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Proteolytic maturation of vaccinia virus structural proteinsVanSlyke, Judy K. 05 November 1992 (has links)
Vaccinia virus (VV) is a large DNA virus belonging to the Orthopoxvirus
family. The viral replicative life cycle takes place solely within the cytoplasm
of a mammalian host cell. The VV genome contains 196 open reading frames
which are expressed in a highly regulated and temporal fashion in order to
bring about the production of a mature virion. In the process of viral
replication many VV proteins are synthesized that require posttranslational
modifications to become functional. A few of these modifications include,
glycosylation, ADP-ribosylation, phosphorylation, fatty acid acylation, and
proteolytic processing. This last modification is especially important with
regard to the structural proteins of the virus in that they undergo prysis
for an infectious virus particle to be formed, a common theme in viral
systems. In order to understand these events in more detail, three abundant
virion protein constituents 4a, 4b, and 25K were chosen as models for study.
The three main questions we wanted to answer were: Is there a cleavage
consensus site within the precursors, what protease(s) and/or factors are
necessary for the process, and how are the events regulated in vivo? Our
approach included development of specific immunological reagents to identify
cleavage products as well as to show where these core proteins are located
during virion assembly. We have subsequently identified cleavage products
by N-terminal microsequence from each of the three structural proteins and
this information has elucidated a putative cleavage consensus site of Ala-Gly-
X, where cleavage is proposed to take place between the Gly and X and X is
usually an aliphatic residue. The immunological reagents were used in
conjunction with immunofluorescent and immunogold labeling analyses to
identify the location of these core proteins during virion assembly. Core
proteins were localized to the virosomes in VV infected cells, to the viroplasm
of immature virus particles, and to the center of mature virions. Precursor
specific antiserum indicated that the larger molecular weight precursors of
core proteins are within immature virions as well. From these results the
following conclusions can be made. Identification of a putative cleavage
consensus site suggests that proteolytic processing is an endoproteolytic
event. The observation that precursor structural proteins were found within
immature particles indicates that the proteinase responsible for cleavage is
also present. The fact that assembly has to occur before proteolytic
processing of VV structural proteins suggests that the cleavage events are
dependent upon a specific core protein conformation. However the nature of
this conformational requirement is not known. Further research is underway
to develop a full understanding of the proteolytic events during virion
morphogensis. / Graduation date: 1993
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Human lysyl hydroxylase isoforms:multifunctionality of human LH3 and the amino acids important for its collagen glycosyltransferase activitiesWang, C. (Chunguang) 17 September 2002 (has links)
Abstract
Lysyl hydroxylase (EC1.14.11.4, LH) catalyzes post-translationally the hydroxylation of lysyl residues in collagens and other proteins with collagenous domains. Hydroxylysyl residues may also be glycosylated by hydroxylysyl galactosyltransferase (EC 2.4.1.50, GT) or galactosylhydroxylysyl glucosyltransferase (EC 2.4.1.66, GGT) to form galactosylhydroxylysyl or glucosylgalactosylhydroxylysyl residues, structures unique to collagen.
Three LH isoenzymes (LH1, LH2a/2b, LH3) have been characterized so far. We analyzed mRNA levels of these isoforms, as well as the mRNAs of the main collagen types (I, III, IV, V) and the α subunit of PH-4 in different human cell lines. Large variations were found in mRNA expression of LH1 and LH2 but not LH3. The mRNA levels of LH1, LH2, and the α subunit of PH-4 showed significant correlation with each other whereas LH3 correlated with none. No correlation was observed between the LH isoforms and individual collagen types.
Three human LH isoforms were expressed in different expression systems. The purified recombinant protein produced by LH3 cDNA was found to be the only one possessing LH, GT and GGT activities. The molecular weight of the partially purified LH3 expressed in Sf9 or Cos-7 cells corresponded to about 85 kDa whereas that in E.coli cells was about 81 kDa probably due to a deficiency of glycosylation in bacterial cells. The recombinant protein of C. elegans LH cDNA was expressed in a cell-free translation system and in E.coli cells. The data indicated that the glycosyltransferase activities, GT and GGT, were also associated with this gene product.
The sequence alignment of LH isoforms from different species revealed that there are 29 amino acids conserved between human LH3, mouse LH3 and C. elegans LH sequences and scattered evenly in the molecule, but differing from those of LH1 and LH2. In vitro mutagenesis data showed that the amino acids important for the glycosyltransferase activities were located at the amino-terminal part of the molecule, being separate from the LH active site. Mutation of a conserved LH3 specific, non-disulfide linked cysteine to isoleucine caused a dramatic reduction in GT and GGT activity but had no effect on LH activity. Mutations of the amino-terminal DxD motif (D187-191) characteristic of many glycosyltransferases eliminated both GT and GGT activities, showing the importance of this motif for collagen glycosyltransferases and suggesting that it might serve as the Mn2+ binding site in the molecule.
