β-Lactamases as models for protein-folding studies

This review traces some of the key features of the folding of β-lactamases and their relevance to the way proteins fold in general. Studies on the enzymes have highlighted the nature and role of equilibrium and transient condensed states. The kinetics of folding are multiphasic, and when monitored by acrylamide quenching of the tryptophan fluorescence, an early phase provides evidence for the transient accumulation of a nonnative intermediate involving burial of tryptophan in a nonpolar environment. Intermediate phases can be understood in terms of progressive folding of different parts of the molecule. The later, slow phases are associated with proline isomerization in the TEM-1 enzyme and, in its P167T mutant form, with isomerization from trans to cis of the E166 T167 peptide bond. Coupled with kinetic and X-ray crystallographic studies of the β-lactamase from Staphylococcus aureus and its D179Q mutant, it appears that the final stage of folding is that of collapse and packing of the Ω-loop on to the main body of the protein.     

Chimeras of human lysozyme and α-lactalbumin: an interesting tool for studying partially folded states during protein folding

Protein folding is an extremely active field of research where biology, chemistry, computer science and physics meet. Although the study of protein-folding intermediates in general and equilibrium intermediates in particular has grown considerably in recent years, many questions regarding the conformational state and the structural features of the various partially folded intermediate states remain unanswered. Performing kinetic measurements on proteins that have had their structures modified by site-directed mutagenesis, the so-called protein-engineering method, is an obvious way to gain fine structural information. In the present review, this method has been applied to a variety of proteins belonging to the lysozyme/α-lactalbumin family. Besides recombinants obtained by point mutations of individual critical residues, chimeric proteins in which whole structural elements (10 – 25 residues) from α-lactalbumin were inserted into a human lysozyme matrix are examined. The conformational properties of the equilibrium intermediate states are discussed together with the structural characterization of the partially unfolded states encountered in the kinetic folding pathway. Received 28 May 1998; received after revision 6 July 1998; accepted 6 July 1998     

The perspectives of studying multi-domain protein folding

Most of fundamental studies on protein folding have been performed with small globular proteins consisting of a single domain. In vitro many of these proteins are well characterized by a reversible two-state folding scheme. However, the majority of proteins in the cell belong to the class of larger multi-domain proteins that often unfold irreversibly under in vitro conditions. This makes folding studies difficult or even impossible. In spite of these problems for many multi-domain proteins, folding has been investigated by classical refolding. Co-translational folding of nascent polypeptide chains when synthesized by ribosomes has also been studied. Single molecule techniques represent a promising approach for future studies on the folding of multi-domain proteins, and tremendous advances have been made in these techniques in recent years. In particular, fluorescence-based methods can contribute significantly to an understanding of the fundamental principles of multi-domain protein folding. Received 3 December 2008; accepted 23 December 2008     

Structural determinants of protein folding

The last several decades have seen an explosion of knowledge in the field of structural biology. With critical advances in spectroscopic techniques in examining structures of biomacromolecules, in maturation of molecular biology techniques, as well as vast improvements in computation prowess, protein structures are now being elucidated at an unprecedented rate. In spite of all the recent advances, the protein folding puzzle remains as one of the fundamental biochemical challenges. A facet to this empiric problem is the structural determinants of protein folding. What are the driving forces that pivot a polypeptide chain to a specific conformation amongst the vast conformation space? In this review, we shall discuss some of the structural determinants to protein folding that have been identified in the recent decades.     

Heat-shock protein 90, a chaperone for folding and regulation

Heat-shock protein 90 (Hsp90) is an abundant and highly conserved molecular chaperone that is essential for viability in eukaryotes. Hsp90 fulfills a housekeeping function in contributing to the folding, maintenance of structural integrity and proper regulation of a subset of cytosolic proteins. A remarkable proportion of its substrates are proteins involved in cell cycle control and signal transduction. Hsp90 acts with a cohort of Hsp90 co-chaperones that modulate its substrate recognition, ATPase cycle and chaperone function. The large conformational flexibility of Hsp90 and a multitude of dynamic co-chaperone complexes contribute to generating functional diversity, and allow Hsp90 to assist a wide range of substrates.     

