Browsing by Subject "biochemistry"

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  • Raulinaitis, Vytautas (Helsingin yliopisto, 2020)
    Staphylococcus aureus is a pervasive pathogen, whose infections frequently result in serious medical complications and death. Its encounters are yet more perilous in clinical settings where professional care and financial resources alone do not suffice to ensure successful treatment results. The virulence of the bacteria is enforced by numerous cellular mechanisms that have allowed it to develop resistance to every drug used to this date. The bacterial cell wall (CW) is the primary line of defense, the most common target in treatment strategies, and is likely to remain the prioritized candidate for future therapeutic solutions. The main structural component of bacterial CW is peptidoglycan (PG) that forms protective layers. PG is administered by a large number of enzymes that are involved in its synthesis, maintenance, and cleavage. One family of enzymes, M23 peptidases, cleaves pentaglycine bridges that link chains of PG and are specific to S. aureus. These enzymes can be used by the bacteria to manage its own PG in a controlled manner or, alternatively, by hostile microorganisms and cause cell death. Therefore, M23 peptidases of S. aureus are important as potential targets for drugs as well as pharmacological tools themselves that are already employed by nature. Substrate recognizing SH3b domains enhance the effectiveness of M23 endopeptidases. Previous research had identified a putative M23 peptidase gene, transcription of which is upregulated under S. aureus exposure to compounds harmful to cell wall. We examined and characterized the product of the gene. The protein, which we named LytU, is an M23 family zinc-dependent enzyme that cleaves pentaglycine. It is anchored in plasma membrane and is extracytoplasmic, residing in a periplasm-like space. The physiological role of LytU is not confirmed, but evidence suggest it can recycle PG fragments and participate in daughter cell separation. A distinct feature of the enzyme is its ability to strongly bind a second zinc ion, which incapacitates catalytic residues. We propose that together with pH, the binding of second ion serves a regulatory function in situ. Solution structure of the LytU catalytic domain has been determined. Binding of substrate pentaglycine to catalytic M23 domain is very transient at least in vitro. The binding, nevertheless, is accomplished by SH3b domain of enzymes bearing it. Contrarily to previous beliefs, we found that SH3b domain binding to substrate is primarily driven by interactions with PG branching peptides, rather than by weaker interaction with pentaglycine. The binding of SH3b to substrate is independent of catalytic domain and it targets and binds the PG peptide moieties that are proximal to but different from the pentaglycine cleaved by catalytic domain. In summary, we have introduced and characterized a new M23 family endopeptidase, proposed a regulation mechanism, and changed the paradigm of substrate binding by M23 peptidases. Our results are expected to contribute to a better understanding of S. aureus physiology and provide means for the development of cures.
  • Kulesskiy, Evgeny (Helsingin yliopisto, 2015)
    Syndecans are cell surface heparan sulfate proteoglycans which are present in all tissues and cell types and have distinct temporal and spatial expression patterns. They play important roles in embryonic development of the organism and control relocation and alteration of extracellular matrix components. Syndecans regulate cell migration, adhesion and proliferation and are engaged in tissue injury, inflammation processes, pathogenesis of infectious diseases and tumor biology. This thesis summarizes the results of studies on one of the syndecan family receptors syndecan-3 (also known as N-syndecan). This proteoglycan is abundantly expressed in developing brain. Syndecan-3 acts as a signaling receptor upon binding of its ligand, heparin-binding growth associated molecule (HB-GAM; also known as pleiotrophin), which activates the cortactin c-Src signaling pathway. This leads to rapid neurite extension in neuronal cells, which makes syndecan-3 an interesting transmembrane receptor in neuronal development and regeneration. However, little is known about the signaling mechanism of syndecan-3. Here I show formation of ligand-syndecan-3 signaling complexes at the cell surface using fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET). Ligand binding leads to dimerization of syndecan-3 at the cell surface. The dimerized syndecan-3 colocalizes with actin in the filopodia of cells. Lysine 383 in the juxtamembrane (ERKE) sequence and G392 and G396 from GXXXG canonical motif are shown to be important for the ligand-induced dimerization, whereas the cytosolic domain are not required for the dimerization. In addition to acting as a signaling receptor, syndecan-3 acts as a co-receptor in epidermal growth factor receptor (EGFR) ligand binding. FRET analysis suggests that interactions of syndecan-3 and EGFR depend on a shared ligand such as heparin-binding EGF-like growth factor (HB-EGF). Furthermore, it was shown that syndecan-3 may act as a receptor for other ligands, like glial cell line-derived neurotrophic factor (GDNF). In addition, I have found a new receptor for HB-GAM glypican-2 which may be involved in regulation of HB-GAM signaling by competing with syndecan-3 for ligand binding.
