PubMed Health⌕ Search

Biomedical subjects

Frederick Harris

Publications and source records attributed to Frederick Harris.

30 records · Page 2Linked to original sources
PubMed2004

Investigation of hydrophobic moment and hydrophobicity properties for transmembrane alpha-helices.

Integral membrane proteins are the primary targets of novel drugs but are largely without solved structures. As a consequence, hydrophobic moment plot methodology is often used to identify putative transmembrane alpha-helices of integral membrane proteins, based on their local maximum mean hydrophobic moment ( ) and the corresponding mean hydrophobicity ( ). To calculate these properties, the methodology identifies an optimal eleven residue window (L = 11), assuming an amino acid angular frequency, theta, fixed at 100 degrees. Using a data set of 403 transmembrane alpha-helix forming sequences, the relationship between and , and the effect of varying of L and / or theta on this relationship, was investigated. Confidence intervals for correlations between and are established. It is shown, using bootstrapping procedures that the strongest statistically significant correlations exist for small windows where 7 < or = L < or = 16. Monte Carlo analysis suggests that this correlation is dependent upon amino acid residue primary structure, implying biological function and indicating that smaller values of L give better characterisation of transmembrane sequences using . However, varying window size can also lead to different regions within a given sequence being identified as the optimal window for structure / function predictions. Furthermore, it is shown that optimal periodicity varies with window size; the optimum, based on over the range of window sizes, (7 < or = L < o= 16), was at theta = 102 degrees for the transmembrane alpha-helix data set.

Humans↗
PubMed2004

Calpains: targets of cataract prevention?

There is emerging evidence to suggest that the unregulated Ca(2+)-mediated proteolysis of essential lens proteins by calpains might be a major contributor to some forms of cataract in both animals and humans. Moreover, recently solved calpain structures have revealed molecular-level details of the activation mechanism used by these proteases, enabling the structure-based design of potent calpain inhibitors with the potential to act as anti-cataract agents. These agents offer the first real hope of an urgently needed alternative to the surgical treatment of at least some forms of cataract and relief from a life-depreciating condition on a global scale.

Animals↗
PubMed2004

Role of calpains in diabetes mellitus-induced cataractogenesis: a mini review.

Premature visual impairment due to lens opacification is a debilitating characteristic of untreated diabetes. Lens opacification is primarily due to the insolubilization of crystallins, proteins essential for lens optical properties, and recent studies have suggested that a major cause of this insolubilization may be the unregulated proteolysis of crystallins by calpains. These are intracellular cysteine proteases whose activation requires the presence of calcium (Ca2+) and elevated levels of lens Ca2+ is a condition associated with both diabetic cataractogenesis and other forms of the disorder. A number of calpains have been identified in the lens, including calpain 2, calpain 10 and two isozymes of calpain 3: Lp82 and Lp85. The use of animal hereditary cataract models have suggested that calpain 2 and/or Lp82 may be the major calpains involved in murine cataractogenesis with contributions from calpain 10 and Lp85. However, calpain 2 appears to be the major calpain involved in murine diabetic cataractogenesis and the strongest candidate of the calpains for a role in human types of cataractogenesis. Here, we present an overview of recent evidence on which these observations are based with an emphasis on the ability of calpains to proteolyse lens crystallins and calpain structural features, which appear to be involved in the Ca2+-mediated activation of these enzymes.

Calpain↗
PubMed2004

The in vitro retardation of porcine cataractogenesis by the calpain inhibitor, SJA6017.

Calpain inhibitors show the potential to serve as non-surgical alternatives in treating diabetic cataract and other types of these disorders. Here, we have tested the recently developed calpain inhibitor, SJA6017, for its ability to inhibit cataractogenesis in porcine lenses. These lenses were incubated in increasing levels of extralenticular calcium (Ca2+; 5-30 mM). Atomic absorption spectroscopy was used to determine total internal lens Ca2+ and a correlation between porcine lens Ca2+ uptake and levels of lens opacification were found with a total internal lens Ca2+ level of 5.8 microM Ca2+ g(-1) wet lens weight corresponding to the onset of catarctogenesis. A total internal lens Ca2+ level of 8.0 microM Ca2+ g(-1) wet lens weight corresponded to cataract occupying approximately 70% of the lens cell volume. This degree of cataract was reduced by approximately 40%, when SJA6017 (final concentration 0.8 microM) was included in the extralenticular medium, suggesting that the Ca2+-mediated activation of calpains may be involved in the observed opacification. Supporting this suggestion atomic absorption spectroscopy showed that the effect of SJA6017 (final concentration 0.8 microM) on lens opacification was not due to the compound restricting porcine lens Ca2+ uptake. The results indicate that calpain-induced cataractogenesis is dependent on extracellular Ca2+ and the calpain inhibitor SJA6017 (0.8 microM) had no significant effect on Ca2+ uptake by lens. Its inhibitory effect on lens opacification may be due to a direct action on the activity of calpain.