Peptidyl-prolyl cis-trans isomerases, a superfamily of ubiquitous folding catalysts

Cyclosporine A therapy for prophylaxis against graft rejection revolutionized human organ transplantation. The immunosuppressant drugs cyclosporin A (CsA), FK506 and rapamycin block T-cell activation by interfering with the signal transduction pathway. The target proteins for CsA and FK506 were found to be cyclophilins and FK506-binding proteins, (FKBPs), respectively. They are unrelated in primary sequence, although both are peptidyl-prolyl cis-trans isomerases catalyzing the interconversion of peptidyl-prolyl imide bonds in peptide and protein substrates. However, the prolyl isomerase activity of these proteins is not essential for their immunosuppressive effects. Instead, the specific surfaces of the cyclophilin-CsA and FKBP-FK506 complexes mediate the immunosuppressive action. Moreover, the natural cellular functions of all but a few remain elusive. In some cases it could be demonstrated that prolyl isomerization is the rate-limiting step in protein folding in vitro, but many knockout mutants of single and multiple prolyl isomerases were viable with no detectable phenotype. Even though a direct requirement for in vivo protein folding could not be demonstrated, some important natural substrates of the prolyl isomerases are now known, and they demonstrate the great variety of prolyl isomerization functions in the living cell: (i) A human cyclophilin binds to the Gag polyprotein of the human immunodeficiency virus-1 (HIV-1) virion and was found to be essential for infection with HIV to occur, probably by removal of the virion coat. (ii) Together with heat shock protein (HSP) 90, a member of the chaperone family, high molecular weight cyclophilins and FKBPs bind and activate steroid receptors. This example also demonstrates that prolyl isomerases act together with other folding enzymes, for example the chaperones, and protein disulfide isomerases. (iii) An FKBP was found to act as a modulator of an intracellular calcium release channel. (iv) Along with the cyclophilins and FKBPs, a third class of prolyl isomerases exist, the parvulins. The human parvulin homologue Pin1 is a mitotic regulator essential for the G2/M transition of the eukaryotic cell cycle. These findings place proline isomerases at the intersection of protein folding, signal transduction, trafficking, assembly and cell cycle regulation. Received 18 September 1998; received after revision 4 November 1998; accepted 23 November 1998     

Protein farnesylation in mammalian cells: effects of farnesyltransferase inhibitors on cancer cells

Protein farnesylation, catalyzed by protein farnesyltransferase, plays important roles in the membrane association and protein-protein interaction of a number of eukaryotic proteins. Recent development of farnesyltransferase inhibitors (FTIs) has led to further insight into the biological significance of farnesylation in cancer cells. A number of reports point to the dramatic effects FTIs exert on cancer cells. In addition to inhibiting anchorage-independent growth, FTIs cause changes in the cell cycle either at the G1/S or at the G2/M phase. Furthermore, induction of apoptosis by FTIs has been reported. FTIs also affects the actin cytoskeleton and cell morphology. This review summarizes these reports and discusses implications for farnesylated proteins responsible for these FTI effects. Received 17 April 2001; received after revision 28 May 2001; accepted 28 May 2001     

Molecular guardians for newborn proteins: ribosome-associated chaperones and their role in protein folding

A central dogma in biology is the conversion of genetic information into active proteins. The biosynthesis of proteins by ribosomes and the subsequent folding of newly made proteins represent the last crucial steps in this process. To guarantee the correct folding of newly made proteins, a complex chaperone network is required in all cells. In concert with ongoing protein biosynthesis, ribosome-associated factors can interact directly with emerging nascent polypeptides to protect them from degradation or aggregation, to promote folding into their native structure, or to otherwise contribute to their folding program. Eukaryotic cells possess two major ribosome-associated systems, an Hsp70/Hsp40-based chaperone system and the functionally enigmatic NAC complex, whereas prokaryotes employ the Trigger Factor chaperone. Recent structural insights into Trigger Factor reveal an intricate cradle-like structure that, together with the exit site of the ribosome, forms a protected environment for the folding of newly synthesized proteins. Received 29 June 2005; received after revision 4 August 2005; accepted 18 August 2005     

Protein misfolding and disease: the case of prion disorders

Recent findings strongly support the hypothesis that diverse human disorders, including the most common neurodegenerative diseases, arise from misfolding and aggregation of an underlying protein. Despite the good evidence for the involvement of protein misfolding in disease pathogenesis, the mechanism by which protein conformational changes participate in the disease is still unclear. Among the best-studied diseases of this group are the transmissible spongiform encephalopathies or prion-related disorders, in which misfolding of the normal prion protein plays a key role in the disease. In this article we review recent data on the link between prion protein misfolding and the pathogensis of spongiform encephalopathies. Received 15 July 2002; received after revision 19 August 2002; accepted 23 August 2002 RID="*" ID="*"Corresponding author.     

The folding process of hen lysozyme: a perspective from the ‘new view’

How a conformationally disordered polypeptide chain rapidly and efficiently achieves its well-defined native structure is still a major question in modern structural biology. Although much progress has been made towards rationalizing the principles of protein structure and dynamics, the mechanism of the folding process and the determinants of the final fold are not yet known in any detail. One protein for which folding has been studied in great detail by a combination of diverse techniques is hen lysozyme. In this article we review the present state of our knowledge of the folding process of this enzyme and focus in particular on recent experiments to probe some of its specific features. These results are then discussed in the context of the ‘new view’ of protein folding based on energy surfaces and land scapes. It is shown that a schematic energy surface for lysozyme folding, which is broadly consistent with our experimental data, begins to provide a unified model for protein folding through which experimental and theoretical ideas can be brought together.     