  • Cypryk, Wojciech (Helsingin yliopisto, 2016)
    Extracellular vesicles (EVs) are small, membranous entities secreted from most eukaryotic cells at both homeostatic and stress conditions. Carrying active biological molecules nucleic acids, lipids and proteins EVs serve as important means of intercellular communication. In the immune system, EVs circulting in body fluids play important modulatory roles in coordination of responses. EVs are integral part of secretome all proteins secreted by cells. EVs provide means for secretion of proteins that are not trafficked through conventional, ER/Golgi-mediated mechanisms. Analysis of EV proteome provides basis for fundamental discoveries in understanding the biogenesis, secretion and delivery of these nanosized messengers to target cells. Macrophages are principal tissue-resident effector cells of innate immune system, performing surveillance of their neighborhood in search for danger. Macrophages express pattern recognition receptors (PRRs) which recognize conserved pathogen-associated molecular patterns (PAMPs) conserved range of molecules expressed by pathogens. PRRs also recognize endogeneous ligands that appear in the human body in other dangerous conditions and diseases. These are collectively called danger-associated molecular patterns (DAMPs). Recognition of PAMPs and DAMPs by PRRs triggers intracellular signaling, leading to activation of macrophage defense mechanisms. These begin with inflammation and secretion of pro-inflammatory cytokines, but many other proteins are also actively secreted by activated macrophages. Although inflammatory cytokine secretion is a well-studied process, very few studies investigating overall and EV-mediated protein secretion from human macrophages were performed. This thesis aims at broadening our understanding of EV-mediated protein secretion from human macrophages activated in response to selected innate immune activators, namely extracellular adenosine triphosphate (ATP), a potent DAMP released during cell damage; (1,3)-β-glucan, a polysaccharide component of fungal cell wall, activating dectin-1-mediated signaling and influenza A virus (IAV) - common seasonal pathogenic virus targeting lung epithelia and macrophages. Total secretome and purified EVs were analyzed using different high-throughput mass spectrometry-based methods and further explored using bioinformatic and biochemical studies. The presented results provide evidence that EV-mediated protein secretion is an integral part of responses of human macrophages to studied stimuli. Its activation is demonstrated with quantitative and comparative proteomic approaches. The secreted vesicles are characterized by a broad size range consistent with both exosomes and microvesicles, demonstrating that both types of vesicular structures are involved in protein secretion. The EVs carry distinct set of immunologically important proteins, and bioinformatic analysis suggests that the secreted EVs may exert immunomodulatory effects on recipient cells. It is shown that during IAV infection, EVs are mediators of pro-inflammatory and antiviral cytokine release, thus they may serve as protective capsules of targeted cytokine delivery. Proteomic analysis identified also a broad set of DAMPs unconventionally secreted in association with EVs, further extrapolating their function towards danger signaling in cellular immune responses. The study on ATP-mediated responses further investigates the intracellular signaling involving calpains, abundant cytosolic proteases, identifying their crucial roles downstream P2X7 receptor: in EV release, as well as inflammasome activation and IL-1β secretion. The data presented here indicate that EVs serve as unconventional means for secretion of a broad range of proteins secreted in PRR-mediated responses in human macrophages. Bioinformatic and functional analysis identifies potential processes involved in their generation as well as their roles in intercellular communication. Together, the presented thesis contributes to our understanding of unconventional, EV-mediated protein secretion in macrophage responses towards common, physiologically releveant threats. The studies presented here will serve as basis for further detailed functional analysis of the roles of EVs in communication between macrophages and other immune system cells. It will also lay the grounds for future studies involving EV-mediated macrophage responses in patients with fungal and viral infections.
