The alpha-, beta- and gamma-crystallins are the major protein components of the vertebrate eye lens, alpha-crystallin as a molecular chaperone as well as a structural protein, beta- and gamma-crystallins as structural proteins. For the lens to be able to retain life-long transparency in the absence of protein turnover, the crystallins must meet not only the requirement of solubility associated with high cellular concentration but that of longevity as well. For proteins, longevity is commonly assumed to be correlated with long-term retention of native structure, which in turn can be due to inherent thermodynamic stability, efficient capture and refolding of non-native protein by chaperones, or a combination of both. Understanding how the specific interactions that confer intrinsic stability of the protein fold are combined with the stabilizing effect of protein assembly, and how the non-specific interactions and associations of the assemblies enable the generation of highly concentrated solutions, is thus of importance to understand the loss of transparency of the lens with age. Post-translational modification can have a major effect on protein stability but an emerging theme of the few studies of the effect of post-translational modification of the crystallins is one of solubility and assembly. Here we review the structure, assembly, interactions, stability and post-translational modifications of the crystallins, not only in isolation but also as part of a multi-component system. The available data are discussed in the context of the establishment, the maintenance and finally, with age, the loss of transparency of the lens. Understanding the structural basis of protein stability and interactions in the healthy eye lens is the route to solve the enormous medical and economical problem of cataract.

/pdf/ageing-and-vision-structure-stability-and-function-of-lens-539zffk8d5.pdf

Ageing and vision: structure, stability and function of lens crystallins

Forty-five distinct subfamilies of EF-hand proteins have been identified. They contain from two to eight EF-hands that are recognizable by amino acid sequence as being statistically similar to other EF-hand domains. All proteins within one subfamily are congruent to one another, i.e. the dendrogram computed from one of the EF-hand domains is similar, within statistical error, to the dendrogram computed from another(s) domain. Thirteen subfamilies--including Calmodulin, Troponin C, Essential light chain, Regulatory light chain--referred to collectively as CTER, are congruent with one another. They appear to have evolved from a single ur-domain by two cycles of gene duplication and fusion. The subfamilies of CTER subsequently evolved by gene duplications and speciations. The remaining 32 subfamilies do not show such general patterns of congruence; however, some--such as S100, intestinal calcium binding protein (calbindin 9 kd), and trichohylin--do not form congruent clusters of subfamilies. Nearly all of the domains 1, 3, 5, and 7 are most similar to other ODD domains. Correspondingly the EVEN numbered domains of all 45 subfamilies most closely resemble EVEN domains of other subfamilies. Many sequence and chemical characteristics do not show systemic trends by subfamily or species of host organisms; such homoplasy is widespread. Eighteen of the subfamilies are heterochimeric; in addition to multiple EF-hands they contain domains of other evolutionary origins.

Classification and evolution of EF-hand proteins

This review summarizes the recent discovery of the cupin superfamily (from the Latin term "cupa," a small barrel) of functionally diverse proteins that initially were limited to several higher plant proteins such as seed storage proteins, germin (an oxalate oxidase), germin-like proteins, and auxin-binding protein. Knowledge of the three-dimensional structure of two vicilins, seed proteins with a characteristic beta-barrel core, led to the identification of a small number of conserved residues and thence to the discovery of several microbial proteins which share these key amino acids. In particular, there is a highly conserved pattern of two histidine-containing motifs with a varied intermotif spacing. This cupin signature is found as a central component of many microbial proteins including certain types of phosphomannose isomerase, polyketide synthase, epimerase, and dioxygenase. In addition, the signature has been identified within the N-terminal effector domain in a subgroup of bacterial AraC transcription factors. As well as these single-domain cupins, this survey has identified other classes of two-domain bicupins including bacterial gentisate 1, 2-dioxygenases and 1-hydroxy-2-naphthoate dioxygenases, fungal oxalate decarboxylases, and legume sucrose-binding proteins. Cupin evolution is discussed from the perspective of the structure-function relationships, using data from the genomes of several prokaryotes, especially Bacillus subtilis. Many of these functions involve aspects of sugar metabolism and cell wall synthesis and are concerned with responses to abiotic stress such as heat, desiccation, or starvation. Particular emphasis is also given to the oxalate-degrading enzymes from microbes, their biological significance, and their value in a range of medical and other applications.

Microbial Relatives of the Seed Storage Proteins of Higher Plants: Conservation of Structure and Diversification of Function during Evolution of the Cupin Superfamily

When tissue sections are extracted with 0.1 N HCl, cellular nuclear proteins, including histones, are removed but nuclear DNA is retained. Histones can be reconstituted back to nuclear DNA in acid-extracted tissue sections so that the resulting nuclear substrate is composed only of DNA and histones and does not contain acidic nuclear protein antigens. The resulting DNA-histone tissue substrate can be used in the immunofluorescent method for specific detention of antibodies to histones. Sera from 23 patients with drug-induced lupus erythematosus (procainamide 19, isoniazid 2, nitrofurantoin 2) and 20 patients with idiopathic (not drug-induced) systemic lupus erythematosus (SLE) were studied. All 23 patients with drug-induced lupus erythematosus (LE) lost nuclear staining on acid-extracted sections. In contrast, only 12 of 20 with idiopathic SLE lost nuclear staining on acid-extracted tissues, and in the remaining 8, there was no significant fall in titer. In the drug-induced LE group, loss of nuclear staining was due to the absence of histones on the substrate because with histone-reconstituted sections, 22 of 23 again became positive for nuclear staining at titers equal to or at one doubling dilution below titers on unextracted tissues. In contrast, of the 12 idiopathic SLE sera which lost nuclear staining, only 5 regained nuclear staining on histone-reconstituted tissue sections. In idiopathic SLE, antinuclear antibodies are heterogeneous in specificities and may consist of antibodies to native DNA, histones, or nonhistone proteins. In contrast, antinuclear antibodies in drug-induced LE are less heterogeneous and mainly consist of antibodies to histones.