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Identification of Non-histone Acetylation Targets in Saccharomyces cerevisiaePourhanifeh-Lemeri, Roghayeh 06 June 2012 (has links)
Lysine acetylation is a conserved post-translational modification (PTM) which was traditionally believed to be limited to histones and the regulation of gene expression. However, recent proteomic studies have identified lysine acetylation on proteins implicated in virtually all cellular processes indicating that this PTM plays a global regulatory role. Indeed, in humans, aberrance of lysine acetyltransferase (KAT) activity is associated with various pathogenesis. To date, over 2500 human proteins are known to be acetylated in vivo, but very few acetylations have been linked to specific KATs. Hence, to understand the biological relevance of KATs and acetylation in human pathology, it is important to learn about the mechanism regulating KAT activity and the identity of their in vivo targets. This is a complex task and will require the use of model organisms and system biology approaches. The work presented here explores the significance of self-acetylation in regulating KAT function by focusing on the highly NuA4 lysine acetyltransferase in the model organism Saccharomyces cerevisiae or budding yeast. Using genetics and biochemical assays I have identified NuA4 subunit Epl1 as a novel in vivo NuA4 substrate. I have also shown that Epl1 acetylation regulates NuA4 function at elevated temperatures. In an attempt to identify new biological processes regulated by yeast KATs and putative novel substrates, I have also performed a genome-wide synthetic dosage lethality screen with six non-essential yeast KATs; Hat1, Rtt109, Hpa2, Sas3, Sas2, and Elp3. My screen identified largely distinct sets of genetic interactions for each KAT suggesting that each KAT has specific cellular functions. Together, this study demonstrates the importance of auto-acetylation in regulating KAT function and the diversity of cellular processes impacted by KAT activity in vivo.
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Kinome-wide RNAi Screening to Identify Kinases Involved in Post-translational Modification of FUSLiu, Serena E. B. January 2016 (has links)
Amyotrophic lateral sclerosis (ALS) is a devastating adult onset neurodegenerative
disorder characterized by the selective degeneration of upper and lower motor neurons.
Patients typically die from respiratory failures within 2-5 years after diagnosis. One of
the milestones in ALS research is the discovery Fused in Sarcoma (FUS), an ALS causative gene. FUS is an RNA/DNA-binding protein and predominantly resides in the nucleus.
Majority of the FUS mutations are located in the C-terminus and causing aberrant
misdistribution to the cytoplasm. Currently, only a few binding partners of FUS are
known, which makes it difficult to speculate on the function and interaction of the
protein. In this study, we conducted a kinome-wide RNAi screen to identify kinases that
affect the localization of FUS. A dual specificity protein kinase named CDC2-like kinase
(CLK1) from the screen was found to be responsible for in post-translational modification of
FUS and affects the localization of FUS in the nucleus. The identification of CLK1 as FUSmodifying kinase is consistent with roles ascribed to both in the binding and regulation of RNA.
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Protein kinase A inhibits tumor mutator APOBEC3B through phosphorylation / プロテインキナーゼAはがんの変異源であるAPOBEC3Bをリン酸化することで抑制するMatsumoto, Tadahiko 25 November 2019 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22118号 / 医博第4531号 / 新制||医||1039(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 道行, 教授 小柳 義夫, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM
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Regulation of Neuroblastoma Malignant Properties by Pannexin 1 Channels: Role of Post-Translational Modifications and MutationsHolland, Stephen Henry 17 January 2020 (has links)
Neuroblastoma (NB) is the most common extracranial solid tumour in childhood. NB is thought to arise from the failed differentiation of neural crest progenitor cells that would normally form tissues of the adrenal gland and sympathetic nervous system. These neural crest progenitors then uncontrollably proliferate forming a tumour. Despite aggressive surgery and chemotherapy, the cure rate of high-risk NB patients remains below 30%. Our laboratory has shown that human NB tumour specimens and high-risk patient derived cell lines express pannexin 1 (PANX1), and that treatment with the PANX1 channel blockers carbenoxolone or probenecid constitute reduce NB progression in vitro and in vivo. PANX1 is a glycoprotein that forms single membrane channels best known to serve as conduits for ATP release. Interestingly, while PANX1 was also detected in control neurons by western blotting, its banding pattern was strikingly different as a band at around 50 kDa was found in all NB cell lines, but not in neurons. Using shRNA targeting PANX1 and deglycosylation enzymes, I have shown that this band corresponds to a PANX1 glycosylated species. PANX1 has been reported to be phosphorylated in NB at amino acid Y10. PANX1 is also predicted to be glycosylated at N255. In order to study the role of these post-translational modifications, myc-tagged Y10F and N255A PANX1 mutants were engineered by site-directed mutagenesis. Immunolocalization and cell surface biotinylation assays suggest that the localization both mutants at the cell surface is reduced compared to that of myc-PANX1. Dye uptake assays revealed that myc-Y10F has significantly reduced channel activity. Expression of myc-Y10F and myc-N255A in NB cells inhibited cell proliferation and decreased metastatic potential in vitro. Further analysis of NB tumour specimens revealed that there is a missense mutation in PANX1 resulting in the formation of truncated peptide (amino acid 1-99). Interestingly, I have found that when co-expressed with myc-PANX1, PANX11-99, reduced PANX1 channel activity. Taken together, these findings indicate that phosphorylation on Y10 and glycosylation on N255 regulate PANX1 channel activity and exacerbate NB malignancy, while the expression of PANX11-99 in NB may be beneficial.