  • Batchu, Krishna chaithanya (Helsingin yliopisto, 2016)
    The aim of this thesis was to study the substrate specificity of A-type phospholipases (PLAs) that belong to different sub-families in order to understand the key factors that regulate their activity. The experimental part of this thesis consists of three studies each one focusing on PLAs that belong to a specific subgroup. In the first study, we developed a mass-spectrometric (MS) assay and implemented it to study in detail the effect of acyl chain length and unsaturation of glycerophospholipids (GPLs) on their hydrolysis by three different secretory PLA2s (sPLA2s) from various sources. The key finding of this study was that efflux of the GPL substrate from the bilayer largely determines the rate of hydrolysis by these PLAs. In micelles, accommodation of the GPL acyl chains in the catalytic active site seems to be more important for substrate specificity. In the next study we used the MS assay developed in study I, to investigate whether substrate efflux propensity regulates the activity of the Ca2+ -independent PLA-Beta (iPLAβ). Our results strongly suggest that the activity of iPLAβ is also determined by the efflux of the GPL substrate from the membrane bilayer. Our last study was on the cytoplasmic PLA-alpha (cPLA2α) that has been implicated in the initiation of the inflammatory lipid-mediator cascade generating eicosanoids and platelet-activating factor. The study was carried out to understand to what extent accommodation in the catalytic site determines specificity for arachidonic acid (AA) and if efflux propensity plays a role in the substrate specificity of cPLA2α. Our results indicate that while accommodation of the substrate in the active site greatly contributes to the preferential hydrolysis of AA-containing GPLs by cPLA2α, efflux of the substrate from a membrane bilayer also plays a significant role. In summary, these studies not only provide information on the factors regulating the substrate specificity of various PLAs but also indicate that lateral arrangement of GPLs could be a key regulator of homeostatic PLAs like iPLAβ.
  • Liu, Yixin (Helsingin yliopisto, 2021)
    The RET receptor tyrosine kinase is a versatile receptor which responds to multiple signalling cues. The signalling of RET plays a central role in cell proliferation, differentiation and maintenance. Its dysregulation contributes to various human diseases through gain- and loss-of-function, such as Hirschsprung’s disease and thyroid and lung cancers. Mutations at the cysteine-rich domain (CRD) of RET lead to constitutive activation through receptor dimerization via an intermolecular disulfide bond. However, due to the lack of functional and structural studies of RET and its ligands, the molecular mechanisms of RET activation under normal and pathological conditions is unclear. In this thesis project, a combination of biochemical and biophysical tools are used to characterize the activation of RET with an emphasize on three aspects: studying (1) its extracellular domain (ECD) complex formation with ephrin As (efn-As) and the growth and differentiation factor 15 (GDF15)/the glial cell-derived neurotrophic factor receptor a-like (GFRAL), (2) the oncogenic C634R mutation, which accounts for the majority of the multiple endocrine neoplasia type 2A (MEN2A) cases and (3) the solubilization and purification of full-length RET. To explore the binding between RET and efn-As, I first demonstrated that functional zebrafish RETECD (zRETECD) and efn-A5 (zefn-A5) can be expressed using insect cells. In contrast to previous cellular studies, I showed that zRETECD does not interact directly with zefn-As and postulated that another binding partner is required to mediate their interaction for the reverse signalling of efn-As. Unlike zRETECD, I found that human RETECD (hRETECD) expressed in insect cells is non-functional; therefore, I established a mammalian expression platform for functional expression of hRETECD, demonstrating its superiority to insect cell expression in which hRET is prone to misfold. Using recombinantly expressed proteins, I reconstituted the wild-type (WT) hRETECD/hGDF15/hGFRALECD complex and was able to reconstruct an 8-Å cryo-EM map of the complex, which revealed a “butterfly”-shaped tripartite complex. For the expression of the hRETECD(C634R) mutant, I used a C-terminal Fc tag to successfully drive the receptor dimer formation, as the mutant protein is otherwise monomeric. I studied the resulting apo-hRETECD(C634R) dimer and its complex with GDF15/GFRALECD using structural tools. Comparison of the cryo-EM structure of the hRETECD(WT)/hGDF15/hGFRALECD complex and the negative stain EM maps of the apo-hRETECD(C634R) dimer and the mutant hRETECD(C634R)/hGDF15/hGFRALECD complex revealed significant conformational changes that provide a structural model for the oncogenic activation of RET. Finally, I established a membrane protein production pipeline for full-length RET that will enable future structural studies in a more biologically native system.
