Glucokinase regulatory protein is essential for the proper subcellular localisation of liver glucokinase.

Glucokinase (GK), a key enzyme in the glucose homeostatic responses of the liver, changes its intracellular localisation depending on the metabolic status of the cell. Rat liver GK and Xenopus laevis GK, fused to the green fluorescent protein (GFP), concentrated in the nucleus of cultured rat hepatocytes at low glucose and translocated to the cytoplasm at high glucose. Three mutant forms of Xenopus GK with reduced affinity for GK regulatory protein (GKRP) did not concentrate in the hepatocyte nuclei, even at low glucose. In COS-1 and HeLa cells, a blue fluorescent protein (BFP)-tagged version of rat liver GK was only able to accumulate in the nucleus when it was co-expressed with GKRP-GFP. At low glucose, both proteins concentrated in the nuclear compartment and at high glucose, BFP-GK translocated to the cytosol while GKRP-GFP remained in the nucleus. These findings indicate that the presence of and binding to GKRP are necessary and sufficient for the proper intracellular localisation of GK and directly involve GKRP in the control of the GK subcellular distribution.     

Evidence for protein phosphorylation as a regulatory mechanism for hepatic microsomal glucose 6 phosphatase

Incubation of microsomal vesicles with ATP and protein kinase results in a fivefold increased glucose-6-phosphatase activity. Evidence is presented that this effect is mediated via a moiety of the outer membrane surface. Evidence is also presented for the presence of an endogenous, peripheral membrane protein also capable of activating glucose-6-phosphatase in an ATP dependent reaction. It is suggested that the glucose-6-phosphate transmembrane carrier system may be the target of phosphorylation.     

Multidrug ATP‐binding cassette transporters are essential for hepatic development of Plasmodium sporozoites

Multidrug resistance‐associated proteins (MRPs) belong to the C‐family of ATP‐binding cassette (ABC) transport proteins and are known to transport a variety of physiologically important compounds and to be involved in the extrusion of pharmaceuticals. Rodent malaria parasites encode a single ABC transporter subfamily C protein, whereas human parasites encode two: MRP1 and MRP2. Although associated with drug resistance, their biological function and substrates remain unknown. To elucidate the role of MRP throughout the parasite life cycle, Plasmodium berghei and Plasmodium falciparum mutants lacking MRP expression were generated. P. berghei mutants lacking expression of the single MRP as well as P. falciparum mutants lacking MRP1, MRP2 or both proteins have similar blood stage growth kinetics and drug‐sensitivity profiles as wild type parasites. We show that MRP1‐deficient parasites readily invade primary human hepatocytes and develop into mature liver stages. In contrast, both P. falciparum MRP2‐deficient parasites and P. berghei mutants lacking MRP protein expression abort in mid to late liver stage development, failing to produce mature liver stages. The combined P. berghei and P. falciparum data are the first demonstration of a critical role of an ABC transporter during Plasmodium liver stage development.     

PremPS: Predicting the impact of missense mutations on protein stability

Computational methods that predict protein stability changes induced by missense mutations have made a lot of progress over the past decades. Most of the available methods however have very limited accuracy in predicting stabilizing mutations because existing experimental sets are dominated by mutations reducing protein stability. Moreover, few approaches could consistently perform well across different test cases. To address these issues, we developed a new computational method PremPS to more accurately evaluate the effects of missense mutations on protein stability. The PremPS method is composed of only ten evolutionary- and structure-based features and parameterized on a balanced dataset with an equal number of stabilizing and destabilizing mutations. A comprehensive comparison of the predictive performance of PremPS with other available methods on nine benchmark datasets confirms that our approach consistently outperforms other methods and shows considerable improvement in estimating the impacts of stabilizing mutations. A protein could have multiple structures available, and if another structure of the same protein is used, the predicted change in stability for structure-based methods might be different. Thus, we further estimated the impact of using different structures on prediction accuracy, and demonstrate that our method performs well across different types of structures except for low-resolution structures and models built based on templates with low sequence identity. PremPS can be used for finding functionally important variants, revealing the molecular mechanisms of functional influences and protein design. PremPS is freely available at https://lilab.jysw.suda.edu.cn/research/PremPS/, which allows to do large-scale mutational scanning and takes about four minutes to perform calculations for a single mutation per protein with ~ 300 residues and requires ~ 0.4 seconds for each additional mutation.     