Animals↗
PubMed2003

A statistical investigation of amphiphilic properties of C-terminally anchored peptidases.

A number of DD-peptidases have been reported to interact with the membrane via C-terminal amphiphilic alpha-helices, but experimental support for this rests with a few well-characterized cases. These show the C-terminal interactions of DD-carboxypeptidases to involve high levels of membrane penetration, DD-endopeptidases to involve membrane surface binding and class C penicillin-binding proteins to involve membrane binding with intermediate properties. Here, we have characterized C-terminal alpha-helices from each of these peptidase groups according to their amphiphilicity, as measured by mean , and the corresponding mean hydrophobicity, . Regression and statistical analyses showed these properties to exhibit parallel negative linear relationships, which resulted from the spatial ordering of alpha-helix amino acid residues. Taken with the results of compositional and graphical analyses, our results suggest that the use of C-terminal alpha-helices may be a universal feature of the membrane anchoring for each of these groups of DD-peptidases. Moreover, to accommodate differences between these mechanisms, each group of C-terminal alpha-helices optimizes its structural amphiphilicity and hydrophobicity to fulfil its individual membrane-anchoring function. Our results also show that each anchor type analysed requires a similar overall balance between amphiphilicity for membrane interaction, which we propose is necessary to stabilize their initial membrane associations. In addition, we present a methodology for the prediction of C-terminal alpha-helical anchors from the classes of DD-peptidases analysed, based on a parallel linear model.

Amino Acid Sequence↗
PubMed2003

Factors determining the efficacy of alpha-helical antimicrobial peptides.

A database of alpha-helical antimicrobial peptides (AMP) was established and their minimum inhibitory concentrations (MIC) were compared with their physiochemical characteristics in an attempt to establish those features that determine efficacy. There is no significant difference in AMP sensitivity between Gram-positive and Gram-negative bacteria but fungi did require higher concentrations to achieve the same degree of growth inhibition. For antibacterial peptides there appears to be a positive correlation between MIC and hydrophobic arc size and a negative correlation between MIC and net charge.

Animals↗
PubMed2003

Is use of the hydrophobic moment a sound basis for predicting the structure-function relationships of membrane interactive alpha-helices?

Amphiphilic alpha-helices play a fundamental role in protein membrane association and show a segregation of polar and apolar amino acid residues. Based on correlations between amphiphilic properties and biological function, a number of theoretical approaches have been developed, which quantify alpha-helix amphiphilicity and then attempt to assign function. The most commonly used measure of amphiphilicity is the hydrophobic moment, < microH >, which, when used in conjunction with an alpha-helix's mean hydrophobicity, < H >, has been used to classify membrane interactive amphiphilic alpha-helices as either surface active or transmembrane. Here, the predictive efficacy of plot methodology is reviewed by examining published data, which compare the function of known membrane interactive amphiphilic alpha-helices to that assigned by this methodology. The results of this review are discussed in relation to the reliability of < microH > as a quantifier of alpha-helical amphiphilicity, and the ability of < microH > and < H > to describe alpha-helical structure / function relationships. It is concluded that hydrophobic moment plot methodology is not a generally reliable predictor of alpha-helical structure / function relationships. It appears that the inefficacy of plot methodology is primarily due to the inability of the plot diagram to accommodate the heterogeneity of the alpha-helical classes it attempts to define. However, the predictive efficacy of the methodology appears to be improved if other alpha-helical parameters are also considered when assigning alpha-helical function. It is suggested that the conventional methodology should be seen only as an indicator for the assignation of structure / function relationships, providing a guide to future experimental investigations.

Amino Acid Sequence↗
PubMed2002

Domain V of m-calpain shows the potential to form an oblique-orientated alpha-helix, which may modulate the enzyme's activity via interactions with anionic lipid.

