Spelling suggestions: "subject:"2proteins anda 5peptides"" "subject:"2proteins anda dipeptides""
21 |
SURE PROTEIN FOR PEPTIDE CYCLIZATIONBrianne S Nunez (11185875) 26 July 2021 (has links)
<div>Cyclic peptides are important sources of medicines. </div><div>They are advantageous compared to linear peptides because they possess lower flexibility, which allows for high-affinity target binding and enhanced proteolytic stability. Unfortunately, achieving head-to-tail cyclization of peptides is quite challenging, as it is hard to control efficiency and regiospecificity of peptide macrocyclization. Many have attempted to improve peptide cyclization, including the use of different synthetic reagents as well as synthetic techniques to allow amide-bond formation and promote cyclization. While these strategies have offered great potential solutions, the aim of this study is to explore an alternative strategy that utilizes biocatalysis as a method of achieving successful peptide cyclization. Biocatalysis is the use of enzymes as natural process catalysts under artificial in vitro conditions. Biocatalysis is often more environmentally friendly and safer compared to traditional organic synthesis methods. Non-ribosomal peptide synthetases (NRPSs) are one of the major sources of cyclic peptides in nature. These are systems of large multifunctional proteins are organized into functional domains that act as an assembly line to generate peptide natural products. Normally, the thioesterase domain is responsible for hydrolysis and cyclization of the peptide. Recently, a novel cyclase (SurE) that is physically discrete from the NRPS was discovered. Based on this unique quality, we hypothesized that SurE would be easier to express compared to thioesterase domains and, for this reason, SurE could be a fantastic biocatalyst for the cyclization of peptides. To test this, we designed and generated an expression vector for SurE. We then expressed and purified the SurE protein. We also synthesized three linear peptides of varying lengths. To test for SurE activity, we attempted to add N-acetylcysteamine (SNAC) to mimic its native substrate. Unfortunately, we were unable to successfully attach the SNAC to our linear peptide. To combat this issue, a new synthesis strategy is currently being developed. This work is currently ongoing in the Parkinson lab, with the aim being to test the SurE protein, as well as other PBP-like cyclases, on other modified linear peptides and demonstrate whether the protein has the ability to cyclase a wide scope of peptides.</div><div><br></div>
|
22 |
Development of Biocompatible Polymer Monoliths for the Analysis of Proteins and PeptidesLi, Yun 12 August 2009 (has links) (PDF)
Biocompatibility is an important issue for the development of chromatographic stationary phases for the analysis of biomolecules (including proteins and peptides). A biocompatible stationary phase material is a material that resists nonspecific adsorption of biomolecules and does not interact with them in a way that would alter or destroy their structures or biochemical functions. The monolithic column format is a good alternative to typical spherical particle packed columns for capillary liquid chromatography of biomacromolecules. Several novel anion-exchange polymer monoliths for the analysis of proteins were synthesized for improved biocompatibility. Two novel polymeric monoliths were prepared in a single step by a simple photoinitiated copolymerization of 2-(diethylamino)ethyl methacrylate and polyethylene glycol diacrylate (PEGDA), or copolymerization of 2-(acryloyloxy)ethyl trimethylammonium chloride (AETAC) and PEGDA, in the presence of selected porogens. The resulting monoliths contained functionalities of diethylaminoethyl (DEAE) as a weak anion exchanger and quaternary amine as a strong anion exchanger, respectively. An alternative weak anion exchange monolith with DEAE functionalities was also synthesized by chemical modification after photoinitiated copolymerization of glycidyl methacrylate (GMA) and PEGDA. The dynamic binding capacities of the three monoliths were comparable or superior to values that have been reported for various other monoliths. Chromatographic performances were also similar to those provided by a modified poly(GMA-co-ethylene glycol dimethacrylate) monolith. Separations of standard proteins were achieved under gradient elution conditions using these monolithic columns. This work represents a successful attempt to prepare functionalized monoliths via direct copolymerization of monomers with desired functionalities. Compared to earlier publications, laborious surface modifications were avoided and the PEGDA crosslinker improved the biocompatibility of the monolithic backbone. Protein separations by capillary size exclusion chromatography (SEC) require a monolith that is biocompatible, has sufficient pore volume, has the appropriate pore size distribution, and is rigid. Most polymer monoliths have not possessed a biomodal pore-size distribution, i.e., especially with one distribution in the macropore region and the other in the