The C-terminal helices of heat shock protein 70 are essential for J-domain binding and ATPase activation

The J-domain co-chaperones work together with the heat shock protein 70 (HSP70) chaperone to regulate many cellular events, but the mechanism underlying the J-domain-mediated HSP70 function remains elusive. We studied the interaction between human-inducible HSP70 and Homo sapiens J-domain protein (HSJ1a), a J domain and UIM motif-containing co-chaperone. The J domain of HSJ1a shares a conserved structure with other J domains from both eukaryotic and prokaryotic species, and it mediates the interaction with and the ATPase cycle of HSP70. Our in vitro study corroborates that the N terminus of HSP70 including the ATPase domain and the substrate-binding β-subdomain is not sufficient to bind with the J domain of HSJ1a. The C-terminal helical α-subdomain of HSP70, which was considered to function as a lid of the substrate-binding domain, is crucial for binding with the J domain of HSJ1a and stimulating the ATPase activity of HSP70. These fluctuating helices are likely to contribute to a proper conformation of HSP70 for J-domain binding other than directly bind with the J domain. Our findings provide an alternative mechanism of allosteric activation for functional regulation of HSP70 by its J-domain co-chaperones.     

C-terminal residues 621-635 of protein S are essential for binding to factor Va

Protein S is anticoagulant in the absence of activated protein C because of direct interactions with coagulation Factors Xa and Va. Synthetic peptides corresponding to amino acid sequences of protein S were tested for their ability to inhibit prothrombinase activity. The peptide containing the C-terminal sequence of protein S, residues 621-635 (PSP14), reversibly inhibited prothrombinase activity in the presence but not in the absence of Factor Va (K(i) approximately 2 microM). PSP14 inhibition of prothrombinase was independent of phospholipids but could be competitively overcome by increasing Factor Xa concentrations, suggesting that the C-terminal region of protein S may compete for a Factor Xa binding site on Factor Va. Studies using peptides with amino acid substitutions suggested that lysines 630, 631, and 633 were critical residues. PSP14 inhibited Factor Va activity in Factor Xa-one-stage clotting assays. PSP14 inhibited protein S binding to immobilized Factor Va. When preincubated with protein S, antibodies raised against PSP14 inhibited binding of protein S to Factor Va and blocked inhibition of prothrombinase activity by protein S. These results show that the C-terminal region of protein S containing residues 621-635 is essential for binding of protein S to Factor Va and that this interaction contributes to anticoagulant action.     

Members of the fatty acid binding protein family are differentiation factors for the mammary gland

Mammary gland development is controlled by systemic hormones and by growth factors that might complement or mediate hormonal action. Peptides that locally signal growth cessation and stimulate differentiation of the developing epithelium have not been described. Here, we report that recombinant and wild-type forms of mammary-derived growth inhibitor (MDGI) and heart-fatty acid binding protein (FABP), which belong to the FABP family, specifically inhibit growth of normal mouse mammary epithelial cells (MEC), while growth of stromal cells is not suppressed. In mammary gland organ culture, inhibition of ductal growth is associated with the appearance of bulbous alveolar end buds and formation of fully developed lobuloalveolar structures. In parallel, MDGI stimulates its own expression and promotes milk protein synthesis. Selective inhibition of endogenous MDGI expression in MEC by antisense phosphorothioate oligonucleotides suppresses appearance of alveolar end buds and lowers the beta-casein level in organ cultures. Furthermore, MDGI suppresses the mitogenic effects of epidermal growth factor, and epidermal growth factor antagonizes the activities of MDGI. Finally, the regulatory properties of MDGI can be fully mimicked by an 11-amino acid sequence, represented in the COOH terminus of MDGI and a subfamily of structurally related FABPs. This peptide does not bind fatty acids. To our knowledge, this is the first report about a growth inhibitor promoting mammary gland differentiation.     

Artificial intelligence challenges for predicting the impact of mutations on protein stability

Stability is a key ingredient of protein fitness, and its modification through targeted mutations has applications in various fields, such as protein engineering, drug design, and deleterious variant interpretation. Many studies have been devoted over the past decades to build new, more effective methods for predicting the impact of mutations on protein stability based on the latest developments in artificial intelligence. We discuss their features, algorithms, computational efficiency, and accuracy estimated on an independent test set. We focus on a critical analysis of their limitations, the recurrent biases toward the training set, their generalizability, and interpretability. We found that the accuracy of the predictors has stagnated at around 1 kcal/mol for over 15 years. We conclude by discussing the challenges that need to be addressed to reach improved performance.     

