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     

Genetic and functional linkage between ADAMTS superfamily proteins and fibrillin-1: a novel mechanism influencing microfibril assembly and function

Tissue microfibrils contain fibrillin-1 as a major constituent. Microfibrils regulate bioavailability of TGFβ superfamily growth factors and are structurally crucial in the ocular zonule. FBN1 mutations typically cause the Marfan syndrome, an autosomal dominant disorder manifesting with skeletal overgrowth, aortic aneurysm, and lens dislocation (ectopia lentis). Infrequently, FBN1 mutations cause dominantly inherited Weill–Marchesani syndrome (WMS), isolated ectopia lentis (IEL), or the fibrotic condition, geleophysic dysplasia (GD). Intriguingly, mutations in ADAMTS [a disintegrin-like and metalloprotease (reprolysin-type) with thrombospondin type 1 motif] family members phenocopy these disorders, leading to recessive WMS (ADAMTS10), WMS-like syndrome (ADAMTS17), IEL (ADAMTSL4 and ADAMTS17) and GD (ADAMTSL2). An ADAMTSL2 founder mutation causes Musladin–Lueke syndrome, a fibrotic disorder in beagle dogs. The overlapping disease spectra resulting from fibrillin-1 and ADAMTS mutations, interaction of ADAMTS10 and ADAMTSL2 with fibrillin-1, and evidence that these ADAMTS proteins accelerate microfibril biogenesis, constitutes a consilience suggesting that some ADAMTS proteins evolved to provide a novel mechanism regulating microfibril formation and consequently cell behavior.     

Conformational constraints on side chains in protein residues increase their information content

Like all other complex biological systems, proteins exhibit properties not seen in free amino acids (i.e., emergent properties). The present investigation arose from the deduction that proteins should offer a good model to approach the reverse phenomenon, namely top-down constraints experienced by protein residues compared to free amino acids. The crystalline structure of profilin Ib, a contractile protein of Acanthamoeba castellanii, was chosen as the object of study and submitted to 2-ns molecular dynamics simulation. The results revealed strong conformational constraints on the side chain of residues compared to the respective free amino acids. A Shannon entropy (SE) analysis of the conformational behavior of the side chains showed in most cases a strong decrease in the SE of the 1 and 2 dihedral angles compared to free amino acids. This is equivalent to stating that conformational constraints on the side chain of residues increase their information content and hence recognition specificity compared to free amino acids. In other words, the vastly increased information content of a protein relative to its free monomers is embedded not only in the tertiary structure of the backbone, but also in the conformational behavior of the side chains. The postulated implication is that both backbone and side chains, by virtue of being conformationally constrained, contribute to the recognition specificity of the protein toward other macromolecules and ligands.Received 13 July 2003; received after revision 18 August 2003; accepted 4 September 2003     

Molecular basis of autosomal-dominant polycystic kidney disease

Autosomal-dominant polycystic kidney disease (ADPKD) is one of the most common monogenetic diseases in humans. The discovery that mutations in the PKD1 and PKD2 genes are responsible for ADPKD has sparked extensive research efforts into the physiological and pathogenetic role of polycystin-1 and polycystin-2, the proteins encoded by these two genes. While polycystin-1 may mediate the contact among cells or between cells and the extracellular matrix, a lot of evidence suggests that polycystin-2 represents an endoplasmic reticulum-bound cation channel. Cyst development has been compared to the growth of benign tumors and this view is highlighted by the model that a somatic mutation in addition to the germline mutation is responsible for cystogenesis (two-hit model of cyst formation). Since in vitro polycystin-1 and polycystin-2 interact through their COOH termini, the two proteins possibly act in a common pathway, which controls the width of renal tubules. The loss of one protein may lead to a disruption of this pathway and to the uncontrolled expansion of tubules. Our increasing knowledge of the molecular events in ADPKD has also started to be useful in designing novel diagnostic and therapeutic strategies. Received 12 September 2001; received after revision 7 November 2001; accepted 7 November 2001     

Claudins: multifunctional players in epithelial tight junctions and their role in cancer

The molecular architecture of tight junctions has been a subject of extensive studies that have shown tight junctions to be composed of many peripheral and integral membrane proteins. Claudins have been considered the main tight junction-forming proteins; however, the role they play in a series of pathophysiological events, including human carcinoma development, is only now beginning to be understood. Increasing evidence from in vitro and in vivo studies have identified the influence of claudins on tight junction structure and function, although claudins also participate in cellular contexts other than tight junctions. The aim of this review is to summarize and discuss the conceptual framework concerning claudins, focusing on the involvement of these proteins in epithelial cell polarity establishment, paracellular transport control, signal transduction and tumorigenesis. Received 5 July 2006; received after revision 29 August 2006; accepted 29 September 2006     

