| 1. |
- Andersson, E., et al.
(författare)
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Isolation of human cationic antimicrobial protein-18 from seminal plasma and its association with prostasomes.
- 2002
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Ingår i: Human Reproduction. - : Oxford University Press (OUP). - 0268-1161 .- 1460-2350. ; 17:10, s. 34-2529
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Cathelicidins are a group of antibiotic peptides with broad antimicrobial activity. They are considered to be an essential part of the innate immune system. The only known human cathelicidin is the human cationic antimicrobial protein (hCAP-18), from which the antimicrobial peptide LL-37 is released. METHODS AND RESULTS: In the present study, we purified hCAP-18 from seminal plasma and confirmed its identity by N-terminal amino acid sequencing. Gel filtration of seminal plasma showed the presence of hCAP-18 in both a low and a high molecular weight peak. Fractions corresponding to the high molecular form of hCAP-18 also contained dipeptidyl peptidase IV (CD26), a prostasome marker. This finding suggested that hCAP-18 found in fractions corresponding to high molecular weight molecules, is prostasome-associated. Flow cytometry confirmed the association of hCAP-18 with prostasomes and indicated that the molecule is surface bound. Western blot showed the presence of intact hCAP-18 in sperm, prostasomes and ultracentrifuged seminal plasma. CONCLUSIONS: These findings suggest that hCAP-18 may have an important role in antimicrobial defence during human reproduction. The binding of hCAP-18 to prostasomes indicates that protasomes can serve as a reservoir of this precursor of the antibiotic peptide LL-37.
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| 2. |
- Arosio, Paolo, et al.
(författare)
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Quantification of the Concentration of A beta 42 Propagons during the Lag Phase by an Amyloid Chain Reaction Assay
- 2014
-
Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 136:1, s. 219-225
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Tidskriftsartikel (refereegranskat)abstract
- The aggregation of the amyloid beta peptide, A beta 42, implicated in Alzheimer's disease, is characterized by a lag phase followed by a rapid growth phase. Conventional methods to study this reaction are not sensitive to events taking place early in the lag phase promoting the assumption that only monomeric or oligomeric species are present at early stages and that the lag time is defined by the primary nucleation rate only. Here we exploit the high sensitivity of chemical chain reactions to the reagent composition to develop an assay which improves by 2 orders of magnitude the detection limit of conventional bulk techniques and allows the concentration of fibrillar A beta 42 propagons to be detected and quantified even during the lag time. The method relies on the chain reaction multiplication of a small number of initial fibrils by secondary nucleation on the fibril surface in the presence of monomeric peptides, allowing the quantification of the number of initial propagons by comparing the multiplication reaction kinetics with controlled seeding data. The quantitative results of the chain reaction assay are confirmed by qualitative transmission electron microscopy analysis. The results demonstrate the nonlinearity of the aggregation process which involves both primary and secondary nucleation events even at the early stages of the reaction during the lag-phase.
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| 3. |
- Bauer, Mikael, et al.
(författare)
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Zn2+ binding to human calbindin D(28k) and the role of histidine residues.
- 2008
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Ingår i: Protein Science. - : Wiley. - 1469-896X .- 0961-8368. ; 17:4, s. 760-767
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Tidskriftsartikel (refereegranskat)abstract
- We have studied the binding of Zn2+ to the hexa EF-hand protein, calbindin D(28k)-a strong Ca2+-binder involved in apoptosis regulation-which is highly expressed in brain tissue. By use of radioblots, isothermal titration calorimetry, and competition with a fluorescent Zn2+ chelator, we find that calbindin D(28k) binds Zn2+ to three rather strong sites with dissociation constants in the low micromolar range. Furthermore, we conclude based on spectroscopic investigations that the Zn2+-bound state is structurally distinct from the Ca2+-bound state and that the two forms are incompatible, yielding negative allosteric interaction between the zinc- and calcium-binding events. ANS titrations reveal a change in hydrophobicity upon binding Zn2+. The binding of Zn2+ is compatible with the ability of calbindin to activate myo-inositol monophosphatase, one of the known targets of calbindin. Through site-directed mutagenesis, we address the role of cysteine and histidine residues in the binding of Zn2+. Mutation of all five cysteines into serines has no effect on Zn2+-binding affinity or stoichiometry. However, mutating histidine 80 into a glutamine reduces the binding affinity of the strongest Zn2+ site, indicating that this residue is involved in coordinating the Zn2+ ion in this site. Mutating histidines 5, 22, or 114 has significantly smaller effects on Zn2+-binding affinity.
