| 1. |
- Al-Eryani, Yusra, et al.
(författare)
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Exploring structure and interactions of the bacterial adaptor protein YjbH by crosslinking mass spectrometry
- 2016
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Ingår i: Proteins: Structure, Function and Genetics. - : Wiley. - 0887-3585. ; 84:9, s. 1234-1245
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Tidskriftsartikel (refereegranskat)abstract
- Adaptor proteins assist proteases in degrading specific proteins under appropriate conditions. The adaptor protein YjbH promotes the degradation of an important global transcriptional regulator Spx, which controls the expression of hundreds of genes and operons in response to thiol-specific oxidative stress in Bacillus subtilis. Under normal growth conditions, the transcription factor is bound to the adaptor protein and therefore degraded by the AAA+ protease ClpXP. If this binding is alleviated during stress, the transcription factor accumulates and turns on genes encoding stress-alleviating proteins. The adaptor protein YjbH is thus a key player involved in these interactions but its structure is unknown. To gain insight into its structure and interactions we have used chemical crosslinking mass spectrometry. Distance constraints obtained from the crosslinked monomer were used to select and validate a structure model of YjbH and then to probe its interactions with other proteins. The core structure of YjbH is reminiscent of DsbA family proteins. One lysine residue in YjbH (K177), located in one of the α-helices outside the thioredoxin fold, crosslinked to both Spx K99 and Spx K117, thereby suggesting one side of the YjbH for the interaction with Spx. Another lysine residue that crosslinked to Spx was YjbH K5, located in the long and presumably very flexible N-terminal arm of YjbH. Our crosslinking data lend support to a model proposed based on site-directed mutagenesis where the YjbH interaction with Spx can stabilize and present the C-terminal region of Spx for protease recognition and proteolysis. Proteins 2016; 84:1234–1245.
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| 2. |
- Ampah-Korsah, Henry, et al.
(författare)
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The aquaporin splice variant NbXIP1;1α is permeable to boric acid and is Phosphorylated in the N-terminal domain
- 2016
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Ingår i: Frontiers in Plant Science. - : Frontiers Media SA. - 1664-462X. ; 7:JUNE2016
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Tidskriftsartikel (refereegranskat)abstract
- Aquaporins (AQPs) are membrane channel proteins that transport water and uncharged solutes across different membranes in organisms in all kingdoms of life. In plants, the AQPs can be divided into seven different subfamilies and five of these are present in higher plants. The most recently characterized of these subfamilies is the XIP subfamily, which is found in most dicots but not in monocots. In this article, we present data on two different splice variants (α and β) of NbXIP1;1 from Nicotiana benthamiana. We describe the heterologous expression of NbXIP1;1α and β in the yeast Pichia pastoris, the subcellular localization of the protein in this system and the purification of the NbXIP1;1α protein. Furthermore, we investigated the functionality and the substrate specificity of the protein by stopped-flow spectrometry in P. pastoris spheroplasts and with the protein reconstituted in proteoliposomes. The phosphorylation status of the protein and localization of the phosphorylated amino acids were verified by mass spectrometry. Our results show that NbXIP1;1α is located in the plasma membrane when expressed in P. pastoris, that it is not permeable to water but to boric acid and that the protein is phosphorylated at several amino acids in the N-terminal cytoplasmic domain of the protein. A growth assay showed that the yeast cells expressing the N-terminally His-tagged NbXIP1;1α were more sensitive to boric acid as compared to the cells expressing the C-terminally His-tagged isoform. This might suggest that the N-terminal His-tag functionally mimics the phosphorylation of the N-terminal domain and that the N-terminal domain is involved in gating of the channel.
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| 3. |
- Awad, Wael, et al.
(författare)
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Structural and Biophysical Characterization of Human EXTL3 : Domain Organization, Glycosylation, and Solution Structure
- 2018
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 57:7, s. 1166-1177
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Tidskriftsartikel (refereegranskat)abstract
- Heparan sulfate proteoglycans are proteins substituted with one or more heparan sulfate (HS) polysaccharides, found in abundance at cell surfaces. HS chains influence the activity of many biologically important molecules involved in cellular communication and signaling. The exostosin (EXT) proteins are glycosyltransferases in the Golgi apparatus that assemble HS chains on HSPGs. The EXTL3 enzyme mainly works as an initiator in HS biosynthesis. In this work, human lumenal N-glycosylated EXTL3 (EXTL3ΔN) was cloned, expressed in human embryonic kidney cells, and purified. Various biophysical and biochemical approaches were then employed to elucidate the N-glycosylation sites and the function of their attached N-glycans. Furthermore, the stability and conformation of the purified EXTL3ΔN protein in solution have been analyzed. Our data show that EXTL3ΔN has N-glycans at least at two positions, Asn290 and Asn592, which seem to be critical for proper protein folding and/or release. EXTL3ΔN is quite stable, as high temperature (∼59 °C) was required for denaturation. Deconvolution of the EXTL3ΔN far-UV CD spectrum revealed a substantial fraction of β sheets (25%) with a minor proportion of α-helices (14%) in the secondary structure. Solution small-angle X-ray scattering and dynamic light scattering revealed an extended structure suggestive of a dimeric arrangement and consisting of two distinct regions, narrow and broad, respectively. This is consistent with bioinformatics analyses suggesting a 3-domain structure with two glycosyltransferase domains and a coiled-coil domain.