  • Ning, Lin (Helsingin yliopisto, 2013)
    In the central nervous system, neurons are connected through local contacting structures called synapses. The initial contacts between axons and dendrites undergo changes in morphology and protein composition, and differentiate into fully functional synapses. Precise regulation on synapse formation is essential for normal brain functioning. Cell adhesion molecules are the key regulators during this process, by connecting the membranes of synapses and initiating signaling cascades that mediate synapse formation. A neuron-specific cell adhesion molecule ICAM-5 (telencephalin) controls immune responses by suppressing T-cell activation or by mediating neuron-microglia interaction. On the other hand, ICAM-5 is so far the only identified CAM that slows synaptic development. Deletion of ICAM-5 from mice leads to accelerated synapse formation, enhanced synaptic capacity, and improved memory and learning. Clinically, changes in ICAM-5 levels are associated with various diseases, such as acute encephalitis, epilepsy, and Alzheimer s disease. The major goal of my thesis is to address the molecular mechanisms by which ICAM-5 regulates synapse formation as well as maturation of dendritic spines, the post-synaptic components of excitatory synapses. In my study, ICAM-5 was observed as a substrate for MMP-2 and -9. Activation NMDA receptors in neurons elevated the level of MMP activity, and subsequently induced ICAM-5 ectodomain cleavage, which in turn promoted spine maturation. In addition, I identified the pre-synaptic β1-integrins as counter-receptors for ICAM-5. This trans-synaptic interaction inhibited the MMP-induced ICAM-5 cleavage, and thereby prevented spine maturation. At the intracellular side, α-actinin, an actin cross-linking protein, was found as a binding partner for ICAM-5. This binding linked ICAM-5 to the actin cytoskeleton and was important for the membrane distribution of ICAM-5 and neurite outgrowth. The GluN1 subunit of the NMDAR is also known to bind to α-actinin. We found here that GluN1 and ICAM-5 competed for the same binding region in α-actinin. Activation of NMDAR changed α-actinin binding property to ICAM-5, resulting in α-actinin accumulation and actin reorganization. In conclusion, my thesis defines a novel, ICAM-5-dependent mechanism, which regulates synapse formation, spine maturation and remodeling.