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| 4. |
- Behnen, Petra, et al.
(författare)
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Calcium-Dependent Interaction of Calmodulin with Human 80S Ribosomes and Polyribosomes.
- 2012
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 51:34, s. 6718-6727
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Tidskriftsartikel (refereegranskat)abstract
- Ribosomes are the protein factories of every living cell. The process of protein translation is highly complex and tightly regulated by a large number of diverse RNAs and proteins. Earlier studies indicate that Ca(2+) plays a role in protein translation. Calmodulin (CaM), a ubiquitous Ca(2+)-binding protein, regulates a large number of proteins participating in many signaling pathways. Several 40S and 60S ribosomal proteins have been identified to interact with CaM, and here, we report that CaM binds with high affinity to 80S ribosomes and polyribosomes in a Ca(2+)-dependent manner. No binding is observed in buffer with 6 mM Mg(2+) and 1 mM EGTA that chelates Ca(2+), suggesting high specificity of the CaM-ribosome interaction dependent on the Ca(2+) induced conformational change of CaM. The interactions between CaM and ribosomes are inhibited by synthetic peptides comprising putative CaM-binding sites in ribosomal proteins S2 and L14. Using a cell-free in vitro translation system, we further found that these synthetic peptides are potent inhibitors of protein synthesis. Our results identify an involvement of CaM in the translational activity of ribosomes.
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| 5. |
- Cedervall, Tommy, et al.
(författare)
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Food chain transport of nanoparticles affects behaviour and fat metabolism in fish.
- 2012
-
Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:2
-
Tidskriftsartikel (refereegranskat)abstract
- Nano-sized (10(-9)-10(-7) m) particles offer many technical and biomedical advances over the bulk material. The use of nanoparticles in cosmetics, detergents, food and other commercial products is rapidly increasing despite little knowledge of their effect on organism metabolism. We show here that commercially manufactured polystyrene nanoparticles, transported through an aquatic food chain from algae, through zooplankton to fish, affect lipid metabolism and behaviour of the top consumer. At least three independent metabolic parameters differed between control and test fish: the weight loss, the triglycerides∶cholesterol ratio in blood serum, and the distribution of cholesterol between muscle and liver. Moreover, we demonstrate that nanoparticles bind to apolipoprotein A-I in fish serum in-vitro, thereby restraining them from properly utilising their fat reserves if absorbed through ingestion. In addition to the metabolic effects, we show that consumption of nanoparticle-containing zooplankton affects the feeding behaviour of the fish. The time it took the fish to consume 95% of the food presented to them was more than doubled for nanoparticle-exposed compared to control fish. Since many nano-sized products will, through the sewage system, end up in freshwater and marine habitats, our study provides a potential bioassay for testing new nano-sized material before manufacturing. In conclusion, our study shows that from knowledge of the molecular composition of the protein corona around nanoparticles it is possible to make a testable molecular hypothesis and bioassay of the potential biological risks of a defined nanoparticle at the organism and ecosystem level.
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| 6. |
- Cohen, Samuel I A, et al.
(författare)
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A molecular chaperone breaks the catalytic cycle that generates toxic Aβ oligomers.