Missense mutations resulting in type 1 lissencephaly

Proper human brain formation is dependent upon the integrated activity of multiple genes. Malfunctioning of key proteins results in brain developmental abnormalities. Mutation(s) in the LIS1 gene or the X-linked gene doublecortin (DCX) results in a spectrum of disorders including lissencephaly, or smooth brain, and subcortical band heterotopia, or doublecortex. Here, we will focus on a particular subset of missense mutations in these two genes and their effect on protein structure and function.Received 4 August 2004; received after revision 26 September 2004; accepted 5 October 2004     

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     

Interactions of the cell adhesion molecule nectin with transmembrane and peripheral membrane proteins for pleiotropic functions

Cell adhesion molecules (CAMs) have been implicated in the control of a wide variety of cellular processes, such as cell adhesion, polarization, survival, movement, and proliferation. Nectins have emerged as immunoglobulin-like CAMs that participate in calcium-independent cell-cell adhesion by homophilic and heterophilic trans-interactions with nectins and nectin-like molecules. Nectin-based cell-cell adhesion exerts its function independently or in cooperation with other CAMs including cadherins and is essential for the formation of intercellular junctions, including adherens junctions, tight junctions, and puncta adherentia junctions. Nectins cis-interact with integrin α_vβ₃ and platelet-derived growth factor receptor and facilitate their signals to regulate the formation and integrity of intercellular junctions and cell survival. Nectins intracellularly associate with peripheral membrane proteins, including afadin and Par-3. This review focuses on recent progress in understanding the interactions of nectins with other transmembrane and peripheral membrane proteins to exert pleiotropic functions. Received 27 June 2007; received after revision 14 August 2007; accepted 12 September 2007     

High affinity recognition of a <Emphasis Type="Italic">Phytophthora</Emphasis> protein by <Emphasis Type="Italic">Arabidopsis</Emphasis> via an RGD motif

The RGD tripeptide sequence, a cell adhesion motif present in several extracellular matrix proteins of mammalians, is involved in numerous plant processes. In plant-pathogen interactions, the RGD motif is believed to reduce plant defence responses by disrupting adhesions between the cell wall and plasma membrane. Photoaffinity cross-linking of [¹²⁵I]-azido-RGD heptapeptide in the presence of purified plasma membrane vesicles of Arabidopsis thaliana led to label incorporation into a single protein with an apparent molecular mass of 80 kDa. Incorporation could be prevented by excess RGD peptides, but also by the IPI-O protein, an RGD-containing protein secreted by the oomycete plant pathogen Phytophthora infestans. Hydrophobic cluster analysis revealed that the RGD motif of IPI-O (positions 53–56) is readily accessible for interactions. Single amino acid mutations in the RGD motif in IPI-O (of Asp⁵⁶ into Glu or Ala) resulted in the loss of protection of the 80-kDa protein from labelling. Thus, the interaction between the two proteins is mediated through RGD recognition and the 80-kDa RGD-binding protein has the characteristics of a receptor for IPI-O. The IPI-O protein also disrupted cell wall-plasma membrane adhesions in plasmolysed A. thaliana cells, whereas IPI-O proteins mutated in the RGD motif (D56A and D56E) did not.Received 23 October 2003; received after revision 5 December 2003; accepted 12 December 2003     

The copper-transporting capacity of ATP7A mutants associated with Menkes disease is ameliorated by COMMD1 as a result of improved protein expression

Menkes disease (MD) is an X-linked recessive disorder characterized by copper deficiency resulting in a diminished function of copper-dependent enzymes. Most MD patients die in early childhood, although mild forms of MD have also been described. A diversity of mutations in the gene encoding of the Golgi-resident copper-transporting P_1B-type ATPase ATP7A underlies MD. To elucidate the molecular consequences of the ATP7A mutations, various mutations in ATP7A associated with distinct phenotypes of MD (L873R, C1000R, N1304S, and A1362D) were analyzed in detail. All mutants studied displayed changes in protein expression and intracellular localization parallel to a dramatic decline in their copper-transporting capacity compared to ATP7A the wild-type. We restored these observed defects in ATP7A mutant proteins by culturing the cells at 30°C, which improves the quality of protein folding, similar to that which as has recently has been demonstrated for misfolded ATP7B, a copper transporter homologous to ATP7A. Further, the effect of the canine copper toxicosis protein COMMD1 on ATP7A function was examined as COMMD1 has been shown to regulate the proteolysis of ATP7B proteins. Interestingly, in addition to adjusted growth temperature, binding of COMMD1 partially restored the expression, subcellular localization, and copper-exporting activities of the ATP7A mutants. However, no effect of pharmacological chaperones was observed. Together, the presented data might provide a new direction for developing therapies to improve the residual exporting activity of unstable ATP7A mutant proteins, and suggests a potential role for COMMD1 in this process.     