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| 4. |
- Bllaci, Loreta, et al.
(författare)
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Fast Surface Acoustic Wave-Matrix-Assisted Laser Desorption Ionization Mass Spectrometry of Cell Response from Islets of Langerhans.
- 2013
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 85:5, s. 2623-2629
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Tidskriftsartikel (refereegranskat)abstract
- A desire for higher speed and performance in molecular profiling analysis at a reduced cost is driving a trend in miniaturization and simplification of procedures. Here we report the use of a surface acoustic wave (SAW) atomizer for fast sample handling in matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) peptide and protein profiling of Islets of Langerhans, for future type 2 diabetes (T2D) studies. Here the SAW atomizer was used for ultrasound (acoustic) extraction of insulin and other peptide hormones released from freshly prepared islets, stimulated directly on a membrane. A high energy propagating SAW atomizes the membrane-bound liquid into approximately 2 μm diameter droplets, rich in cell-released molecules. Besides acting as a sample carrier, the membrane provides a purification step by entrapping cell clusters and other impurities within its fibers. A new SAW-based sample-matrix deposition method for MALDI MS was developed and characterized by a strong insulin signal, and a limit of detection (LOD) lower than 100 amol was achieved. Our results support previous work reporting the SAW atomizer as a fast and inexpensive tool for ultrasound, membrane-based sample extraction. When interfaced with MALDI MS, the SAW atomizer constitutes a valuable tool for rapid cell studies. Other biomedical applications of SAW-MALDI MS are currently being developed, aiming at fast profiling of biofluids. The membrane sampling is a simplistic and noninvasive collection method of limited volume biofluids such as the gingival fluid and the tearfilm.
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| 5. |
- Butler, Daniel S.C., et al.
(författare)
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A bacterial protease depletes c-MYC and increases survival in mouse models of bladder and colon cancer
- 2021
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Ingår i: Nature Biotechnology. - : Springer Science and Business Media LLC. - 1087-0156 .- 1546-1696. ; 39:6, s. 754-764
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Tidskriftsartikel (refereegranskat)abstract
- Is the oncogene MYC upregulated or hyperactive? In the majority of human cancers, finding agents that target c-MYC has proved difficult. Here we report specific bacterial effector molecules that inhibit cellular MYC (c-MYC) in human cells. We show that uropathogenic Escherichia coli (UPEC) degrade the c-MYC protein and attenuate MYC expression in both human cells and animal tissues. c-MYC protein was rapidly degraded by both cell-free bacterial lysates and the purified bacterial protease Lon. In mice, intravesical or peroral delivery of Lon protease delayed tumor progression and increased survival in MYC-dependent bladder and colon cancer models, respectively. These results suggest that bacteria have evolved strategies to control c-MYC tissue levels in the host and that the Lon protease shows promise for therapeutic targeting of c-MYC in cancer.
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| 6. |
- De Marchi, Tommaso, et al.
(författare)
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Proteomic profiling reveals that ESR1 mutations enhance cyclin-dependent kinase signaling
- 2024
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Ingår i: Scientific Reports. - 2045-2322. ; 14, s. 1-16
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Tidskriftsartikel (refereegranskat)abstract
- Three quarters of all breast cancers express the estrogen receptor (ER, ESR1 gene), which promotes tumor growth and constitutes a direct target for endocrine therapies. ESR1 mutations have been implicated in therapy resistance in metastatic breast cancer, in particular to aromatase inhibitors. ESR1 mutations promote constitutive ER activity and affect other signaling pathways, allowing cancer cells to proliferate by employing mechanisms within and without direct regulation by the ER. Although subjected to extensive genetic and transcriptomic analyses, understanding of protein alterations remains poorly investigated. Towards this, we employed an integrated mass spectrometry based proteomic approach to profile the protein and phosphoprotein differences in breast cancer cell lines expressing the frequent Y537N and Y537S ER mutations. Global proteome analysis revealed enrichment of mitotic and immune signaling pathways in ER mutant cells, while phosphoprotein analysis evidenced enriched activity of proliferation associated kinases, in particular CDKs and mTOR. Integration of protein expression and phosphorylation data revealed pathway-dependent discrepancies (motility vs proliferation) that were observed at varying degrees across mutant and wt ER cells. Additionally, protein expression and phosphorylation patterns, while under different regulation, still recapitulated the estrogen-independent phenotype of ER mutant cells. Our study is the first proteome-centric characterization of ESR1 mutant models, out of which we confirm estrogen independence of ER mutants and reveal the enrichment of immune signaling pathways at the proteomic level.