Protein oxidation and 20S proteasome-dependent proteolysis in mammalian cells

The generation of reactive oxygen species is an inevitable aspect of aerobic life. In addition to being exposed to free radicals in the environment, aerobic organisms must also deal with oxygen radicals generated as byproducts of a number of physiological mechanisms - for example, by the mitochondrial and endoplasmic reticulum electron transport chains, and by cells of the immune system. Although most organisms are equipped with several lines of defense against oxidative stress, these defensive mechanisms are not 100% effective, and oxidatively modified forms of proteins accumulate during aging, and in many pathological conditions.?Oxidatively modified proteins can form large aggregates due to covalent cross-linking or increased surface hydrophobicity. Unless repaired or removed from cells, these oxidized proteins are often toxic and can threaten cell viability. Mammalian cells exhibit only limited direct repair mechanisms, and oxidatively damaged proteins appear to undergo selective proteolysis, primarily by the major cytosolic proteinase, the proteasome. Interestingly, it appears that the 20S 'core' proteasome conducts the recognition and elimination of oxidized proteins in an ATP-independent and ubiquitin-independent pathway. Received 31 May 2001; accepted 26 June 2001     

Distribution of tightened end fragments of globular proteins statistically matches that of topohydrophobic positions: towards an efficient punctuation of protein folding?

Using a set of 372 proteins representative of a variety of 56 distinct globular folds, a statistical correlation was observed between two recently revealed features of protein structures: tightened end fragments or 'closed loops', i. e. sequence fragments that are able in three-dimensional (3D) space to nearly close their ends (a current parameter of polymer physics), and 'topohydrophobic positions', i. e. positions always occupied in 3D space by strong hydrophobic amino acids for all members of a fold family. Indeed, in sequence space, the distribution of preferred lengths for tightened end fragments and that for topohydrophobic separation match. In addition to this statistically significant similarity, the extremities of these 'closed loops' may be preferentially occupied by topohydrophobic positions, as observed on a random sample of various folds. This observation may be of special interest for sequence comparison of distantly related proteins. It is also important for the ab initio prediction of protein folds, considering the remarkable topological properties of topohydrophobic positions and their paramount importance within folding nuclei. Consequently, topohydrophobic positions locking the 'closed loops' belong to the deep cores of protein domains and might have a key role in the folding process. Received 1 February 2001; accepted 7 February 2001     

Flavodoxins: sequence, folding, binding, function and beyond

Flavodoxins are electron-transfer proteins involved in a variety of photosynthetic and non-photosynthetic reactions in bacteria, whereas, in eukaryotes, a descendant of the flavodoxin gene helps build multidomain proteins. The redox activity of flavodoxin derives from its bound flavin mononucleotide cofactor (FMN), whose intrinsic properties are profoundly modified by the host apoprotein. This review covers the very exciting last decade of flavodoxin research, in which the folding pathway, the structure and stability of the apoprotein, the mechanism of FMN recognition, the interactions that stabilize the functional complex and tailor the redox potentials, and many details of the binding and electron transfer to partner proteins have been revealed. The next decade should witness an even deeper understanding of the flavodoxin molecule and a greater comprehension of its many physiological roles. The fact that flavodoxin is essential for the survival of some human pathogens could make it a drug target on its own. Received 26 October 2005; received after revision 20 November 2005; accepted 14 December 2005     

Caveolin-1 interacts with the chaperone complex TCP-1 and modulates its protein folding activity

We report that caveolin-1, one of the major structural protein of caveolae, interacts with TCP-1, a hetero-oligomeric chaperone complex present in all eukaryotic cells that contributes mainly to the folding of actin and tubulin. The caveolin-TCP-1 interaction entails the first 32 amino acids of the N-terminal segment of caveolin. Our data show that caveolin-1 expression is needed for the induction of TCP-1 actin folding function in response to insulin stimulation. Caveolin-1 phosphorylation at tyrosine residue 14 induces the dissociation of caveolin-1 from TCP-1 and activates actin folding. We show that the mechanism by which caveolin-1 modulates TCP-1 activity is indirect and involves the cytoskeleton linker filamin. Filamin is known to bind caveolin-1 and to function as a negative regulator of insulin-mediated signaling. Our data support the notion that the caveolin-filamin interaction contributes to restore insulin-mediated phosphorylation of caveolin, thus allowing the release of active TCP-1. Received 17 November 2005; received after revision 1 December 2005; accepted 17 February 2006     