mesopore region. Furthermore, non-specific adsorption of proteins in these stationary phases has persisted as a major unresolved problem. To overcome these difficulties, a porous poly[polyethylene glycol methyl ether acrylate (PEGMEA)-co-PEGDA] monolith which can resist adsorption of both acidic and basic proteins when using an aqueous buffer without any organic solvent additives was developed. Based on this biocompatible monolith, surfactants were introduced as porogens with the hope of significantly increasing the mesopore volume within the polymer. Two types of surfactants were studied, including poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) or PPO-PEO-PPO and Brij. Pore size distributions were examined using a well-defined molecular weight range series of proteins and peptides by inverse size exclusion chromatography, which indicated relatively large volume percentages of mesopores and micropores. The two new monoliths demonstrated different SEC behaviors, low nonspecific adsorption of proteins, and high mechanical rigidity. High density lipoprotein (HDL) is a heterogeneous class of lipoprotein particles with subspecies that differ in apolipoprotein and lipid composition, size, density, and charge. In this work, I developed a new capillary SEC method for size separation of native HDL particles from plasma using a capillary packed with BioSep-SEC-4000 particles, Three major sizes of HDL particles were separated. Additionally, capillary SEC and capillary strong anion-exchange chromatography of non-delipidated HDL were accomplished using poly(PEGMEA-co-PEGDA) and poly(AETAC-co-PEGDA) monoliths. These new LC methods using packed and monolithic stationary phases provided rapid separation of HDLs and excellent reproducibility.
|
23 |
HIGH-THROUGHPUT IDENTIFICATION OF ONCOGENIC TYROSINE KINASE SUBSTRATE PREFERENCES TO IMPROVE METHODS OF DETECTIONMinervo Perez (5930141) 14 January 2021 (has links)
<div>The use of computational approaches to understand kinase substrate preference has been a powerful tool in the search to develop artificial peptide probes to monitor kinase activity, however, most of these efforts focus on a small portion of the human kinome. The use of high throughput techniques to identify known kinase substrates plays an important role in development of sensitive protein kinase activity assays.</div><div>The KINATEST-ID pipeline is an example of a computational tool that uses known kinase substrate sequence information to identify kinase substrate preference. This approach was used to design three artificial substrates for ABL, JAK2 and SRC family kinases. These biosensors were used to design ELISA and lanthanide-based assays to monitor in vitro kinase activity. The KINATEST-ID pipeline relies on a high number of reported kinase substrates to predict artificial substrate sequences, however, not all kinases have the sufficient number of known substrates to make an accurate prediction. </div><div>The adaptation of kinase assay linked with phosphoproteomics technique was used to increase the number of known FLT3 kinase variant substrate sequences. Subsequently, a set of data formatting tools were developed to curate the mass spectrometry data to become compatible with a command line version of the KINATEST-ID pipeline modules. This approach was used to design seven pan-FLT3 artificial substrate (FAStides) sequences. The pair of FAStides that were deemed the most sensitive toward FLT3 kinase phosphorylation were assayed in increasing concentrations of clinically relevant tyrosine kinase inhibitors. </div><div>To improve the automation of the mass spectrometry data analysis and formatting for use with the KINATEST-ID pipeline, a streamlined process was developed within a bioinformatic platform, GalaxyP. The data formatting tools used to process the FLT3 mass spectrometry data were converted into compatible versions to execute within the GalaxyP framework. This process was used to design four BTK artificial substrates (BAStide) to monitor kinase activity. Additionally, one of the BAStide sequences was designed in the lanthanide chelating motif to develop an antibody-free activity assay for BTK. </div><div>Lastly, a multicolored time resolved lanthanide assay was designed by labeling SYK artificial substrate and a SRC family artificial substrate to measure the activity of both kinases in the same kinase reaction. This highlighted the functionality of lanthanide-based time resolved assays for potential multiplexing assay development. </div><div><br></div>
|
24 |
Clarifying the Role of the CST Complex in DNA Replication and RepairBrandon Carter Wysong (11519407) 20 December 2021 (has links)
<p>Ends of linear chromosomes are maintained by specialized
structures known as telomeres. These
structures are protected by a number of essential protein complexes including
the shelterin complex and CST (CTC1 – STN1 – TEN1) complex. CST is an RPA-like ssDNA binding protein that
is vital for telomere length maintenance <i>via</i> inhibition of telomerase
and stimulation of DNA polymerase α -primase during C-strand fill-in synthesis.