Structural and functional analyses of disease-causing missense mutations in Bloom syndrome protein

Bloom syndrome (BS) is an autosomal recessive disorder characterized by genomic instability and the early development of many types of cancer. Missense mutations have been identified in the BLM gene (encoding a RecQ helicase) in affected individuals, but the molecular mechanism and the structural basis of the effects of these mutations remain to be elucidated. We analysed five disease-causing missense mutations that are localized in the BLM helicase core region: Q672R, I841T, C878R, G891E and C901Y. The disease-causing mutants had low ATPase and helicase activities but their ATP binding abilities were normal, except for Q672, whose ATP binding activity was lower than that of the intact BLM helicase. Mutants C878R, mapping near motif IV, and G891E and C901Y, mapping in motif IV, displayed severe DNA-binding defects. We used molecular modelling to analyse these mutations. Our work provides insights into the molecular basis of BLM pathology, and reveals structural elements implicated in coupling DNA binding to ATP hydrolysis and DNA unwinding. Our findings will help to explain the mechanism underlying BLM catalysis and interpreting new BLM causing mutations identified in the future.     

Retinol and extracellular collagen matrices modulate hepatic Ito cell collagen phenotype and cellular retinol binding protein levels

The hepatic vitamin A-storing Ito cell has been implicated as a causative cell in hepatic fibrogenesis. Using a modification of a recent method (Friedman, S. L., Roll, F. J., Boyles, J., and Bissell, D. M. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 8681-8685), rat Ito cells were isolated and passaged in vitro on collagen-coated plastic dishes through cell generation 40-50. The collagen synthetic phenotype for Ito cells grown on various extracellular matrices was demonstrated by immunofluorescence and quantitated by competition enzyme-linked immunosorbent assays. When grown on a type I collagen matrix, Ito cells produced type IV greater than type III greater than type I collagen. When grown on a type IV collagen matrix, the cells produced relatively equal amounts of types I and III collagen. The absolute amounts of type I collagen produced were greater when cells were grown on type IV versus type I matrix. When 10(-5) M retinol was added to cell cultures, there was a uniform increase in type III collagen regardless of matrix type but a decrease in type I collagen when cells were grown on a type IV matrix and a large increase in type I collagen when cells were grown on a type I collagen matrix. The levels of cellular retinol binding protein, a key cytosolic retinol transport protein, were quantitated by high performance liquid chromatography and compared for cells grown on type I versus type IV collagen matrices. It was found that cells on a type I matrix contain 4.96 +/- 2.8 times more cellular retinol binding protein than do cells grown on a type IV matrix. In conclusion, Ito cell collagen synthesis may be altered by underlying extracellular matrix and exogenous retinol. This in vitro culture system should allow the study of regulatory factors and possible therapeutic anti-fibrogenic mediators.     

Oxysterol binding protein and its homologues: new regulatory factors involved in lipid metabolism

PURPOSE OF REVIEW: Oxysterol binding protein was discovered in the 1980s as a cytosolic high-affinity receptor for oxysterols, but its function has remained enigmatic. Families of genes/proteins with sequence homology to oxysterol binding protein have been identified in eukaryotes from yeast to man, indicating that these proteins, denoted as oxysterol binding protein-related proteins (ORPs), serve a fundamental purpose conserved in evolution. This review discusses recent findings that provide important clues to the mode of action of these proteins. RECENT FINDINGS: The long variant of ORP1 is induced upon differentiation of monocytes to macrophages and has capacity to enhance the trans-activation potential of liver X receptors, indicating a function in macrophage lipid metabolism. Important clues to ORP function were provided by the finding that most family members carry an endoplasmic reticulum targeting motif, while the amino-terminal regions of the proteins have targeting specificities for other organelles. Extensive splice variation occurs within the gene family, suggesting that a large number of distinct protein products are encoded. Further implications were obtained for a possible role of a family member in tumor cell metastasis. SUMMARY: ORPs constitute a novel family of proteins implicated in cellular lipid metabolism and different aspects of cell regulation. The function of several family members is connected with cellular sterol metabolism, and there is evidence for a role of oxysterol binding protein in lipid transport from the endoplasmic reticulum. Recently, a model on the function of these proteins at membrane contact sites, specialized zones of communication between two different organelles, has been presented.     