Long-term changes in tyrosine phosphorylation of the abundant nuclear proteins during granulocytic differentiation of HL-60 cells

The two-dimensional electrophoretic patterns of nuclear proteins and their tyrosine phosphorylation were compared for HL-60 cells before and after differentiation induction to granulocytes by dimethyl sulfoxide, all-trans retinoic acid and N ⁶,O ²-dibutyryl adenosine 3′5′-cyclic monophosphate. Regardless of the inducer used, some nuclear proteins, which are tyrosine-phosphorylated in proliferating HL-60 cells, undergo gradual dephosphorylation 12–72 h after induction of differentiation, followed by drastic dephosphorylation during maturation to granulocytes. At least 13 nuclear proteins with a molecular mass of 35–110 kDa are dephosphorylated, and 6 nuclear proteins undergo tyrosine phosphorylation. Analysis of the nuclear proteins differentially extracted by salt and detergents indicates that changes in their tyrosine phosphorylation during the maturation stage of differentiating granulocytes occur mainly in proteins which are abundant in nucleoplasm, chromatin and residual nuclear structures. The abundance of these proteins, residing in the nuclear structures, and their long-term modification in phosphorylation during the maturation stages of differentiation strongly suggest that tyrosine phosphorylation of these proteins is involved in reorganization of the differentiating cell nucleus. Received 21 September 1998; received after revision 24 November 1998; accepted 3 December 1998     

The search for the right partner: Homologous pairing and DNA strand exchange proteins in eukaryotes

Finding the right partner is a central problem in homologous recombination. Common to all models for general recombination is a homologous pairing and DNA strand exchange step. In prokaryotes this process has mainly been studied with the RecA protein ofEscherichia coli. Two approaches have been used to find homologous pairing and DNA strand exchange proteins in eukaryotes. A biochemical approach has resulted in numerous proteins from various organisms. Almost all of these proteins are biochemically fundamentally different from RecA. The in vivo role of these proteins is largely not understood. A molecular-genetical approach has identified structural homologs to theE. coli RecA protein in the yeastSaccharomyces cerevisiae and subsequently in other organisms including other fungi, mammals, birds, and plants. The biochemistry of the eukaryotic RecA homologs is largely unsolved. For the fungal RecA homologs (S. cerevisiae RAD51, RAD55, RAD57, DMC1; Schizosaccharomyces pombe rad51; Neurospora crassa mei3) a role in homologous recombination and recombinational repair is evident. Besides recombination, homologous pairing proteins might be involved in other cellular processes like chromosome pairing or gene inactivation.     

Protein-O-mannosyltransferases in virulence and development

Protein-O-mannosyltransferases (Pmt proteins) catalyse the addition of mannose to serine or threonine residues of secretory proteins. This modification was described first for yeast and later for other fungi, mammals, insects and recently also for bacteria. O-mannosylation depends on specific isoforms of the three Pmt1, 2 and 4 subfamilies. In fungi, O-mannosylation determines the structure and integrity of cell walls, as well as cellular differentiation and virulence. O-mannosylation of specific secretory proteins of the human fungal pathogen Candida albicans and of the bacterial pathogen Mycobacterium tuberculosis contributes significantly to virulence. In mammals and insects, Pmt proteins are essential for cellular differentiation and development, while lack of Pmt activity causes Walker-Warburg syndrome (muscular dystrophy) in humans. The susceptibility of human cells to certain viruses may also depend on O-mannosyl chains. This review focuses on the various roles of Pmt proteins in cellular differentiation, development and virulence. Received 6 September 2007; received after revision 3 October 2007; accepted 5 October 2007     

Die Säurefällung der Serumproteine

Summary Diluted serum was treated with diluted acid. Between pH 7·8 and 6·5 a part of the globulines is precipitated whose composition changes in favour of the more rapidly migrating fractions with rising pH. Sera denaturated by heat are flocculated quantitatively and behave like one monodisperse protein also in electrophoresis and chromatography. This leads to the conclusion, other than after denaturation, that negative loads of native proteins possess different strengths of absorption toward H.

---

---

K. Simon, Exper.8, 55 (1952).     

Redundant and unique roles of coronin proteins in <Emphasis Type="Italic">Dictyostelium</Emphasis>

Dictyostelium discoideum harbors a short (CRN12) and a long coronin (CRN7) composed of one and two beta-propellers, respectively. They are primarily present in the cell cortex and cells lacking CRN12 (corA ⁻) or CRN7 (corB ⁻) have defects in actin driven processes. We compared the characteristics of a mutant cell line (corA ⁻ /corB ⁻) lacking CRN12 and CRN7 with the single mutants focusing on cytokinesis, phagocytosis, chemotaxis and development. Cytokinesis, uptake of small particles, and developmental defects were not enhanced in the corA ⁻ /corB ⁻ strain as compared to the single mutants, whereas motility and phagocytosis of yeast particles were more severely impaired. It appears that although both proteins affect the same processes they do not act in a redundant manner. Rather, they often act antagonistically, which is in accordance with their proposed roles in the actin cytoskeleton where CRN12 acts in actin disassembly whereas CRN7 stabilizes actin filaments and protects them from disassembly.