- 2015
-
Ingår i: Nature Structural & Molecular Biology. - : Springer Science and Business Media LLC. - 1545-9985 .- 1545-9993. ; 22:3, s. 207-213
-
Tidskriftsartikel (refereegranskat)abstract
- Alzheimer's disease is an increasingly prevalent neurodegenerative disorder whose pathogenesis has been associated with aggregation of the amyloid-β peptide (Aβ42). Recent studies have revealed that once Aβ42 fibrils are generated, their surfaces effectively catalyze the formation of neurotoxic oligomers. Here we show that a molecular chaperone, a human Brichos domain, can specifically inhibit this catalytic cycle and limit human Aβ42 toxicity. We demonstrate in vitro that Brichos achieves this inhibition by binding to the surfaces of fibrils, thereby redirecting the aggregation reaction to a pathway that involves minimal formation of toxic oligomeric intermediates. We verify that this mechanism occurs in living mouse brain tissue by cytotoxicity and electrophysiology experiments. These results reveal that molecular chaperones can help maintain protein homeostasis by selectively suppressing critical microscopic steps within the complex reaction pathways responsible for the toxic effects of protein misfolding and aggregation.
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| 7. |
- Covin, Michael T., et al.
(författare)
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High Resolution Structural Characterization of A beta(42) Amyloid Fibrils by Magic Angle Spinning NMR
- 2015
-
Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 137:23, s. 7509-7518
-
Tidskriftsartikel (refereegranskat)abstract
- The presence of amyloid plaques composed of amyloid beta (A beta) fibrils is a hallmark of Alzheimer's disease (AD). The A beta peptide is present as several length variants with two common alloforms consisting of 40 and 42 amino acids, denoted A beta(1-40) and A beta(1-42), respectively. While there have been numerous reports that structurally characterize fibrils of A beta(1-40), very little is known about the structure of amyloid fibrils of A beta(1-42), which are considered the more toxic alloform involved in AD. We have prepared isotopically C-13/N-13 labeled A beta(M01-42) fibrils in vitro from recombinant protein and examined their C-13-C-13 and C-13-N-15 magic angle spinning (MAS) NMR spectra. In contrast to several other studies of A beta fibrils, we observe spectra with excellent resolution and a single set of chemical shifts, suggesting the presence of a single fibril morphology. We report the initial structural characterization of A beta(M01-42) fibrils utilizing C-13 and N-15 shift assignments of 38 of the 43 residues, including the backbone and side chains, obtained through a series of cross-polarization based 2D and 3D C-13-C-13, C-13-N-15 MAS NMR experiments for rigid residues along with J-based 2D TOBSY experiments for dynamic residues. We find that the first similar to 5 residues are dynamic and most efficiently detected in a J-based TOBSY spectrum. In contrast, residues 16-42 are easily observed in cross-polarization experiments and most likely form the amyloid core. Calculation of psi and phi dihedral angles from the chemical shift assignments indicate that beta-strands are present in the fibril's secondary structure.
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| 8. |
- Cukalevski, Risto, et al.
(författare)
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Role of Aromatic Side Chains in Amyloid β-Protein Aggregation.
- 2012
-
Ingår i: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 3:12, s. 1008-1016
-
Tidskriftsartikel (refereegranskat)abstract
- Aggregation of the amyloid β-protein (Aβ) is believed to be involved in Alzheimer's disease pathogenesis. Here we have investigated the importance of the aromatic rings at positions 19 and 20 for the aggregation rate and mechanism by substituting phenylalanine with leucine. Aggregation kinetics were monitored as a function of time and peptide concentration by thioflavin T (ThT) fluorescence, the aggregation equilibrium by sedimentation assay, structural changes using circular dichroism spectroscopy and the presence of fibrillar material was detected with cryo-transmission electron microscopy. All peptides convert from monomer to amyloid fibrils in a concentration-dependent manner. Substituting F19 with leucine results in a peptide that aggregates significantly slower than the wild type, while substitution of F20 produces a peptide that aggregates faster. The effects of the two substitutions are additive, since simultaneous substitution of F19 and F20 produces a peptide with aggregation kinetics intermediate between F19L and F20L. Our results suggest that the aromatic side-chain of F19 favors nucleation of the aggregation process and may be an important target for therapeutic intervention.
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| 9. |
- Cukalevski, Risto, et al.