  • Quintero, Ileana B. (Helsingin yliopisto, 2015)
    Prostatic acid phosphatase (PAP) has been linked to prostate cancer since the mid-1930s. The main research approach of PAP over that time has been based on its role in the human prostate. The regulatory mechanisms of expression of the PAP gene have also been studied, giving us information about the regulatory elements in the gene and the transcription factors that affect the gene expression in the prostatic tissue. However, little was known until recently about this protein s role and physiological function in other tissues. Our group generated and used a PAP-deficient mouse and was able to show that PAP is expressed in dorsal root ganglia (DRG) and spinal cord in mice. This is the same protein as thiamine monophosphatase (TMPase) whose enzymatic activity has been used for five decades to mark primary sensory neurons. In these tissues, PAP acts as an ecto-5'-nucleotidase and is able to dephosphorylate AMP to adenosine, and therefore produce an anti-nociceptive effect due to the binding of adenosine to the A1-adenosine receptor. We analyzed the ACPP gene, which enabled us to describe a new transmembrane isoform for PAP (TMPAP). This novel PAP isoform is produced by alternative splicing of the 11th exon of the ACPP gene. The alternative splicing is present in species such as the human, mouse and rat. The newly discovered isoform is widely expressed in human and mouse tissues and contains a tyrosine sequence (YxxΦ) in its carboxyl-terminus, which directs the protein to endocytosis. We have also corroborated its functionality by co-localization studies with different organelle markers in the endosomal/lysosomal pathway (I). The generation of a PAP-deficient mouse also enabled us to study the function/s of PAP in vivo. The lack of PAP in this mouse model led to the gradual changes in the mouse prostate that finally culminated with the development of prostate adenocarcinoma at the age of 12 months. Microarray analyses of different tissues that compared the PAP deficient mouse with the wild type (WT) mouse revealed changes in genes related to the vesicular trafficking, which support our previous results and led us to the conclusion that TMPAP could be involved in the regulation of the vesicular trafficking. We also detected the interaction between TMPAP and snapin, a SNARE (Soluble NSF Attachment Protein Receptor) associated protein, by yeast two-hybrid screening, co-localization and FRET (Förster resonance energy transfer) analysis. We concluded that, the disruption of this interaction in the PAP-deficient mouse leads to a disturbance in the vesicular transport of the cell and to the development of prostate adenocarcinoma in the PAP-deficient mouse prostate (II). Furthermore, we showed that the PAP-deficient mice present multiple behavioral and neurochemical alterations including increased size of brain lateral ventricles, hyperdopaminergic deregulation, and altered GABAergic transmission, symptoms that indicate that PAP also disturbes the normal function of the central nervous system (III). Snapin protein in the brain has been described as a protein important for the vesicular transport and for the fusion of vesicles with the plasma membrane, and we observed that the lack of PAP in GABAergic neurons leads to a change in the localization of snapin in the PAP-deficient mouse (III), which could indicate that as in the prostate a dysregulated vesicular trafficking could be the reason for the detected phenotype. The discovery of the new TMPAP and its localization in the endosomal/lysosomal pathway enabled an understanding of the phenotypic changes that occur in the PAP-deficient mouse. We hypothesized that TMPAP regulates vesicular trafficking by interacting with snapin, and its deficiency leads to a dysregulation of the endo-/exocytosis cycle, which produces the observed alterations in the mouse organs and tissues. The results obtained throughout this research project have opened up new lines of research related to PAP physiological function, and a deeper understanding of the expression, regulation and function of this protein could lead to new clinical applications.
  • Araujo, César (Helsingin yliopisto, 2016)
    Prostatic acid phosphatase (PAP) was discovered during the mid-1930s, but the molecular mechanisms in which this protein is involved remain poorly understood. This enzyme was originally described as a highly-expressed protein in the human prostate that was secreted to the seminal fluid. It has always been associated to prostate cancer, since high levels of acid phosphatase activity were found in the sera of patients with metastatic disease. Therefore, for 40 years, research was focused on the improvement of biochemical assays in an effort to find specific substrates for clinical application. However, in the 1980s PSA (prostate specific antigen) superseded PAP as a biomarker for early detection of the disease and became the preferred marker for diagnosis. Later, in the mid-1990s with the advent of new molecular techniques