Null mutations in the SNF3 gene of Saccharomyces cerevisiae cause a different phenotype than do previously isolated missense mutations.

Missense mutations in the SNF3 gene of Saccharomyces cerevisiae were previously found to cause defects in both glucose repression and derepression of the SUC2 (invertase) gene. In addition, the growth properties of snf3 mutants suggested that they were defective in uptake of glucose and fructose. We have cloned the SNF3 gene by complementation and demonstrated linkage of the cloned DNA to the chromosomal SNF3 locus. The gene encodes a 3-kilobase poly(A)-containing RNA, which was fivefold more abundant in cells deprived of glucose. The SNF3 gene was disrupted at its chromosomal locus by several methods to create null mutations. Disruption resulted in growth phenotypes consistent with a defect in glucose uptake. Surprisingly, gene disruption did not cause aberrant regulation of SUC2 expression. We discuss possible mechanisms by which abnormal SNF3 gene products encoded by missense alleles could perturb regulatory functions.     

Short circulatory residence of the hepatic binding protein

We evaluated the distribution and fate of homologous radioactive Hepatic-binding-protein (125I-HBP) in the rabbit after intravenous (iv) injection and the possibility that this protein may induce interferon production. We demonstrated that only 9% of the injected 125I-HBP remained in the circulation 30 min after injection. The 125I-HBP-Asialoorosomucoid complex displayed a longer half-life than the HBP alone, while 125I-Asialoorosomucoid had a very short half-life and only 1% of the dose was found in the circulation after iv administration. Ten min after iv injection of 125I-HBP the major amount of radioactivity was present in the liver and less in the kidneys and lung. HBP, after iv administration, does not stimulate interferon production and this fact is probably due to its rapid catabolism.     

Spontaneous missense mutations in the rplX gene for ribosomal protein L24 from Escherichia coli.

Temperature-resistant pseudorevertants of the temperature-sensitive Escherichia coli mutant KNS19, harboring a mutation in rplX, the gene for ribosomal protein L24, were isolated, cloned, and sequenced. The codon GAC for the amino acid Asp in the temperature-sensitive mutant corresponding to position 84 in the protein chain mutated either back to the wild type (Gly) or to codons for the amino acids Tyr and Glu. Furthermore, rplX genes from two other mutants with an altered protein L24 were cloned and sequenced. The mutations were localized at position 56 (Gly to Asp) and at position 62 (Glu to Lys) in the rplX gene. The latter two mutants lacked a conditional lethal phenotype. The results suggest that the amino acid Gly at positions 56 and 84 in the protein might be involved in loop formations.     

Predicting the effect of missense mutations on protein function: analysis with Bayesian networks

Background  

A number of methods that use both protein structural and evolutionary information are available to predict the functional consequences of missense mutations. However, many of these methods break down if either one of the two types of data are missing. Furthermore, there is a lack of rigorous assessment of how important the different factors are to prediction.     

The salt dependence of the interferon regulatory factor 1 DNA binding domain binding to DNA reveals ions are localized around protein and DNA

The equilibrium dissociation constant of the DNA binding domain of interferon regulatory factor 1 (IRF1 DBD) for its DNA binding site depends strongly on salt concentration and salt type. These dependencies are consistent with IRF1 DBD binding to DNA, resulting in the release of cations from the DNA and both release of anions from the protein and uptake of a cation by the protein. We demonstrated this by utilizing the fact that the release of fluoride from protein upon complex formation does not contribute to the salt concentration dependence of binding and by studying mutants in which charged residues in IRF1 DBD that form salt bridges with DNA phosphates are changed to alanine. The salt concentration dependencies of the dissociation constants of wild-type IRF1 DBD and the mutants R64A, D73A, K75A, and D73A/K75A were measured in buffer containing NaF, NaCl, or NaBr. The salt concentration and type dependencies of the mutants relative to wild-type IRF1 DBD provide evidence of charge neutralization by solution ions for R64 and by a salt bridge between D73 and K75 in buffer containing chloride or bromide salts. These data also allowed us to determine the number, type, and localization of condensed ions around both IRF1 DBD and its DNA binding site.