(författare)
-
The A beta 40 and A beta 42 peptides self-assemble into separate homomolecular fibrils in binary mixtures but cross-react during primary nucleation
- 2015
-
Ingår i: Chemical Science. - : Royal Society of Chemistry (RSC). - 2041-6539 .- 2041-6520. ; 6:7, s. 4215-4233
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Tidskriftsartikel (refereegranskat)abstract
- The assembly of proteins into amyloid fibrils, a phenomenon central to several currently incurable human diseases, is a process of high specificity that commonly tolerates only a low level of sequence mismatch in the component polypeptides. However, in many cases aggregation-prone polypeptides exist as mixtures with variations in sequence length or post-translational modifications; in particular amyloid beta (A beta) peptides of variable length coexist in the central nervous system and possess a propensity to aggregate in Alzheimer's disease and related dementias. Here we have probed the co-aggregation and cross-seeding behavior of the two principal forms of A beta, A beta 40 and A beta 42 that differ by two hydrophobic residues at the C-terminus. We find, using isotope-labeling, mass spectrometry and electron microscopy that they separate preferentially into homomolecular pure A beta 42 and A beta 40 structures during fibril formation from mixed solutions of both peptides. Although mixed fibrils are not formed, the kinetics of amyloid formation of one peptide is affected by the presence of the other form. In particular monomeric A beta 42 accelerates strongly the aggregation of A beta 40 in a concentration-dependent manner. Whereas the aggregation of each peptide is catalyzed by low concentrations of preformed fibrils of the same peptide, we observe a comparably insignificant effect when A beta 42 fibrils are added to A beta 40 monomer or vice versa. Therefore we conclude that fibril-catalysed nucleus formation and elongation are highly sequence specific events but A beta 40 and A beta 42 interact during primary nucleation. These results provide a molecular level description of homomolecular and heteromolecular aggregation steps in mixtures of polypeptide sequence variants.
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| 10. |
- Edström, Anneli M L, et al.
(författare)
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The major bactericidal activity of human seminal plasma is zinc-dependent and derived from fragmentation of the semenogelins.
- 2008
-
Ingår i: Journal of immunology. - 1550-6606. ; 181:5, s. 3413-3421
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Tidskriftsartikel (refereegranskat)abstract
- One of the major roles of seminal plasma is to provide antimicrobial protection for the spermatozoa in the female reproductive tract. We found that the bactericidal activity of seminal plasma was highest after resolution of the seminal clot and that this antibacterial activity subsequently became greatly diminished. The antibacterial activity was derived from peptides generated by fragmentation of the semenogelins while the semenogelin holoproteins displayed no antibacterial activity. After ejaculation the semenogelin-derived peptides were fragmented to smaller and smaller fragments over time and thereby lost antibacterial activity. This paralleled the loss of antibacterial activity of whole seminal plasma both in vitro and after sexual intercourse. Moreover, the antibacterial activity of the semenogelin-derived peptides generated in seminal plasma was strictly zinc-dependent both at neutral and low pH. These data provide novel roles for the resolution of seminal clots and for the high zinc concentration in human seminal plasma.
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| 11. |
- Ferreira, Silvia A., et al.
(författare)
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Biocompatibility of mannan nanogel-safe interaction with plasma proteins
- 2012
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Ingår i: Biochimica et Biophysica Acta. General Subjects. - : Elsevier BV. - 0304-4165. ; 1820:7, s. 1043-1051
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Self-assembled mannan nanogels are designed to provide a therapeutic or vaccine delivery platform based on the bioactive properties of mannan to target mannose receptor expressed on the surface of antigen-presenting cells, combined with the performance of nanogels as carriers of biologically active agents. Methods: Proteins in the corona around mannan nanogel formed in human plasma were identified by mass spectrometry after size exclusion chromatography or centrifugation followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Structural changes and time dependent binding of human apolipoprotein A-I (apoA-I) and human serum albumin (HSA) to mannan nanogel were studied using intrinsic tryptophan fluorescence and circular dichroism spectroscopy. The mannan nanogel effect on blood coagulation and fibrillation of Alzheimer's disease-associated amyloid beta peptide and hemodialysis-associated amyloidosis beta 2 microglobulin was evaluated using thrombin generation assay or thioflavin T fluorescence assay, respectively. Results: The protein corona around mannan nanogel is formed through a slow process, is quite specific comprising apolipoproteins B-100, A-I and E and HSA, evolves over time, and the equilibrium is reached after hours to days. Structural changes and time dependent binding of apoA-I and HSA to mannan nanogel are minor. The mannan nanogel does not affect blood coagulation and retards the fibril formation. Conclusions: Mannan nanogel has a high biosafety and biocompatibility, which is mandatory for nanomaterials to be used in biomedical applications. General Significance: Our research provides a molecular approach to evaluate the safety aspects of nanomaterials, which is of general concern in society and science. (C) 2012 Elsevier B.V. All rights reserved.