such as cloning and high-scale protein purification it was possible to obtain high-quality crystals for 3D-structural determinations of PAP. In addition, new concepts emerged concerning the physiological role of the enzyme, with renewed speculation regarding the molecular mechanisms in which this protein could be involved. The fact that the serum levels of the enzyme are increased in prostate cancer patients with metastatic disease rendered this enzyme an attractive target for immunotherapies against advanced prostate cancer. However, this hypothesis has thus far neglected the existence of any potential isoforms that could be expressed in other organs and tissues. Therefore, as part of this thesis, the molecular mechanisms where PAP is involved will be investigated. For this purpose, two biological tools were employed: a PAP-knockout mouse model and stable virus-transfected LNCaP cell lines. A novel transmembrane type-I isoform of PAP (TMPAP) was first characterized as a product of alternative splicing of the same gene (ACPP) that encodes for the well-known secretory isoform (SPAP). TMPAP is distributed throughout mouse tissues, including prostate, lung, kidney, endometrium, salivary glands, and dorsal-root ganglia. The enzyme comprises an N-terminal domain containing the catalytic active site, a transmembrane helical domain, and a short C-terminal cytosolic domain that carries a tyrosine-based motif (Yxxφ) that targets the enzyme to the endosomal-lysosomal/exosomal pathway. This was confirmed by co-localization studies that revealed that PAP localizes to the plasma membrane as well as to the intracellular membranes of vesicles, lysosomes, and intraluminal vesicles of the multivesicular endosomes. Microarray experiments were performed on mouse tissues to study the differential gene expression profile between wild-type and PAP-knockout mice. The differential gene expression that was observed between the prostates of wild-type and PAP-knockout mice suggested that PAP is involved in secretory mechanisms, as many genes related to this process appeared dysregulated. Moreover, the results obtained from two-hybrid system experiments suggested that snapin (a SNARE-associated protein) was a likely candidate protein that could interact with TMPAP. This interaction was recently proved by co-localization and florescence resonance energy transfer (FRET) studies. The PAP-knockout mice developed prostate adenocarcinoma and showed dysregulation of genes related to vesicular traffic. Consequently, this investigation was focused on murine submandibulary glands (SMG) as a model of an exocrine organ. The expression of PAP in SMG was found to be even higher than in mouse prostate. In addition to microarrays and miRNA analyses, physiological and biochemical determinations help to demonstrate that there is an increased salivation volume in PAP-knockout mice upon stimulation with secretagogue drugs. This supports the hypothesis that PAP is involved in the regulation of secretory and exocytic processes. PAP was found to account for 50% of the total acid phosphatase activity in male mouse saliva and it is expressed by the granular convoluted tubular cells of the male SMG but not by the acinar cells. Unlike prostate gland, however, the mouse SMG does not develop signs of hyperplasia or adenocarcinoma in spite of an observed increased acinar cell proliferation. This discrepancy was explained by studying the degree of lymphocyte infiltration, the dysregulation of miRNAs, and the differentially expressed genes in microarray data. In SMGs of PAP-knockout mice, the innate immune system was shown to be responsive and able to remove proliferating acinar cells, which may explain the absence of adenocarcinoma. In addition, the upregulation of anti-inflammatory molecules may prevent the extension of tissue damage. Finally, we compared the effect of the overexpression of SPAP and TMPAP in LNCaP cells with empty-vector cells. As a result, the TMPAP-LNCaP cells exhibited slower growth than SPAP-LNCaP or empty-vector cells. Cells overexpressing either SPAP or TMPAP isoform showed increased 2D-projection area and increased HRP-uptake when compared with empty-vector cells. These two observations suggested an increased vesicular traffic in endo/exocytic pathways to maintain cell membrane homeostasis. Thus, vesicles loaded with TMPAP are most likely sorted to lysosomes by means of its Yxxφ motif. Consequently, there is an increased degradation of cargo molecules such as receptor tyrosine kinases expressed on the cell surface that could explain the observed slow growth of LNCaP cells that overexpress TMPAP. The molecular mechanisms identified in this study will definitely contribute to a better understanding of the physiological role of PAP in diseases and to a critical re-evaluation of existing immunotherapies. The knowledge of the molecular determinants responsible for the presence of TMPAP in the endo/exocytic pathway can also be exploited for the future development of radio-imaging and drug delivery protocols.