The activity of m-calpain, a heterodimeric, Ca2+-dependent cysteine protease appears to be modulated by membrane interactions involving oblique-orientated alpha-helix formation by a segment, GTAMRILGGVI, in the protein's smaller subunit. Here, graphical and hydrophobic moment-based analyses predicted that this segment may form an alpha-helix with strong structural resemblance to the influenza virus peptide, HA2, a known oblique-orientated alpha-helix former. Fourier transform infrared spectroscopy showed that a peptide homologue of the GTAMRILGGVI segment, VP1, adopted low levels of alpha-helical structure ( approximately 20%) in the presence of zwitterionic lipid and induced a minor decrease (3 degrees C) in the gel to liquid-crystalline phase transition temperature, TC, of the hydrocarbon chains of zwitterionic membranes, suggesting interaction with the lipid headgroup region. In contrast, VP1 adopted high levels of alpha-helical structure (65%) in the presence of anionic lipid, induced a large increase (10 degrees C) in the TC of anionic membranes, and showed high levels of anionic lipid monolayer penetration (DeltaSP = 5.5 mN.m-1), suggesting deep levels of membrane penetration. VP1 showed strong haemolytic ability (LD50 = 1.45 mm), but in the presence of ionic agents, this ability, and that of VP1 to penetrate anionic lipid monolayers, was greatly reduced. In combination, our results suggest that m-calpain domain V may penetrate membranes via the adoption of an oblique-orientated alpha-helix and electrostatic interactions. We speculate that these interactions may involve snorkelling by an arginine residue located in the polar face of this alpha-helix.

Amino Acid Sequence↗
PubMed2002

Investigations into the mechanisms used by the C-terminal anchors of Escherichia coli penicillin-binding proteins 4, 5, 6 and 6b for membrane interaction.

Escherichia coli low molecular mass penicillin-binding proteins (PBPs) include PBP4, PBP5, PBP6 and PBP6b. Evidence suggests that these proteins interact with the inner membrane via C-terminal amphiphilic alpha-helices. Nonetheless, the membrane interactive mechanisms utilized by the C-terminal anchors of PBP4 and PBP6b show differences to those utilized by PBP5 and PBP6. Here, hydrophobic moment-based analyses have predicted that, in contrast to the PBP4 and PBP6b C-termini, those of PBP5 and PBP6 are candidates to form oblique orientated alpha-helices. Consistent with these predictions, Fourier transform infrared spectroscopy (FTIR) has shown that peptide homologs of the PBP4 and PBP5 C-terminal regions, P4 and P5, respectively, both possessed the ability to adopt alpha-helical structure in the presence of lipid. However, whereas P4 appeared to show a preference for interaction with the surface regions of dimyristoylglycerophosphoethanolamine and dimyristoylglycerophosphoglycerol membranes, P5 appeared to show deep penetration of both these latter membranes and dimyristoylglycerophosphocholine membranes. Based on these results, we have suggested that in contrast to the membrane anchoring of the PBP4 and PBP6b C-terminal alpha-helices, the PBP5 and PBP6 C-terminal alpha-helices may possess hydrophobicity gradients and penetrate membranes in an oblique orientation.

Amino Acid Sequence↗
PubMed2002

The gene genie?

Gene therapy has the potential to cure currently incurable conditions, including some cancers and inherited disorders. It might even be used in the womb to prevent congenital abnormalities. The potential was greeted with great excitement ten years ago, when gene therapy first appeared to be viable, but little progress is perceived. Just how close are we to solving the obstacles in the way of successful gene implantation / replacement?

Animals↗
PubMedDate not supplied

The hydrophobic moment and its use in the classification of amphiphilic structures (review).

Amphiphilic alpha-helices play a major role in membrane dependent processes and are manifested in the primary structure of a protein by the periodic appearance of hydrophobic residues. Based on these periodic sequences, the hydrophobic moment was introduced, , which essentially treats the hydrophobicity of amino acid residues as a two-dimensional vector sum and provides a measure of amphiphilicity within regular repeat structures. To identify putative amphiphilic alpha-helix forming sequences, hydrophobic moment analysis assumes an amino acid residue periodicity of 100 and scans protein primary structures to find the 11-residue window with maximal . Taken with the window's mean hydrophobicity, , hydrophobic moment plot analysis uses the coordinate pair, [ , ] to classify alpha-helices as either surface active, globular or transmembrane. More recently, this latter analysis has been extended to recognize candidate oblique orientated alpha-helices. Here, the hydrophobic moment is reviewed and data to query the logic of using a fixed window length and a fixed residue angular periodicity in hydrophobic moment analysis are provided. In addition, problems associated with the use of such analysis to predict alpha-helix structure/function relationships are considered.

Amino Acid Sequence↗