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| 12. |
- Frankel, Rebecca, et al.
(författare)
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Autocatalytic amplification of Alzheimer-associated Aβ42 peptide aggregation in human cerebrospinal fluid
- 2019
-
Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 2:1
-
Tidskriftsartikel (refereegranskat)abstract
- Alzheimer’s disease is linked to amyloid β (Aβ) peptide aggregation in the brain, and a detailed understanding of the molecular mechanism of Aβ aggregation may lead to improved diagnostics and therapeutics. While previous studies have been performed in pure buffer, we approach the mechanism in vivo using cerebrospinal fluid (CSF). We investigated the aggregation mechanism of Aβ42 in human CSF through kinetic experiments at several Aβ42 monomer concentrations (0.8–10 µM). The data were subjected to global kinetic analysis and found consistent with an aggregation mechanism involving secondary nucleation of monomers on the fibril surface. A mechanism only including primary nucleation was ruled out. We find that the aggregation process is composed of the same microscopic steps in CSF as in pure buffer, but the rate constant of secondary nucleation is decreased. Most importantly, the autocatalytic amplification of aggregate number through catalysis on the fibril surface is prevalent also in CSF.
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| 13. |
- Frohm, Birgitta, et al.
(författare)
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A peptide from human semenogelin I self-assembles into a pH-responsive hydrogel.
- 2015
-
Ingår i: Soft Matter. - : Royal Society of Chemistry (RSC). - 1744-6848 .- 1744-683X. ; 11:2, s. 414-421
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Tidskriftsartikel (refereegranskat)abstract
- The peptide GSFSIQYTYHV derived from human semenogelin I forms a transparent hydrogel through spontaneous self-assembly in water at neutral pH. Linear rheology measurements demonstrate that the gel shows a dominating elastic response over a large frequency interval. CD, fluorescence and FTIR spectroscopy and cryo-TEM studies imply long fibrillar aggregates of extended β-sheet. Dynamic light scattering data indicate that the fibril lengths are of the order of micrometers. Time-dependent thioflavin T fluorescence shows that fibril formation by GSFSIQYTYHV is a nucleated reaction. The peptide may serve as basis for development of smart biomaterials of low immunogenicity suitable for biomedical applications, including drug delivery and wound healing.
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| 14. |
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| 15. |
- Jonsson, Magnus, et al.
(författare)
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Binding of Semenogelin I to Intact Human Spermatozoa Studied by Flow Cytometry and Surface Plasmon Resonance
- 2010
-
Ingår i: Journal of Andrology. - : Wiley. - 0196-3635. ; 31:6, s. 560-565
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Tidskriftsartikel (refereegranskat)abstract
- Approximately 1 in 10 couples is infertile No definite cause can be found in about 25% of those cases Studies have indicated that seminal vesicle secretion functions as an optimizer of fertilization The Zn2+ binding protein semenogelin I (SgI) represents a major fraction of the proteins present in seminal vesicle fluid and it serves as a structural component of the coagulum that is formed after ejaculation Cleavage of SgI by prostate specific antigen results in liquefaction of the coagulum Fragmented SgI has antibacterial effects and inhibits spermatozoa mobility SgI has also been found complexed to eppin on spermatozoa and this complex has been suggested to be of importance for fertility Here we used flow cytometry and surface plasmon resonance to study Sgl regarding its association with spermatozoa and the interaction dependency on Zn2+ The concentration of Zn2+ in seminal plasma is approximately 100 times higher than in blood plasma and the metal ion is known to change the structure of SgI We found that Sgl binds to spermatozoa in a concentration dependent and saturable manner In solution Sgl bound to spermatozoa in a non Zn2+ dependent way whereas immobilized Sgl interacts with spermatozoa only in the presence of Zn2+ It indicates that Sgl must exhibit a specific structure or free flexibility to be able to interact with that ligand Our results indicate that the association of Sgl to spermatozoa is conformation dependent and specific These findings could constitute a basis for the development of a male contraceptive
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| 16. |
- Jonsson, Magnus, et al.