  • Ciragan, Annika (2017)
    At the present time resistance to every main class of antibiotic has been observed. Therefore, the continuous development of new-generation antibiotics is crucial to combat the rise of antibiotic resistant strains. Identification of potential antibiotic targets and investigation of their structure and function represent a rational approach to developing a better understanding of the essential processes in which they are involved, and may lead to finding a mechanism to inhibit these processes. The first part of this thesis covers structural characterization and functional studies of the potential antibiotic targets TonB protein and the cell shape determining protein MreC. TonB is needed for TonB-dependent uptake of scarce nutrients, such as iron and vitamin B 12. The cell shape determining protein MreC is involved in cell wall synthesis, which is the target of penicillin and its derivatives. In this study, the three dimensional structures of the C-terminal domain of Helicobacter pylori TonB of different lengths and the C-terminal domain of Bacillus subtilis MreC were determined using nuclear magnetic resonance (NMR) spectroscopy. Additionally, interaction studies of MreC with penicillin-binding proteins were done using NMR spectroscopy and a bacterial two-hybrid system. NMR spectroscopy is a versatile tool to investigate protein structure, dynamics and interactions. One of the advantages of NMR is that proteins can be studied in solution under nearly physiological conditions. However, with increasing molecular size (> 25 – 30 kDa), structural investigation by NMR becomes more complex and often requires specific labelling techniques and alternative methodologies for NMR measurements. Segmental isotopic labelling, where only a part of the protein is stable isotopic labelled, is an attractive method to overcome the challenges of studying large proteins by NMR. Segmental isotopic labelling allows, e.g. investigation of individual protein domains in a full-length context. Furthermore, NMR is a powerful tool to study the integrity of a protein. Certain requirements, however, have to be fulfilled: the protein has to be soluble at high concentrations and stable over the whole measurement time. Therefore it is important to optimize protein production in order to obtain soluble, properly folded proteins at high concentrations. In the second part of this study, TonB has been used as a model protein to show traceless intein-mediated segmental isotopic labelling by salt induced protein trans splicing using a halophilic intein. This approach facilitates structural investigations of TonB by NMR. TonB consists of a well-structured C-terminal domain and a flexible proline-rich region, which would severely interfere with spectral quality in a uniformly labelled sample. Furthermore TonB was used as a model protein to show the benefit in protein expression of using tandem SUMO fusion vectors as tools for the expression of more soluble proteins, which tend to be expressed in an insoluble form. Both of these applications are beneficial for structural investigation of proteins by NMR and can be applied to other proteins.
  • Øemig, Jesper S (Helsingin yliopisto, 2013)
    Inteins are proteins that have an unusual function. Intein are intervening protein sequences that catalysis there own excitation from a protein precursor sequence. Simultaneously, the intein coordinate the ligation of the flanking sequences. This reaction is called protein splicing. The role of inteins is unclear because they do not provide any benefit or disadvantage to the host organism but inteins has been found to have numerous biotechnological applications. In this thesis NMR spectroscopy and X-ray crystallography have been used to determine the 3D-structures inteins. The combination of the two different techniques has provided important insight about intein mechanism and how inteins could be engineered for application purposes. Proteins are dynamic molecules and NMR spectroscopy was used to indentify flexible sites of studied inteins. The study provided basics insight how to engineer intein for biotechnological application like site-specific modifications. High-resolution crystal structure provided novel insight in possible mechanism of inteins. The data suggest there exist an open and closed form of inteins where the closed conformation has been described in this work. The closed conformation is one of several possible conformations the intein experience while performing the reaction. The structure-functional relationship of inteins is not well understood but this thesis has gain further understanding of the structure aspect of inteins. The work provides further knowledge and guide how inteins can be optimized for possible biotechnological applications.