/pdf/antibodies-to-histones-in-drug-induced-and-idiopathic-lupus-11ixrbz6i6.pdf

Antibodies to Histones in Drug-Induced and Idiopathic Lupus Erythematosus

In the first report in this series we described the relationships and evolution of 152 individual proteins of the EF-hand subfamilies. Here we add 66 additional proteins and define eight (CDC, TPNV, CLNB, LPS, DGK, 1F8, VIS, TCBP) new subfamilies and seven (CAL, SQUD, CDPK, EFH5, TPP, LAV, CRGP) new unique proteins, which we assume represent new subfamilies. The main focus of this study is the classification of individual EF-hand domains. Five subfamilies--calmodulin, troponin C, essential light chain, regulatory light chain, CDC31/caltractin--and three uniques--call, squidulin, and calcium-dependent protein kinase--are congruent in that all evolved from a common four-domain precursor. In contrast calpain and sarcoplasmic calcium-binding protein (SARC) each evolved from its own one-domain precursor. The remaining 19 subfamilies and uniques appear to have evolved by translocation and splicing of genes encoding the EF-hand domains that were precursors to the congruent eight and to calpain and to SARC. The rates of evolution of the EF-hand domains are slower following formation of the subfamilies and establishment of their functions. Subfamilies are not readily classified by patterns of calcium coordination, interdomain linker stability, and glycine and proline distribution. There are many homoplasies indicating that similar variants of the EF-hand evolved by independent pathways.

Evolution of EF-hand calcium-modulated proteins. II. Domains of several subfamilies have diverse evolutionary histories.

The rate and extent of plasmodium formation were studied in mating tests involving pairs of largely isogenic amoeba1 strains compatible for matingtype ( mt ) alleles. A systematic variability was observed: plasmodia formed either rapidly and extensively or slowly and inefficiently. Plasmodium formation was found to be 10 3 - to 10 4 -fold more extensive in "rapid" crosses than in "slow" crosses. A genetic analysis revealed that the variability reflects the influence of a multiallelic compatibility locus that determines mating efficiency. This compatibility locus (designated matB ), together with the original mating type locus mt (in this work designated matA ), constitute a tetrapolar mating specificity system in Physarum polycephalum .

https://www.genetics.org/content/genetics/91/4/683.full.pdf

A New Mating Compatibility Locus in PHYSARUM POLYCEPHALUM.

The actin gene family in Physarum polycephalum contains four unlinked loci: ardA, ardB, ardC, and ardD. The ardA locus is complex and probably contains two genes which we designated ardA2-...

Expression of the three unlinked isocoding actin genes of Physarum polycephalum.

Plasmodium formation by mating was studied in cultures containing equal numbers of mating-compatible amoebae. A period of amoebal multiplication always preceded plasmodium formation. When the inoculum varied from a total of 3 X 103 to 1.6 X 10° cells per culture, plasmodium formation commenced when the total number of cells reached about 5 X 105 per culture. When the inoculum included an excess of a third strain of amoebae that is unable to mate with the first two, mating commenced when the total number of matable cells reached only 1 x 10' per culture. This effect of the third strain of amoebae was not brought about by their consumption of the bacteria used as food, since directly limiting the number of food bacteria did not cause earlier mating. When amoebae of the third strain were separated from the mating cells by paired 0.2 gm Nuclepore filters, premature mating still occurred. Thus, as is the case with asexual plasmodium formation, one or more extracellular factors appear to influence the time of onset of mating.

https://www.jstor.org/stable/pdfplus/3759222.pdf

Kinetics of mating in Physarum polycephalum.

We constructed cDNA libraries from plasmodia and amoebal poly(A)+ RNA of Physarum polycephalum The libraries were screened by differential hybridization with labeled poly(A)+ RNA of amoebae and pl

Molecular cloning of stage specific mRNAs from amoebae and Plasmodia of Physarum polycephalum

Variation of gene expression during spherulation of Physarum polycephalum microplasmodia has been studied by two-dimensional gel electrophoresis of proteins synthesized in vivo and in vitro. The electrophoretic pattern of total cellular proteins has also been analyzed during the course of differentiation. In all cases, major variations were observed and correlated with the formation of biologically mature spherules. After 24 h of differentiation, numerous specific proteins started to accumulate. After 48 h, a time at which viable spherule formation was complete, the synthesis of a set of late proteins was observed. It has also been shown that the synthesis of actin, the most abundant protein in this organism, was significantly repressed during the period at which the cells accumulated the spherule-specific proteins. The results showed that the spherulation of Physarum involves complex and major changes of protein synthesis, which occur in a temporal sequence. These variations are also observed in the popu...

Dominick Pallotta

Papers

A New Mating Compatibility Locus in PHYSARUM POLYCEPHALUM.

Expression of the three unlinked isocoding actin genes of Physarum polycephalum.

Kinetics of mating in Physarum polycephalum.

Molecular cloning of stage specific mRNAs from amoebae and Plasmodia of Physarum polycephalum

Changes in gene expression during spherulation in Physarum polycephalum