Bernard Laine

A DNA-binding protein fromE. coliisolation, characterization and its relationship with proteins H1 and B1

A protein of Mr 16,000 (protein 16 K) which tightly binds to DNA has been isolated from E. coli. The amino acid composition and amino-terminal amino acid sequence of this protein have been determined. On the basis of its physico-chemical characteristics the protein 16 K was shown to be identical to the protein H1 [Cukier-Kahn et al. (1972) Proc. Natl. Acad. Sci. USA 69, 3643-3647; Spassky and Buc (1977) Eur. J. Biochem. 81, 79-90] which corresponds itself to the protein B1 [Bakaev (1981) Molekulyarnaya Biologya 15, 1350-1362].

Functional properties of the R154X HNF‐4α protein generated by a mutation associated with maturity‐onset diabetes of the young, type 1

Mutations in the hepatocyte nuclear factor 4α (HNF‐4α) gene are associated with one form of maturity‐onset diabetes of the young (MODY1). The R154X mutation generates a protein lacking the E‐domain which is required for normal HNF‐4α functions. Since pancreatic β‐cell dysfunction is a feature of MODY1 patients, we compared the functional properties of the R154X mutant in insulin‐secreting pancreatic β‐cells and non‐β‐cells. The R154X mutation did not affect nuclear localisation in β‐cells and non‐β‐cells. However, it did lead to a greater impairment of HNF‐4α function in β‐cells compared to non‐β‐cells, including a complete loss of transactivation activity and a dominant‐negative behaviour.

In vitro phosphorylation of histones H5, H2A, H2B and of the dimer H2A--H2B by a cyclic AMP-dependent protein kinase from rat pancreas.

Histone phosphorylation is a post-synthetic modification involved in the regulation of metabolic process and genetic expression [ 1,2] and associated with changes in chromatin structure [3]. All histones are phosphorylated but lysine-rich histones are the most active substrates for cyclic AMPdependent protein kinases [4]. Phosphorylation of lysine-rich histone (Hl) has been correlated with the rate of cell division in different rat and mouse tumors [S]. Phosphorylation is more extensive in rapidly growing cells than in non-dividing tissue. Phosphorylation is also related to cell maturation, for example, to erythrocyte maturation [6] during which time histone H5 is synthesized. The phosphorylation of the histones with protein kinases isolated from different mammalian tissues: brain, liver, testis and thymus, takes place at specific sites of the amino acid sequences, generally located in highly basic regions which are major sites of interaction with DNA [7]. Therefore phosphorylation can modulate the histone-DNA interactions. This paper deals with the in vitro phosphorylation of histones by a cyclic AMP-dependent protein kinase isolated from rat pancreas. Histones from various sources were used: H2B from rat thymus, H2A (variant a) from rat chloroleukemia; the complex H2AH2B from calf thymus; HS from chicken erythrocyte. In rat chloroleukemia, histone H2A exhibits a polymorphism depending on the nature of the amino acid residues at positions 16 and 99 [8]. Three molecular species of H2A were identified in this tumoral

Mutations in hepatocyte nuclear factor 4α (HNF4α) gene associated with diabetes result in greater loss of HNF4α function in pancreatic β-cells than in nonpancreatic β-cells and in reduced activation of the apolipoprotein CIII promoter in hepatic cells

Mutations in the HNF4α gene have been correlated with maturity-onset diabetes of the young, which is characterized mainly by pancreatic β-cell dysfunction and is also associated with mild liver abnormalities. HNF4α D126Y and D126H mutations were found in a patient with early-onset type 2 diabetes, and the R324H mutation was found in a common type 2 diabetic nephropathic patient. We investigated whether these mutations, which have not yet been functionally characterized, impair HNF4α function in three cell models: HEK 293 embryonal kidney cells, HepG2 hepatoma cells, and βTC3 pancreatic β-cells. The R324H mutation had no effect on HNF4α function with either the HNF1α and L-type pyruvate kinase (LPK) promoters, but the D126Y and D126H mutations impaired HNF4α transcriptional activities in all tested cell lines. These impairments by D126Y and D126H mutations, which are located in the T box, are not due to a loss of dimerization but to a loss of DNA binding. Interestingly, the strongest functional consequences of these mutations were observed on the HNF1α promoter in βTC3 cells. Given the key role of the transcription factor HNF1α in pancreatic β-cell function, it can be inferred that impairment of HNF4α function by these mutations affects metabolic pathways in pancreatic β-cells and contributes to development of diabetes. Moreover, the HNF4α-mediated activation of the apolipoprotein CIII promoter in HepG2 cells was significantly impaired by D126Y and D126H mutations. These results support clinical findings that liver function can also be impaired in diabetic patients having HNF4α mutations.