CST is also known to possess additional genome-wide
roles in regulating DNA replication and repair including helping facilitate
replication re-start at stalled forks, activating checkpoint signaling at
double-strand breaks, and promoting replication origin firing. Proper and efficient repair of DNA is
critical in order to protect the integrity of the genome and prevent extreme
mutagenesis. Telomeres have a strong
predisposition to oxidative DNA damage in the form of 8-oxoguanine caused by
exposure to reactive oxygen species and free radicals. These oxidative lesions are repaired by the
base-excision repair (BER) pathway.
Previous work has implicated telomeric proteins such as the shelterin
complex in mediating BER. Here we show
for the first time that the CST complex and individual subunits robustly
stimulate a myriad of proteins involved in the BER pathway including Pol β,
APE1, FEN1, and LIGI. CST’s ability to
augment these BER-associated proteins could be instrumental in promoting
efficient DNA repair. Additionally, we
find that CTC1 and STN1 are able to significantly enhance the polymerase
activity of Pol δ and Pol α on both random-sequence and telomeric-sequence DNA
substrates <i>in vitro</i>. What is more, we establish the ability of CST
to resolve G4 structure and promote Pol δ synthesis, which we predict is a key
feature of CST’s involvement in DNA replication at telomeres, which are known
to form replication-inhibiting G4’s. Our
results define important mechanistic insight into CST’s role in DNA replication
and repair, and provide a strong foundation for future studies relating
defective telomere maintenance to aging disorders and cancers which impact
human health.</p>
|
25 |
BIOACTIVE AND ALLERGENIC PROPERTIES OF EDIBLE CRICKET (GRYLLODES SIGILLATUS) PEPTIDESFelicia G Hall (9739430) 10 December 2020 (has links)
<p>Cardiovascular diseases (CVD) and their risk factors remain the leading cause of
morbidity and mortality in North America. Food-derived
bioactive peptides (BAP) have been shown to play a role in regulating
physiological pathways of CVD risk factors including hypertension, diabetes,
and chronic inflammation. Common sources of BAP include dairy and plant
proteins. In addition to being an alternative protein
source, it is now accepted that edible insect proteins can also confer
health benefits beyond nutrition. However, as with any novel protein source,
allergenicity remains a major concern surrounding edible insect consumption. </p>
<p>This
dissertation aimed to: 1) Evaluate the bioactive potential of peptides from an
edible cricket species and; 2) determine the effects of BAP production methods
on immunoreactivity. First, peptide-rich extracts were generated from farmed
food-grade crickets via enzymatic hydrolysis techniques with the commercial
protease Alcalase™. To measure the <i>in
vitro</i> bioavailability, cricket peptides were also subject to simulated
gastrointestinal digestion (SGD). Peptides and their digests were tested for
relevant bioactivities and active groups were further fractionated by
chromatographic methods to identify the major peptides responsible for the
bioactivity. When tested for <i>in vitro</i>
antihypertensive and anti-glycemic properties, cricket peptides were found to
inhibit the activities of angiotensin converting enzyme, dipeptidyl
peptidase-4, α-amylase, and α-glucosidase. The anti-inflammatory potential was
expounded using RAW-264.7 macrophages and human umbilical vein endothelial
cells (HUVEC). Cricket peptides (after SGD) effectively lowered NF-κB, MCP-1,
and IL-6 production in cells without affecting their viability. Proteomic
analyses identified 18 sequences from the enriched cationic peptide fraction
that showed the highest activity. Three novel peptides were identified via