The DnaK/ClpB chaperone system from <Emphasis Type="Italic">Thermus thermophilus</Emphasis>

Proteins of thermophilic organisms are adapted to remain well structured and functional at elevated temperatures. Nevertheless like their 'cousins' that reside at medium temperatures, they require the assistance of molecular chaperones to fold properly and prevent aggregation. This review compares structural and functional properties of the DnaK/ClpB systems of Thermus thermophilus and, mainly, Escherichia coli (Dnak_Tth and Dnak_Eco). Many elemental properties of these systems remain conserved. However, in addition to a general increase of the thermal stability of its components, the Dnak_Tth system shows profound differences in its regulation, and genetic as well as oligomeric organization. Whether these differences are unique or represent general strategies of adaptation to life at elevated temperatures remains to be clarified. RID="*" ID="*"Corresponding author.     

Colloidal drug carriers: achievements and perspectives

Colloidal drug carriers such as liposomes and nanoparticles are able to modify the distribution of an associated substance. They can therefore be used to improve the therapeutic index of drugs by increasing their efficacy and/or reducing their toxicity. If these delivery systems are carefully designed with respect to the target and route of administration, they may provide one solution to some of the delivery problems posed by new classes of active molecules such as peptides, proteins, genes, and oligonucleotides. They may also extend the therapeutic potential of established drugs such as doxorubicin and amphotericin B. This article discusses the use of colloidal, particulate carrier systems (25 nm to 1 μm in diameter) in such applications. In particular, systems which show diminished uptake by mononuclear phagocytes are described. Specific targeting of carriers to particular tissues or cells is also considered. Received 8 April 2002; received after revision 25 June 2002; accepted 26 June 2002     

Cellulose synthesis: mutational analysis and genomic perspectives using Arabidopsis thaliana

Cellulose microfibrils containing crystalline β-1,4-glucan provide the major structural framework in higher-plant cell walls. Genetic analyses of Arabidopsis thaliana now link specific genes to plant cellulose production just as was achieved some years earlier with bacteria. Cellulose-deficient mutants have defects in several members of one family within a complex glycosyltransferase superfamily and in one member of a small family of membrane-bound endo-1,4-β-glucanases. The mutants also accumulate a readily extractable β-1,4-glucan that has short chains which, in at least one case, are lipid linked. Cellulose could be made by direct extension of the glucan chain by the glycosyltransferase or, as the mutant suggests, by an indirect route which makes lipid-linked oligosaccharides. Models discussed incorporate the known enzymes and lipo-glucan and raise the possibility that different CesA glycosyltransferases may catalyse different steps. Received 5 January 2001; received after revision 25 April 2001; accepted 25 April 2001     

Chemotherapy and immunotherapy of malignant glioma: molecular mechanisms and clinical perspectives

Despite the considerable progress in modern tumor therapy, the prognosis for patients with glioblastoma, the most frequent malignant brain tumor, has not been substantially improved. Although cytoreductive surgery and radiotherapy are the mainstays of treatment for malignant glioma at present, novel cytotoxic drugs and immunotherapeutic approaches hold great promise as effective weapons against these malignancies. Thus, great efforts are being made to enhance antitumoral efficacy by combining various cytotoxic agents, by novel routes of drug administration, or by combining anticancer drugs and immune modulators. Immunotherapeutic approaches include cytotoxic cytokines, targeted antibodies, and vaccination strategies. However, the success of most of these experimental therapies is prevented by the marked molecular resistance of glioma cells to diverse cytotoxic agents or by glioma-associated immunosuppression. One promising experimental strategy to target glioma is the employment of death ligands such as CD95 (Fas/Apo1) ligand or Apo2 ligand (TRAIL). Specific proapoptotic approaches may overcome many of the obvious obstacles to a satisfactory management of malignant brain tumors. Received 8 March 1999; received after revision 27 May 1999; accepted 14 June 1999     

Protein flexibility: its role in structure and mechanism revealed by molecular simulations

Computer simulations at the atomic level have arrived at a stage where they provide realistic modeling of flexibility in proteins (and the mobility of their associated solvent) that is important in understanding the nature of molecular motions. This can now be extended to the molecular and atomic motions that are associated with protein mechanisms. Moreover, the derived data agree reasonably accurately with experimental measurements of several kinetic and thermodynamic parameters. Fundamental insights emerge on the roles that this intrinsic flexibility plays in the thermodynamic characteristics of macromolecules in solution; these equip the investigator to probe the consequences of cognate interactions and ligand binding on entropy and enthalpy. Thus simulations can now provide a powerful tool for investigating protein mechanisms that complements the existing and the emerging experimental techniques. Received 29 May 2005; received after revision 23 August 2005; accepted 21 October 2005