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| 7. |
- Don-Doncow, Nicholas, et al.
(författare)
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Galiellalactone is a Direct Inhibitor of STAT3 in Prostate Cancer Cells.
- 2014
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 289:23, s. 15969-15978
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Tidskriftsartikel (refereegranskat)abstract
- The transcription factor STAT3 is constitutively active in several malignancies including castration-resistant prostate cancer and has been identified as a promising therapeutic target. The fungal metabolite galiellalactone, a STAT3 signaling inhibitor, inhibits the growth, both in vitro and in vivo, of prostate cancer cells expressing active STAT3 and induces apoptosis of prostate cancer stem cell-like cells expressing pSTAT3. However, the molecular mechanism of this STAT3 inhibiting effect by galiellalactone has not been clarified. A biotinylated analogue of galiellalactone (GL-biot) was synthesized to be used for identification of galiellalactone target proteins. By adding streptavidin-sepharose beads to GL-biot treated DU145 cell lysates, STAT3 was isolated and identified as a target protein. Confocal microscopy revealed GL-biot in both the cytoplasm and nucleus of DU145 cells treated with GL-biot, appearing to co-localize with STAT3 in the nucleus. Galiellalactone inhibited STAT3 binding to DNA in DU145 cell lysates without affecting phosphorylation status of STAT3. Mass spectrometry analysis of recombinant STAT3 protein pretreated with galiellalactone revealed three modified cysteines (cys-367, cys-468 and cys-542). We here demonstrate with chemical and molecular pharmacological methods that galiellalactone is a cysteine reactive inhibitor that covalently binds to one or more cysteines in STAT3 and that this leads to inhibition of STAT3 binding to DNA and thus blocks STAT3 signaling without affecting phosphorylation. This further validates galiellalactone as a promising direct STAT3 inhibitor for treatment of castration-resistant prostate cancer.
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| 8. |
- Gonzalez, Henrik, et al.
(författare)
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Identification of novel candidate protein biomarkers for the post-polio syndrome — Implications for diagnosis, neurodegeneration and neuroinflammation
- 2009
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Ingår i: Journal of Proteomics. - : Elsevier BV. - 1874-3919 .- 1876-7737. ; 71:6, s. 670-681
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Tidskriftsartikel (refereegranskat)abstract
- Survivors of poliomyelitis often develop increased or new symptoms decades after the acute infection, a condition known as post-polio syndrome (PPS). The condition affects 20-60% of previous polio patients, making it one of the most common causes of neurological deficits worldwide. The underlying pathogenesis is not fully understood and accurate diagnosis is not feasible. Herein we investigated whether it was possible to identify proteomic profile aberrations in the cerebrospinal fluid (CSF) of PPS patients. CSF from 15 patients with well-defined PPS were analyzed for protein expression profiles. The results were compared to data obtained from nine healthy controls and 34 patients with other non-inflammatory diseases which served as negative controls. In addition, 17 samples from persons with secondary progressive multiple sclerosis (SPMS) were added as relevant age-matched references for the PPS samples. The CSF of persons with PPS displayed a disease-specific and highly predictive (p=0.0017) differential expression of five distinct proteins: gelsolin, hemopexin, peptidylglycine alpha-amidating monooxygenase, glutathione synthetase and kallikrein 6, respectively, in comparison with the control groups. An independent ELISA confirmed the increase of kallikrein 6. We suggest that these five proteins should be further evaluated as candidate biomarkers for the diagnosis and development of new therapies for PPS patients.
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| 9. |
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| 10. |
- Hartman, Erik, et al.
(författare)
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Bioinformatic Analysis of the Wound Peptidome Reveals Potential Biomarkers and Antimicrobial Peptides
- 2021
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Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- Wound infection is a common and serious medical condition with an unmet need for improved diagnostic tools. A peptidomic approach, aided by mass spectrometry and bioinformatics, could provide novel means of identifying new peptide biomarkers for wound healing and infection assessment. Wound fluid is suitable for peptidomic analysis since it is both intimately tied to the wound environment and is readily available. In this study we investigate the peptidomes of wound fluids derived from surgical drainages following mastectomy and from wound dressings following facial skin grafting. By applying sorting algorithms and open source third party software to peptidomic label free tandem mass spectrometry data we provide an unbiased general methodology for analyzing and differentiating between peptidomes. We show that the wound fluid peptidomes of patients are highly individualized. However, differences emerge when grouping the patients depending on wound type. Furthermore, the abundance of peptides originating from documented antimicrobial regions of hemoglobin in infected wounds may contribute to an antimicrobial wound environment, as determined by in silico analysis. We validate our findings by compiling literature on peptide biomarkers and peptides of physiological significance and cross checking the results against our dataset, demonstrating that well-documented peptides of immunological significance are abundant in infected wounds, and originate from certain distinct regions in proteins such as hemoglobin and fibrinogen. Ultimately, we have demonstrated the power using sorting algorithms and open source software to help yield insights and visualize peptidomic data.
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