molecular docking, as potent ACE-inhibitors and binding was similar to that of
the commercial drug captopril. Key binding characteristics included interaction
with hydrophobic amino acids (Phe, Pro, Leu) near the C-terminal position and
coordination with Zn (II) ions near the ACE active site.</p>
<p>Immunological
reactivity was measured by IgE-binding from shrimp-allergenic patient sera to
antigens present within cricket peptides. Our studies demonstrate that
immunoreactivity was impacted by enzymatic hydrolysis, depending on the
conditions and heating source used. Tropomyosin (a major shrimp allergen) was
extracted from both untreated crickets and protein hydrolysates, and verified
as the major reactive protein. Tropomyosin reactivity decreased (under both
partial and extensive hydrolysis) or retained (under conditions which prevented
epitope cleavage). However, using microwave-assisted enzymatic hydrolysis was
effective at decreasing tropomyosin reactivity in all immunoassays tested (IgG
and IgE). Proteomic and immunoinformatic analyses revealed prominent allergen
binding regions of cricket tropomyosin available for cleavage during enzymatic
hydrolysis. Conserved antigen regions showed greater homology with other
crustacean species, but not with other well studied allergenic insect proteins
(i.e., cockroach). Lastly, LC-MS/MS and FT-Raman spectrometry suggests that
reactivity was affected due to distinct epitope cleavage within the protein
instead of decreased antigen extractability/solubility. </p>
<p>The
findings of this dissertation support that edible cricket proteins are a
potential source of bioactive peptides for functional food or nutraceutical
development. Additionally, using protein extraction methods such as
microwave-assisted hydrolysis seems a promising tool for minimizing the
immunoreactivity of the allergen present in this edible cricket species.</p>
|
26 |
DEVELOPMENT OF CHEMICAL PROTEOMIC APPROACHES TO STUDY VIRAL ENDOCYTOSIS AND PHOSPHOPROTEOMICSMayank Srivastava (5930294) 16 August 2019 (has links)
<p>A significant
development in mass spectrometry instrumentation and software in the past
decade has led to its application in solving complex biological problems. One
of the emerging areas is Chemical Proteomics that involves design and use of
chemical reagents to probe protein functions in ‘a live cell’ environment. Another
aspect of Chemical Proteomics is the identification of target proteins of a
drug or small molecule. This is assisted by photoreactive groups, which on exposure
to UV light, covalently link the target proteins that can be purified by
affinity-based enrichment followed by mass-spectrometric identification. This
phenomenon of Photoaffinity labeling (PAL) has been widely used in a broad
range of applications. Herein, we have designed chemical tools to study Zika
endocytosis and phosphoproteomics.</p>
<p>Zika virus
has attracted the interest of researchers globally, following its outbreak in
2016. While a significant development has been made in understanding the structure
and pathogenesis, the actual mechanism of Zika entry into host cells is largely
unknown. We designed a chemical probe to tag the live virus, leading to the
identification of the virus receptors and other host factors involved in viral
entry. We further validated neural cell adhesion molecule (NCAM1) as a host
protein involved in early phase entry of Zika virus into Vero cells.</p>
<p>The second aspect is the development of the DIGE
(Difference Gel Electrophoresis) technology for phosphoproteomics. Phosphoproteins
are known to be involved in various signaling pathways and implicated in
multiple diseased states. We designed chemical reagents composed of titanium
(IV) ion, diazirine and a fluorophore, to covalently label the phosphoproteins.