(författare)
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Semenogelins I and II bind zinc and regulate the activity of prostate-specific antigen
- 2005
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Ingår i: Biochemical Journal. - 0264-6021. ; 387:Part 2, s. 447-453
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Tidskriftsartikel (refereegranskat)abstract
- In semen. the gel proteins SgI and SgII (semenogelins I and II) are digested by PSA (prostate-specific antigen), resulting in liquefaction and release of motile spermatozoa. Semen contains a high concentration of Zn2+, which is known to inhibit the protease activity of PSA. We characterized the binding of Zn2+ to SgI and SgII and found evidence that these proteins are involved in regulating the activity of PSA. Intact SgI and SgII and synthetic semenogelin peptides were used in the experiments. Binding of Zn2+ was studied by radioligand blotting, titration with a zinc (II) fluorophore chelator and NMR analysis. A chromogenic substrate was used to measure the enzymatic activity of PSA. SgI and SgII bound Zn2+ with a stoichiometry of at least 10 cool (mol of protein)(-1) and with an average dissociation constant of approx. 5 mu M per site. Moreover, Zn2+-inhibited PSA was activated by exposure to SgI or SgII. Since both proteins have high affinity for Zn2+ and are the dominating proteins in semen, they probably represent the major Zn2+ binders in semen, one function of which may be to regulate the activity of PSA. The system is self-regulating, and PSA is maintained in an active state by its substrate.
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| 17. |
- Jonsson, Magnus, et al.
(författare)
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Truncated semenogelin I binds zinc and is cleaved by prostate-specific antigen
- 2006
-
Ingår i: Journal of Andrology. - : Wiley. - 0196-3635. ; 27:4, s. 542-547
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Tidskriftsartikel (refereegranskat)abstract
- Semenogelins I and II are major coagulum-forming proteins in semen, and they are secreted mainly by the seminal vesicles. These proteins bind Zn2+ and act as substrates for prostate-specific antigen and transglutaminase. A variant semenogelin I lacking 60 amino acids has been described that occurs in different populations with an allele frequency of 1%-3%. To better understand the function of the semenogelins in vivo, our aim was to characterize the properties of the variant form and compare with the wild type. Recombinant proteins were synthesized in insect cells, Binding of Zn2+ was studied by titration of metal ions in the presence of a zinc (11) fluorophore chelator. SDS-PAGE was used to visualize the results of cleavage by prostate-specific antigen and cross-linking with transglutaminase. We found that the truncated and wild-type semenogelin molecules had similar Zn2+-binding properties (ie, a stoichiometry of at least 9-10 mol per mol of protein and an average dissociation constant of 5 mu mol/L per site), and they showed also similar susceptibility for degradation by prostate-specific antigen. Furthermore, like the wild-type form, the truncated semenogelin I was able to serve as a substrate for transglutaminase. These findings imply that the studied characteristics do not depend on a well-defined tertiary structure, or that the deletion has no major effect on the structure responsible for these features.
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| 18. |
- Lindman, Stina, et al.
(författare)
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In vivo protein stabilization based on fragment complementation and a split GFP system.