Purification and characterization of two basic spermatid‐specific proteins isolated from the dog‐fish Scylliorhinus caniculus

In dog‐fish spermatid nuclei two intermediate proteins S1 and S2 replace histones before the setting down of protamines. These spermatid‐specific proteins were isolated by carboxymethyl‐cellulose chromatography and purified by high pressure liquid chromatography. S1 and S2 are characterized by a high content of basic residues and by the lack of cysteine and phenylalanine. The determination of their amino acid composition and of their N‐ and C‐terminal sequences prove that each protein corresponds to a specific molecule which can be considered neither as a histone hydrolytic product nor as a protamines precursor.

Primary structure of chicken erythrocyte histone H2A.

The complete amino acid sequence (128 residues) of the chicken erythrocyte histone H2A was deduced from the data provided by structural studies on the tryptic peptides from the maleylated histone and of the peptides obtained by thermolysin digestion of the native protein. The sequence of chicken histone H2A differs from the calf homologous histone by the deletion of one residue of histidine at position 123 or 124 and three conservative substitutions: a residue of serine replaces a residue of threonine at position 16, a residue of aspartic acid replaces a residue of glutamic acid at position 121 and a residue of alanine replaces a residue of glycine at position 128.

The G115S mutation associated with maturity-onset diabetes of the young impairs hepatocyte nuclear factor 4α activities and introduces a PKA phosphorylation site in its DNA-binding domain

HNF4alpha (hepatocyte nuclear factor 4alpha) belongs to a complex transcription factor network that is crucial for the function of hepatocytes and pancreatic beta-cells. In these cells, it activates the expression of a very large number of genes, including genes involved in the transport and metabolism of glucose and lipids. Mutations in the HNF4alpha gene correlate with MODY1 (maturity-onset diabetes of the young 1), a form of type II diabetes characterized by an impaired glucose-induced insulin secretion. The MODY1 G115S (Gly115-->Ser) HNF4alpha mutation is located in the DNA-binding domain of this nuclear receptor. We show here that the G115S mutation failed to affect HNF4alpha-mediated transcription on apolipoprotein promoters in HepG2 cells. Conversely, in pancreatic beta-cell lines, this mutation resulted in strong impairments of HNF4alpha transcriptional activity on the promoters of LPK (liver pyruvate kinase) and HNF1alpha, with this transcription factor playing a key role in endocrine pancreas. We show as well that the G115S mutation creates a PKA (protein kinase A) phosphorylation site, and that PKA-mediated phosphorylation results in a decreased transcriptional activity of the mutant. Moreover, the G115E (Gly115-->Glu) mutation mimicking phosphorylation reduced HNF4alpha DNA-binding and transcriptional activities. Our results may account for the 100% penetrance of diabetes in human carriers of this mutation. In addition, they suggest that introduction of a phosphorylation site in the DNA-binding domain may represent a new mechanism by which a MODY1 mutation leads to loss of HNF4alpha function.

Characterization and structural study of the DNA-binding protein HRm From Rhizobium meliloti.

Abstract The protein HRm, a DNA-binding HU-like protein isolated from Rhizobium meliloti , is a single polypeptide chain (Mr 9 300) of 90 residues. Protein HRm exhibits lower affinity for double stranded DNA than E. coli protein HU. The sequence of the first 54 amino acid residues was established by automated Edman degradation of the protein. Many substitutions were observed by comparison with amino acid sequences of HU-like proteins from other bacteria. However a sequence of 7 residues is almost invariable and may be important in the function of the protein.

Preparation of the DNA-binding protein HU from Escherichia coli by immuno-affinity chromatography.

The protein HU is a low molecular weight DNAbinding protein which has been characterized in Escherichia coli cells [l-3] . The isolation of this protein requires usually three steps of chromatography [ 1,2,4] , which are time-consuming and led to a low recovery of pure protein. We describe here a very selective method based on immuno-affinity chromatography to obtain in a single step highly purified protein HU. This method uses a column of purified anti-protein HU antibodies coupled to Sepharose 4B.

The DNA-binding protein II from Zymomonas mobilis. Complete amino acid sequence and interaction with DNA.

The primary structure of the DNA-binding protein II from Zymomonas mobilis has been determined from data provided by automated Edman degradation of the intact protein and of peptides derived from cleavage at aspartic acid and arginine residues. When compared with the homologous protein isolated from other bacteria, the DNA-binding protein II from Z mobilis shows many substitutions. Several non-conservative substitutions at positions usually highly conserved in this type of protein probably account for the weaker DNA-binding activity of this protein compared to that of the E coli protein.