Cyanine3 and cyanine5 fluorophores were employed to reveal the difference in
phosphorylation between samples for the comparative proteomics. Thus far, we have
successfully demonstrated the labeling of standard phosphoproteins in both simple
and complex protein mixtures, and the future efforts are towards applying the
technology to identify phosphoproteins in a cell lysate.</p>
|
27 |
ENGINEERING GENETICALLY ENCODED FLUORESCENT BIOSENSORS TO STUDY THE ROLE OF MITOCHONDRIAL DYSFUNCTION AND INFLAMMATION IN PARKINSON’S DISEASEStevie Norcross (6395171) 10 June 2019 (has links)
<p>Parkinson’s disease is a neurodegenerative disorder
characterized by a loss of dopaminergic neurons, where mitochondrial
dysfunction and neuroinflammation are implicated in this process. However, the
exact mechanisms of mitochondrial dysfunction, oxidative stress and
neuroinflammation leading to the onset and development of Parkinson’s disease
are not well understood. There is a lack of tools necessary to dissect these
mechanisms, therefore we engineered genetically encoded fluorescent biosensors
to monitor redox status and an inflammatory signal peptide with high
spatiotemporal resolution. To measure intracellular redox dynamics, we
developed red-shifted redox sensors and demonstrated their application in dual
compartment imaging to study cross compartmental redox dynamics in live cells.
To monitor extracellular inflammatory events, we developed a family of
spectrally diverse genetically encoded fluorescent biosensors for the
inflammatory mediator peptide, bradykinin. At the organismal level, we characterized the locomotor effects of mitochondrial toxicant-induced
dopaminergic disruption in a zebrafish animal model and evaluated a behavioral
assay as a method to screen for dopaminergic dysfunction. Pairing our
intracellular redox sensors and our extracellular bradykinin sensors in a
Parkinson’s disease animal model, such as a zebrafish toxicant-induced model will
prove useful for dissecting the role of mitochondrial dysfunction and
inflammation in Parkinson’s disease. </p>
|
28 |
HILIC-MS analysis of protein glycosylation using nonporous silicaRachel E. Jacobson (5929808) 16 January 2019 (has links)
The objective of this research is to develop and apply a HILIC UHPLC
stationary phase that allows for separation of intact glycoproteins. In
Chapter 1 I give an overview of the problems of current glycosylation
profiling with regards to biotherapeutics, and my strategy to separate
the intact glycoprotein with HILIC. Chapter 2 describes the methods used
to produce the nonporous packing material and stationary phase. In
Chapter 3 I describe previous work in developing a HILIC polyacrylamide
stationary phase, and further improvements I have made. Chapter 4
describes development of an assay in collaboration with Genentech of
therapeutic mAb glycosylation. In Chapter 5, I show HILIC-MS of digested
ribonuclease B as a beginning step to analyze glycosylated biomarkers.
|
29 |
Host ligands and oral bacterial adhesion studies on phosphorylated polypeptides and gp-340 in saliva and milk /Danielsson Niemi, Liza, January 2010 (has links)
Diss. (sammanfattning) Umeå : Umeå universitet, 2010.
|
30 |
INHIBITION OF ERYTHROCYTE BAND 3 TYROSINE PHOSPHORYLATION: CHARACTERIZATION OF A NOVEL THERAPY FOR SICKLE CELL DISEASE AND MALARIAPanae Noomuna (10716546) 29 April 2021 (has links)
While the molecular
defect that cause sickle cell disease has well been established, the cause of
vaso-occlusive crisis remains elusive and largely debated upon. Majority of
studies have linked the painful episodes to polymerization of sickle hemoglobin
following its deoxygenation. The variability of the disease symptoms among
patients, compounds efforts for a holistic therapy. Hydroxyurea, a stimulator
of Hb F induction and a widely used treatment, has ameliorated the complication
of SCD but it is only effective in 50% of the patients. Expression of Hb F
lowers the content of Hb S in blood and hence reduces oxidative stress caused
by Hb S denaturation. Sickle cell disease severity depends on several factors.
