▪ Abstract Stably folded membrane proteins reside in a free energy minimum determined by the interactions of the peptide chains with each other, the lipid bilayer hydrocarbon core, the bilayer interface, and with water. The prediction of three-dimensional structure from sequence requires a detailed understanding of these interactions. Progress toward this objective is summarized in this review by means of a thermodynamic framework for describing membrane protein folding and stability. The framework includes a coherent thermodynamic formalism for determining and describing the energetics of peptide-bilayer interactions and a review of the properties of the environment of membrane proteins—the bilayer milieu. Using a four-step thermodynamic cycle as a guide, advances in three main aspects of membrane protein folding energetics are discussed: protein binding and folding in bilayer interfaces, transmembrane helix insertion, and helix-helix interactions. The concepts of membrane protein stability that emerge p...

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MEMBRANE PROTEIN FOLDING AND STABILITY: Physical Principles

Quantitative proteome profiling using stable isotope protein tagging and automated tandem mass spectrometry (MS/MS) is an emerging technology with great potential for the functional analysis of biological systems and for the detection of clinical diagnostic or prognostic marker proteins. Owing to the enormous complexity of proteomes, their comprehensive analysis is an as-yet-unresolved technical challenge. However, biologically or clinically important information can be obtained if specific, information-rich protein classes, or sub-proteomes, are isolated and analyzed. Glycosylation is the most common post-translational modification. Here we describe a method for the selective isolation, identification and quantification of peptides that contain N-linked carbohydrates. It is based on the conjugation of glycoproteins to a solid support using hydrazide chemistry, stable isotope labeling of glycopeptides and the specific release of formerly N-linked glycosylated peptides via peptide- N-glycosidase F (PNGase F). The recovered peptides are then identified and quantified by MS/MS. We applied the approach to the analysis of plasma membrane proteins and proteins contained in human blood serum.

Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry

Histone H2AX phosphorylation on a serine four residues from the carboxyl terminus (producing gammaH2AX) is a sensitive marker for DNA double-strand breaks (DSBs). DSBs may lead to cancer but, paradoxically, are also used to kill cancer cells. Using gammaH2AX detection to determine the extent of DSB induction may help to detect precancerous cells, to stage cancers, to monitor the effectiveness of cancer therapies and to develop novel anticancer drugs.

γH2AX and cancer

Ionotropic glutamate receptors are composed of homomeric or heteromeric configurations of glutamate receptor subunits. We have cloned a member of a novel class of the rat ionotropic glutamate receptor family, termed chi-1. This subunit exhibits an average identity of 27% to NMDA subunits and 23% to non-NMDA subunits. Regional transcript levels of chi-1 are elevated just prior to and during the first postnatal week, with the highest levels present in the spinal cord, brainstem, hypothalamus, thalamus, CA1 field of the hippocampus, and amygdala. The spatial distribution of chi-1 expression is similar from postnatal day 1 (P1) to adulthood. However, transcript levels decline sharply between P7 and P14 and remain attenuated into adulthood. Functional expression studies in Xenopus oocytes injected with in vitro transcribed chi-1 RNA did not demonstrate agonist-activated currents. Pairwise expression of chi-1 with members of the AMPA, KA, or delta class of glutamate recepto subunits either failed to generate agonist-activated currents or failed to alter the underlying current generated by the coexpressed subunit. However, coexpression of chi-1 with subunits forming otherwise functional NMDA receptors resulted in an inhibition of current responses. Since chi-1 did not alter the currents generated by non-NMDA subunits, this suggests that chi-1 may specifically interact with NMDA receptor subunits. Further characterization will be required to establish the precise role of this glutamate receptor subunit in neuronal signaling.

https://www.jneurosci.org/content/jneuro/15/10/6498.full.pdf

Cloning and characterization of chi-1: a developmentally regulated member of a novel class of the ionotropic glutamate receptor family

DNA mismatch repair (MMR) is an evolutionarily conserved process that corrects mismatches generated during DNA replication and escape proofreading. MMR proteins also participate in many other DNA transactions, such that inactivation of MMR can have wide-ranging biological consequences, which can be either beneficial or detrimental. We begin this review by briefly considering the multiple functions of MMR proteins and the consequences of impaired function. We then focus on the biochemical mechanism of MMR replication errors. Emphasis is on structure-function studies of MMR proteins, on how mismatches are recognized, on the process by which the newly replicated strand is identified, and on excision of the replication error.

DNA mismatch repair

The variant histone H2AX is phosphorylated in response to UV irradiation of primary human fibroblasts in a complex fashion that is radically different from that commonly reported after DNA double-strand breaks. H2AX phosphorylation after exposure to ionizing radiation produces foci, which are detectable by immunofluorescence microscopy and have been adopted as clear and consistent quantitative markers for DNA double-strand breaks. Here we show that in contrast to ionizing radiation, UV irradiation mainly induces H2AX phosphorylation as a diffuse, even, pan-nuclear staining. UV induced pan-nuclear phosphorylation of H2AX is present in all phases of the cell cycle and is highest in S phase. H2AX phosphorylation in G1 cells depends on nucleotide excision repair factors that may expose the S-139 site to kinase activity, is not due to DNA double-strand breaks, and plays a larger role in UV-induced signal transduction than previously realized.

https://www.pnas.org/content/pnas/103/26/9891.full.pdf

H2AX phosphorylation within the G1 phase after UV irradiation depends on nucleotide excision repair and not DNA double-strand breaks