- 2010
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Ingår i: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 107:46, s. 19826-19831
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Tidskriftsartikel (refereegranskat)abstract
- Protein stabilization was achieved through in vivo screening based on the thermodynamic linkage between protein folding and fragment complementation. The split GFP system was found suitable to derive protein variants with enhanced stability due to the correlation between effects of mutations on the stability of the intact chain and the effects of the same mutations on the affinity between fragments of the chain. PGB1 mutants with higher affinity between fragments 1 to 40 and 41 to 56 were obtained by in vivo screening of a library of the 1 to 40 fragments against wild-type 41 to 56 fragments. Colonies were ranked based on the intensity of green fluorescence emerging from assembly and folding of the fused GFP fragments. The DNA from the brightest fluorescent colonies was sequenced, and intact mutant PGB1s corresponding to the top three sequences were expressed, purified, and analyzed for stability toward thermal denaturation. The protein sequence derived from the top fluorescent colony was found to yield a 12 °C increase in the thermal denaturation midpoint and a free energy of stabilization of -8.7 kJ/mol at 25 °C. The stability rank order of the three mutant proteins follows the fluorescence rank order in the split GFP system. The variants are stabilized through increased hydrophobic effect, which raises the free energy of the unfolded more than the folded state; as well as substitutions, which lower the free energy of the folded more than the unfolded state; optimized van der Waals interactions; helix stabilization; improved hydrogen bonding network; and reduced electrostatic repulsion in the folded state.
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| 19. |
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| 20. |
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| 21. |
- Malm, Johan, et al.
(författare)
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Structural properties of semenogelin I.
- 2007
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X. ; 274:17, s. 4503-4510
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Tidskriftsartikel (refereegranskat)abstract
- The zinc-binding protein semenogelin I is the major structural component of the gelatinous coagulum that is formed in freshly ejaculated semen. Semenogelin I is a rapidly evolving protein with a primary structure that consists of six repetitive units, each comprising approximately 60 amino acid residues. We studied the secondary and tertiary structure of semenogelin I by circular dichroism (CD) spectroscopy and Trp fluorescence emission spectroscopy. Fitting to the far-UV CD data indicated that the molecule comprises 5-10%alpha-helix and 20-30%beta-sheet formations. The far-UV spectrum of semenogelin I is clearly temperature dependent in the studied range 5-90 degrees C, and the signal at 222 nm increased with increasing temperature. The presence of Zn2+ did not change the secondary structure revealed by the far-UV CD spectrum, whereas it did alter the near-UV CD spectrum, which implies that rearrangements occurred on the tertiary structure level. The conformational change induced in semenogelin I by the binding of Zn2+ may contribute to the ability of this protein to form a gel.
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| 22. |
- Malm, Johan, et al.
(författare)
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The pentraxin serum amyloid P component is found in the male genital tract and attached to spermatozoa
- 2008
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Ingår i: International Journal of Andrology. - : Wiley. - 0105-6263 .- 1365-2605. ; 31, s. 508-517
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Tidskriftsartikel (refereegranskat)abstract
- Serum amyloid P component (SAP) belongs to the pentraxin family of proteins, members of which are characterized by radial pentameric structure and calcium-dependent ligand binding. SAP is present in all types of amyloidosis and has been shown to bind to several ligands, but the physiological function of this protein has not been fully elucidated. The present study identified and characterized SAP in human semen and immunolocalized it to the male reproductive tract. SAP was also detected in seminal plasma by immunoblotting and purification by affinity chromatography followed by mass spectrometry. According to electroimmunoassay, the concentration of SAP in semen is approximately 2 mg/L, and flow cytometry revealed SAP attached to the surface of spermatozoa. Moreover, immunohistochemistry showed positive staining of spermatozoa, subsets of epithelial cells, and the stroma of accessory male genital glands and testis. Presence of mRNA supports local production of SAP, as shown with reverse transcription polymerase chain reaction. We identified SAP in a new setting - the human male reproductive system. SAP was detected on ejaculated spermatozoa, in seminal plasma and in tissue sections from the male reproductive tract. Further functional studies are needed to explain the role of SAP in human reproduction.
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| 23. |
- Meisl, Georg, et al.
(författare)
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Differences in nucleation behavior underlie the contrasting aggregation kinetics of the Aβ40 and Aβ42 peptides.