  • Frisk, Camilla (Helsingin yliopisto, 2019)
    Feeding raw food has increased in popularity and many advocate the good effects of it. Only few studies on raw food has been done, mainly on negative effects such as the risk of infection when handling raw meat. Therefore, the purpose of this study was to investigate whether different diets, especially raw food, has an impact on blood parameters. The hypothesis was that raw food will have an impact on the blood parameters. A total of 101 dogs were included in the analysis. Both hematologic and serum biochemical analyses were made. The owners were asked to fill in a questionnaire with information about their dog and the percent of each food they give to their dogs. Diets were defined as raw food, dry food, canned food and homemade food. Based on the questionnaire, the dogs were divided into different diet groups. Staffordshire bull terriers were also analysed individually since they consisted the majority of the population (n =80). The diet groups were as follows; 100 % raw food, 100% dry food and mixed diet. The population was also divided into 5 groups according to a set percent of either raw or dry food (1 = 0%, 2 = 1–30 %, 3 = 31–60 %, 4= 61–99 %, and 5 = 100 %). The mean values of the blood parameters in all groups were compared statistically (Kruskal-Wallis test). Differences were found between raw and dry and raw and mixed diets. The blood values that most often differed were erythrocytes, haemoglobin, alkaline phosphatase (ALP), creatinine, cholesterol, sodium and protein. Erythrocytes, haemoglobin, protein and creatinine increased with increased amount of raw food. ALP and cholesterol showed the opposite. Sodium showed high values in groups with high amounts of raw food and low values in mixed diets. This study gave evidence that diet is affecting blood parameters. In which extent it can affect remains unclear since no exact information about the diets were collected. Further studies need to be done to evaluate the real effect of a raw diet on blood parameters and whether it should be incorporated in clinical work.
  • Lak, Behnam (Helsingin yliopisto, 2021)
    The endoplasmic reticulum (ER) is a continuous network of multiple functional and structural subdomains. This largest membrane-bound organelle in eukaryotic cells is comprised of the nuclear envelope and a dynamic network of peripheral interconnected sheets and tubules. ER function requires an intricate balance between its dynamic, structures, and functions. ER sheets and tubules orchestrate a complex yet not fully understood role in giving ER its explicit structure and function. The ratio of sheet and tubule conformations differs significantly within cell types and during the cell cycle. Such balance is possible with a well-functioning set of factors that communicate with each other throughout the cell cycle, and are responsible for shaping the ER sheets and tubules. These factors bring about the dynamic nature of the ER. The ER membrane harbors many key proteins involved in lipid metabolism, protein synthesis, and membrane shaping, maintenance, and remodeling. This PhD thesis is centered on the study of the family with sequence similarity 134, member C protein (FAM134C) using advanced microscopy techniques. FAM134C localizes to high curvature ER: sheet edges and tubules. Similar to Reticulon family proteins (RTNs) and FAM134B, it promotes ER tubulation upon overexpression through its reticulon homology domain (RHD). Importantly, FAM134C maintains the ER shape, and the shape and structure of the ER accommodate its function. Multiple functions are associated with the ER; however, very little is known about the relative distribution of these functions within the ER subdomains. How various functions are spread within the ER network is an intriguing question in cell biology. The thesis dissected the ER structural subdomains using a combination of light microscopy and quantitative immuno-electron microscopy. The distribution of function in the ER network is highly dynamic and most likely shifting upon changes in the cellular environment and nutrient availability. Thus far, it is arguable that no subdomain is responsible for hosting one function exclusively. The ER is a communicative organelle and contacts almost all other organelles in the cell. It is the site for organelle biogenesis, such as phagophore formation. Phagophore, or the isolating membrane, is the central organelle in autophagy (cellular self-eating). Our functional studies on FAM134C revealed its specific role in regulating the size, number, and cargo of autophagosomes upon amino acid starvation. Obviously, structural proteins, either individually or in complex, serve not only structural-maintenance roles but also other crucial functions in the cell. Lastly, we investigated the subcellular localization of the structural protein, Golgi reassembly stacking protein (GRASP55), in autophagy flux. Upon glucose starvation, GRASP55 shifts its localization from medial- and trans-Golgi cisternae to the surface of autophagosome-lysosome, where it facilitates the membrane fusion between autophagosome and lysosome through its interaction with LC3 and LAMP2. The results obtained during this project have strengthened our understanding of the proteins and factors involved in ER structure, function, and biogenesis, and thereby provided valuable insights into normal cell functions. Identification of the proteins and forces that maintain the ER structural and functional subdomains would be a pivotal step towards unraveling the relation between many diseases and the ER. This in turn could be the basis for summoning future strategies with therapeutic potentials.