Unpaired and mispaired bases in DNA can arise by replication errors, spontaneous or induced base modifications, and during recombination. The major pathway for correction of mismatches arising during replication is the MutHLS pathway of Escherichia coli and related pathways in other organisms. MutS initiates repair by binding to the mismatch, and activates together with MutL the MutH endonuclease, which incises at hemimethylated dam sites and thereby mediates strand discrimination. Multiple MutS and MutL homologues exist in eukaryotes, which play different roles in the mismatch repair (MMR) pathway or in recombination. No MutH homologues have been identified in eukaryotes, suggesting that strand discrimination is different to E. coli. Repair can be initiated by the heterodimers MSH2-MSH6 (MutSα) and MSH2-MSH3 (MutSβ). Interestingly, MSH3 (and thus MutSβ) is missing in some genomes, as for example in Drosophila, or is present as in Schizosaccharomyces pombe but appears to play no role in MMR. MLH1-PMS1 (MutLα) is the major MutL homologous heterodimer. Again some, but not all, eukaryotes have additional MutL homologues, which all form a heterodimer with MLH1 and which play a minor role in MMR. Additional factors with a possible function in eukaryotic MMR are PCNA, EXO1, and the DNA polymerases δ and ϵ. MMR-independent pathways or factors that can process some types of mismatches in DNA are nucleotide-excision repair (NER), some base excision repair (BER) glycosylases, and the flap endonuclease FEN-1. A pathway has been identified in Saccharomyces cerevisiae and human that corrects loops with about 16 to several hundreds of unpaired nucleotides. Such large loops cannot be processed by MMR. J. Cell. Physiol. 191: 28–41, 2002. © 2002 Wiley-Liss, Inc.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1002/jcp.10077

DNA mismatch repair and mutation avoidance pathways.

γ-Seminoprotein (γ-SM), a glycoprotein from human seminal plasma, was isolated in highly purified form by ion-exchange chromatography on a Mono Q column. The main form, fraction M, was homogeneous by PAGE at pH 8.3 and by SDS-PAGE. The complete amino acid sequence of γ-SM was determined with the aid of fragments generated by cleavages with cyanogen bromide, clostripain, chymotrypsin and Staphylococcus aureus V8 protease. The fragments were aligned with overlapping sequences. The single polypeptide chain of γ-SM contains 237 amino acids with a calculated Mr of 26079. A single N-linked carbohydrate side chain is attached to Asn45. The complex structure of this oligosaccharide has been determined recently [van Halbeek H. et al. (1985) Biochem. Biophys. Res. Commun. 131, 507–514]. Sequence comparison with serine proteases shows a high degree of homology, especially with the kallikrein family. The residues in the vicinity of the active site of serine proteases are also highly conserved in γ-SM, indicating the participation of His41, Asp96 and Ser189 in its active site.



γ-SM hydrolyzed M-casein with a pH optimum at 8.0, but failed to hydrolyze any of the synthetic substrates tested. This proteolytic activity could be inhibited with diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, Zn2+ or Hg2+ ions.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1987.tb13674.x

Isolation, characterization and amino-acid sequence of gamma-seminoprotein, a glycoprotein from human seminal plasma.

Refolding of bacteriorhodopsin from expressed polypeptide fragments

Optimizing the safety and efficacy of standard chemotherapeutic agents such as cisplatin (CDDP) is of clinical relevance. Serum starvation in vitro and short-term food starvation in vivo both stress cells by the sudden depletion of paracrine growth stimulation. The effects of serum starvation on CDDP toxicity were investigated in normal and cancer cells by assessing proliferation, cell cycle distribution and activation of DNA-damage response and of AMPK, and were compared to effects observed in cells grown in serum-containing medium. The effects of short-term food starvation on CDDP chemotherapy were assessed in xenografts-bearing mice and were compared to effects on tumor growth and/or regression determined in mice with no diet alteration. We observed that serum starvation in vitro sensitizes cancer cells to CDDP while protecting normal cells. In detail, in normal cells, serum starvation resulted in a complete arrest of cellular proliferation, i.e. depletion of BrdU-incorporation during S-phase and accumulation of the cells in the G0/G1-phase of the cell cycle. Further analysis revealed that proliferation arrest in normal cells is due to p53/p21 activation, which is AMPK-dependent and ATM-independent. In cancer cells, serum starvation also decreased the fraction of S-phase cells but to a minor extent. In contrast to normal cells, serum starvation-induced p53 activation in cancer cells is both AMPK- and ATM-dependent. Combination of CDDP with serum starvation in vitro increased the activation of ATM/Chk2/p53 signaling pathway compared to either treatment alone resulting in an enhanced sensitization of cancer cells to CDDP. Finally, short-term food starvation dramatically increased the sensitivity of human tumor xenografts to cisplatin as indicated not only by a significant growth delay, but also by the induction of complete remission in 60% of the animals bearing mesothelioma xenografts, and in 40% of the animals with lung carcinoma xenografts. In normal cells, serum starvation in vitro induces a cell cycle arrest and protects from CDDP induced toxicity. In contrast, proliferation of cancer cells is only moderately reduced by serum starvation whereas CDDP toxicity is enhanced. The combination of CDDP treatment with short term food starvation improved outcome in vivo. Therefore, starvation has the potential to enhance the therapeutic index of cisplatin-based therapy.

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Thomas M. Marti

Papers

H2AX phosphorylation within the G1 phase after UV irradiation depends on nucleotide excision repair and not DNA double-strand breaks

DNA mismatch repair and mutation avoidance pathways.

Isolation, characterization and amino-acid sequence of gamma-seminoprotein, a glycoprotein from human seminal plasma.

Refolding of bacteriorhodopsin from expressed polypeptide fragments

Starvation-induced activation of ATM/Chk2/p53 signaling sensitizes cancer cells to cisplatin