Most importantly, the ability of red cell to sickle dominates all other
determinants. While deoxygenation of sickle hemoglobin may be inevitable, the
duration with which the red cell remains in the deoxygenated state can be
manipulated. Deoxygenation is a transient process that when compared to the
time taken to develop the long filaments of deoxyhemoglobin to causes severe
sickling, the red cell would have been cycled back to the lungs and
re-oxygenated to restore the healthy conditions of the cell. In fact, if sickle
cells would flow as fast as healthy erythrocytes, the detrimental impacts of
sickling such as vaso-occlusive crisis, would not be a concern for this
disease. Unfortunately, the unstable sickle hemoglobin undergoes denaturation
through auto-oxidation, which imposes oxidative stress to the cells. The
oxidative stress inhibits erythrocytes tyrosine phosphatases, a course which
subsequently impair their constitutive action against the tyrosine kinases. In
the end, a net tyrosine phosphorylation state in the red cell membrane
proteins, most notably the transmembrane protein band 3, succeeds. Band 3
tyrosine phosphorylation abrogates the protein’s interaction with ankyrin and
spectrin-actin cytoskeleton, hence the cytoskeleton loses its major anchorage
to the membrane thus engendering membrane destabilization. A destabilized
erythrocyte sheds membrane fragments in form of microvesicles/microparticles
and discharges free hemoglobin into the extra cellular matrix. In consequence,
the microparticles power initiation of coagulation cascade through activation of
thrombin, while free Hb inflicts inflammation, scavenges nitric oxide which is
necessary for vasodilation and induces further oxidative stress within the
microvasculature, and activates expression of adhesion receptors on the
endothelium. Taken together, these events culminate in entrapment of red cells
(not naming leucocytes and platelets) in the microvasculature, blockade of
blood vessels and further damage of erythrocytes through prolonged deoxygenated
state thus terminating in tissue injury, strokes, and organ damage, amid
vaso-occlusive episodes which always require hospitalization and extensive
medical care for survival. Band 3 tyrosine phosphorylation and membrane
weakening is not unique just to SCD, but also a druggable target for malaria.
Malaria, a disease that is touted as the evolutionary cause of sickle cell
disease, surprisingly thrives through the same mechanism. Briefly, malaria
parasite consumes hemoglobin for its DNA synthesis, and in the process generate
reactive oxygen species from denatured hemoglobin that feeds into the oxidative
stress which triggers band 3 tyrosine phosphorylation. In this case however, a
destabilized membrane offers perfect conditions for merozoites’ (malaria
daughter parasites) egress/exit out of the cell to begin infecting other red
cells. Ultimately, the ensuing anemia and organ dysfunction leads to patient’s
death. Treatment of diseased cells with imatinib and other Syk inhibitors
effectively reversed membrane weakening. A stabilized membrane not only
survives longer in circulation to alleviate SCD symptoms but also traps and
starves malaria parasite leading to termination of the parasitic infection.
With band 3 tyrosine phosphorylation at center stage, this dissertation
explores the above events in an effort to unveil a novel therapy for sickle
cell and malaria diseases. First, the therapeutic strategy regarding SCD is
discussed in detail beginning with non-transfused patients and ending in
additional mechanistic study on inactivation of the principal erythrocyte’s protein
tyrosine phosphatase 1 B, PTP1B. The dissertation then provides an initial
proof of concept on efficacy of imatinib in treatment of malaria as a
monotherapy and its efficacy when used in a triple combination therapy with the
standard of care treatment. Finally, I outline an alternative possible
mechanism of action of quinine against malaria.
|
Page generated in 0.0799 seconds