Terje Lund

On the presence of two new high mobility group-like proteins in HeLa S3 cells

Two phosphorylated HMG‐like proteins with M r ≈ 10 000 have been isolated from HeLa S3 cells, one being present in metaphase and one in interphase cells. The amino acid compositions of these proteins are very similar but differ from the known HMG proteins. However, they exhibit similarities being rich in proline, basic and acidic amino acids. A possible role in chromatin condensation of the HMG‐like protein characteristic for metaphase cells is suggested.

On the phosphorylation of low molecular mass HMG (high mobility group) proteins in Ehrlich ascites cells.

This paper shows that the low molecular mass HMG proteins 14 and 17 do not seem to be phosphorylated in Ehrlich ascites cells whereas two other small HMG proteins designated HMG I and Y are. Amino acid analysis and peptide mapping of all four proteins demonstrated that HMG I and Y were not phosphorylated modifications of HMG 14 or 17.

Low Levels of Raf Kinase Inhibitory Protein in Growth Hormone-Secreting Pituitary Adenomas Correlate with Poor Response to Octreotide Treatment

CONTEXT Excessive GH production by pituitary tumors causes acromegaly. Medical treatment of acromegaly with somatostatin analogs (SMSs), like octreotide, is well established, but the clinical effect is variable. One mechanism for octreotide effect is inhibition of the MAPK signaling pathway after binding to the G protein-coupled somatostatin receptor. Nonphosphorylated Raf kinase inhibitory protein (RKIP) binds to and inhibits Raf1 kinase, and thereby attenuates MAPK signaling, whereas phosphorylated RKIP inhibits G protein receptor internalization and degradation due to inhibition of G protein receptor kinase 2. OBJECTIVE Our objective was to study RKIP levels in pituitary somatotroph adenomas, and relate them to clinical characteristics and response to octreotide treatment in patients with acromegaly. PATIENTS AND METHODS RKIP level was analyzed by Western blot of proteins extracted from somatotroph tumors frozen a short time after surgery in 51 patients with active acromegaly. An acute somatostatin test was performed in 46 of the patients, and in 21 the IGF-I level before and 6 months after SMS treatment was available. RESULTS The adenoma RKIP level correlated significantly to both the acute and the long-term octreotide responses on serum levels of GH and IGF-I, respectively. In multiple regression analyses, the RKIP level was a significant determinant for both the GH reduction in the acute test and the IGF-I reduction after approximately 6 months. CONCLUSION The RKIP level in somatotroph adenomas seems to be important for the clinical effect of SMS treatment, in which low levels of RKIP correlate to poor clinical response to SMSs.

The human chromosomal protein HMG I contains two identical palindrome amino acid sequences

The sequence of 105 amino acids of the human high mobility group chromosomal protein HMG I has been determined. The most striking feature of this sequence is two identical palindrome sequences: pro-arg-gly-arg-pro, which together with a third related sequence: gly-arg-pro-arg, may represent the binding sites of HMG I to clusters of A-T base pairs in DNA.

Fibrin(ogen) may be an important target for methylglyoxal-derived AGE modification in elastic arteries of humans

Diabetes is associated with increased risk of cardiovascular disease. Advanced glycation end-products (AGEs) are considered to be a major pathogenic factor for diabetic vascular complications. The levels of AGEs are increased in diabetic patients. We have studied the presence of the major AGE methylglyoxal (MGO)-derived hydroimidazolone in human aorta and carotid arteries, using immunohistochemistry (IHC), western blotting and mass spectrometry. By IHC, MGO-derived modifications were detected mainly associated with cells in intimal thickenings and cells in microvessels in adventitia. In type V lesions MGO-derived AGE was also present, extracellular in the necrotic core and in cells at the border of the core. The highest degree of modification was probably associated with cell nuclei. By western blotting and mass spectrometry fibrin(ogen), the cytoskeleton-associated protein moesin and the nuclear proteins lamin A and C were identified as putative main targets for MGO-derived modification. LC-MS/MS studies of fibrin(ogen) modified in vitro with low concentrations of MGO identified the sites that were most prone to modification. These results indicate that AGE modifications occur preferentially on specific proteins. The modification of these proteins may play a role in vascular dysfunction and development of atherosclerosis in diabetes.

The metaphase specific phosphorylation of HMG I

In vivo labelling of HeLa cells arrested in metaphase with [32P]-phosphate and in vitro phosphorylation of HMG I with the partially purified growth associated H1 kinase was used to study metaphase specific phosphorylation of HMG I. It was found that threonine 53 and 78 became phosphorylated. These amino acids are embedded in respectively the sequence PTPKR and TPGRK which are similar to the sequences phosphorylated by the growth associated H1 kinase.

On the presence of the chromosomal proteins HMG I and HMG Y in rat organs.

Using antiserum raised against HMG I, we have shown that HMG I and HMG Y are present in perchloric acid extracts of kidney, lung, heart, brain, liver and intestine in the rat, suggesting that the expression of these proteins may not be dependent upon proliferative activity. The results also show that the ratio between HMG I and HMG Y varies between different organs.

Metaphase-specific* phosphorylations weaken the association between chromosomal proteins HMG 14 and 17, and DNA

The high‐mobility‐group proteins HMG 14 and 17 have been isolated from human cells arrested in metaphase. The affinity between an unphosphorylated and two phosphorylated forms of these proteins, and DNA has been investigated using columns of single‐stranded and double‐stranded DNA. It was shown that the most phosphorylated forms had much lower affinity for single‐stranded and double‐stranded DNA compared to the unphosphorylated form present in interphase cells. The results are in accordance with the view that HMG 14 and 17 may dissociate transiently from chromatin during mitosis.

The amino acid sequence of the chromosomal protein HMG-Y, its relation to HMG-I and possible domains for the preferential binding of the proteins to stretches of A-T base pairs

The primary structure of the human high mobility group (HMG) protein HMG-Y has been established except for a few amino acids in the N-terminal and the C-terminal part of the protein. It was found that the sequence was identical to that of HMG-I except for a run of eleven amino acids. Like HMG-I the protein was N-terminally blocked and the palindromic sequence Pro-Arg-Gly-Arg-Pro occurred twice as in HMG-I. The binding of peptides derived from HMG-I (after thermolysin cleavage) to poly (dA-dT).poly(dA-dT) suggested that there are at least two different binding domains in the protein and that binding is not dependent upon an intact protein.

HMG 17 in metaphase-arrested and interphase HeLa S3 cells

Proteins HMG 17 and 14 belong to a group of proteins designated high mobility group proteins [ 11. Experiments have indicated that transcribed genes are enriched in HMG 17 and 14 or the homologous protein H6 [2-51. The exact location of these proteins on the nucleosomes of transcribed sequences has not yet been settled. Certain experiments indicate that in the transcribed regions HMG 17 and 14 replace Hl , and it has been suggested that this results in a local unfolding of chromatin, thereby facilitating transcription [6]. On the other hand, these proteins may not only play a role during transcription since they remain attached to extensively digested nucleosomes [6] and reconstitution experiments [7,8] indicate that HMG 17 and 14 readily bind to the 145 base pair core particle at a stoichiometry of 1 and 2 molecules/core. 2.1. Propagation of HeLa S3 cells Interphase cells were grown in suspension culture as in [9]. To obtain cells arrested in metaphase, cells were grown in roller tubes and harvested after treatment with colcemid (0.05 pg/ml) as in [9]; 95% of the cells were in metaphase.