- 2014
-
Ingår i: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 111:26, s. 9384-9389
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Tidskriftsartikel (refereegranskat)abstract
- The two major forms of the amyloid-beta (Aβ) peptide found in plaques in patients suffering from Alzheimer's disease, Aβ40 and Aβ42, only differ by two amino acids in the C-terminal region, yet they display markedly different aggregation behavior. The origins of these differences have remained challenging to connect to specific molecular-level processes underlying the aggregation reaction. In this paper we use a general strategy to apply the conventional workflow of chemical kinetics to the aggregation of the Aβ40 peptide to identify the differences between Aβ40 and Aβ42 in terms of the microscopic determinants of the aggregation reaction. Our results reveal that the major source of aggregates in the case of Aβ40 is a fibril-catalyzed nucleation process, the multistep nature of which is evident through its saturation behavior. Moreover, our results show that the significant differences in the observed behavior of the two proteins originate not simply from a uniform increase in all microscopic rates for Aβ42 compared with Aβ40, but rather are due to a shift of more than one order of magnitude in the relative importance of primary nucleation versus fibril-catalyzed secondary nucleation processes. This analysis sheds light on the microscopic determinants of the aggregation behavior of the principal forms of Aβ and outlines a general approach toward achieving an understanding at the molecular level of the aberrant deposition of insoluble peptides in neurodegenerative disorders.
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| 24. |
- Meisl, Georg, et al.
(författare)
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Quantitative analysis of intrinsic and extrinsic factors in the aggregation mechanism of Alzheimer-associated Aβ-peptide.
- 2016
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6
-
Tidskriftsartikel (refereegranskat)abstract
- Disease related mutations and environmental factors are key determinants of the aggregation mechanism of the amyloid-β peptide implicated in Alzheimer's disease. Here we present an approach to investigate these factors through acquisition of highly reproducible data and global kinetic analysis to determine the mechanistic influence of intrinsic and extrinsic factors on the Aβ aggregation network. This allows us to translate the shift in macroscopic aggregation behaviour into effects on the individual underlying microscopic steps. We apply this work-flow to the disease-associated Aβ42-A2V variant, and to a variation in pH as examples of an intrinsic and an extrinsic perturbation. In both cases, our data reveal a shift towards a mechanism in which a larger fraction of the reactive flux goes via a pathway that generates potentially toxic oligomeric species in a fibril-catalyzed reaction. This is in agreement with the finding that Aβ42-A2V leads to early-onset Alzheimer's disease and enhances neurotoxicity.
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| 25. |
- Richthoff, J, et al.
(författare)
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The impact of testicular and accessory sex gland function ion sperm chromatin integrity as assessed by the sperm chromatin structure assay (SCSA)
- 2002
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Ingår i: Human Reproduction. - : Oxford University Press (OUP). - 0268-1161 .- 1460-2350. ; 17:12, s. 3162-3169
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The sperm chromatin structure assay (SCSA) provides an objective assessment of sperm chromatin integrity, which is essential for normal sperm function. SCSA is valuable as a fertility marker in epidemiological studies and in the clinical situation. Little is known about the impact of testicular and post-testicular function on SCSA parameters. METHODS: Ejaculates from 278 military conscripts of median age 18.1 (range 18-21) years were included. Levels of reproductive hormones, the length of the CAG repeat of the androgen receptor gene, sperm concentration, abstinence period and biochemical parameters of epididymal and accessory sex gland secretions were correlated to the SCSA parameters, DNA fragmentation index (DFI) and highly DNA stainable (HDS) cells. RESULTS: Negative correlations were found between sperm concentration and DFI (r = -0.119, P = 0.049) and HDS (r = -0.513, P < 0.0001). DFI was negatively correlated with levels of estradiol (r = -0.19, P = 0.002) and free testosterone (r = -0.13, P = 0.03). DFI also correlated positively with abstinence time (r = 0.17, P = 0.005), and with seminal concentrations of fructose (r = 0.18, P = 0.004) and zinc (r = 0.12, P = 0.04). CONCLUSIONS: Sex steroid production, spermatogenic function, abstinence time and seminal vesicle function appear to impact on sperm chromatin integrity and thereby on sperm fertilizing capacity. These findings may improve present understanding of the pathophysiology of male infertility.
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