-two-hybrid screen with CK2α and CK2α’. We identified the motor neuron protein KIF5C as a new binding partner for CK2. The interaction found in the yeast-two-hybrid screen was confirmed by co-sedimentation analysis on a sucrose density gradient and by co-immunoprecipitation analysis. Pull-down experiments and surface plasmon resonance spectrometry revealed a direct binding of KIF5C to CK2α’. Co-localization studies with neuroblastoma cells, bone marrow and with primary neurons confirmed the biochemical analysis that KIF5C preferentially bound to CK2α’. Received 8 August 2008; received after revision 3 November 2008; accepted 4 November 2008