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Investigating Polyphosphate Biology: From Post-Translational Modification to Rare DiseaseBentley-DeSousa, Amanda 31 May 2021 (has links)
The first report of polyphosphates (polyP) was in 1890 by L. Liberman and since then, polyP’s role in biology has been explored. PolyPs are chains of phosphoanhydride-linked inorganic phosphates ranging from 3-1000s of units in length. These chains are implicated in many cellular pathways including blood clotting, bacterial virulence, and neuroproteotoxic disease. Given the diversity of polyP, they make an excellent candidate in the development of novel therapeutics. In yeast, polyP is synthesized by the vacuolar transporter chaperone (VTC) complex as a translocation event into the vacuole lumen. In 2015, polyP chains were found to act as a post-translational modification termed polyphosphorylation on yeast proteins (Nsr1 and Top1). This modification occurs non-enzymatically on lysine residues within poly-acidic, serine, and lysine (PASK) motifs and can only be detected via electrophoretic mobility shift on NuPAGE gels. We have since expanded the pool of yeast polyphosphorylated substrates to 25, with an enrichment of proteins with roles related to RNA biology. Additionally, we were the first group to demonstrate polyphosphorylation of 6 human proteins by expressing E. coli PPK1 in HEK293T cells. We next focused on elaborating how polyP is being regulated via the VTC complex by assessing which protein trafficking pathways are critical for VTC localization at the vacuole membrane. We found the adaptor protein 3 (AP-3) complex is responsible for localizing Vtc5 subunit to the vacuole membrane and in AP-3 mutants, Vtc5 becomes mislocalized to the vacuole lumen and degraded. Vtc5 degradation, upon AP-3 mutation, is mediated by the endosomal sorting complex required for transport (ESCRT) complex. The loss of polyP in AP-3 mutants is imparted by Vtc5 mislocalization. In humans, mutations in AP-3 cause a rare genetic disorder termed Hermansky-Pudlak Syndrome (HPS) which has a wide range of symptoms. These include defects in polyP accumulation in platelets, likely related to a loss of polyP. We expect that our work using yeast will provide a framework for understanding fundamental aspects of polyP biology related to HPS and other health conditions.
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CaMKII Phosphorylation of the Voltage-Gated Sodium Channel Nav1.6 Regulates Channel Function and Neuronal ExcitabilityZybura, Agnes Sara 01 1900 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / Voltage-gated sodium channels (Navs) undergo remarkably complex modes of modulation to fine tune membrane excitability and neuronal firing properties. In neurons, the isoform Nav1.6 is highly enriched at the axon initial segment and nodes, making it critical for the initiation and propagation of neuronal impulses. Thus, Nav1.6 modulation and dysfunction may profoundly impact the input-output properties of neurons in normal and pathological conditions. Phosphorylation is a powerful and reversible mechanism that exquisitely modulates ion channels. To this end, the multifunctional calcium/calmodulin-dependent protein kinase II (CaMKII) can transduce neuronal activity through phosphorylation of diverse substrates to serve as a master regulator of neuronal function. Because Nav1.6 and CaMKII are independently linked to excitability disorders, I sought to investigate modulation of Nav1.6 function by CaMKII signaling to reveal an important mechanism underlying neuronal excitability. Multiple biochemical approaches show Nav1.6 is a novel substrate for CaMKII and reveal multi-site phosphorylation within the L1 domain; a hotspot for post-translational regulation in other Nav isoforms. Consistent with these findings, pharmacological inhibition of CaMKII reduces transient and persistent sodium currents in Purkinje neurons. Because Nav1.6 is the predominant sodium current observed in Purkinje neurons, these data suggest that Nav1.6 may be modulated through CaMKII signaling. In support of this, my studies demonstrate that CaMKII inhibition significantly attenuates Nav1.6 transient and persistent sodium currents and shifts the voltage-dependence of activation to more depolarizing potentials in heterologous cells. Interestingly, I show that these functional effects are likely mediated by CaMKII phosphorylation of Nav1.6 at S561 and T642, and that each phosphorylation site regulates distinct biophysical characteristics of the channel. These findings are further extended to investigate CaMKII modulation of disease-linked mutant Nav1.6 channels. I show that different Nav1.6 mutants display distinct responses to CaMKII modulation and reveal that acute CaMKII inhibition attenuates gain-of-function effects produced by mutant channels. Importantly, computational simulations modeling the effects of CaMKII inhibition on WT and mutant Nav1.6 channels demonstrate dramatic reductions in neuronal excitability in Purkinje and cortical pyramidal cell models. Together, these findings suggest that CaMKII modulation of Nav1.6 may be a powerful mechanism to regulate physiological and pathological neuronal excitability. / 2022-02-02
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Regulation of Wingless secretion, distribution and signalingTang, Xiaofang January 2012 (has links)
No description available.
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Hsp70 Phosphorylation: A Case Study of Serine Residues 385 and 400Saini, Sashrika 20 October 2021 (has links) (PDF)
Molecular chaperones play a key role in maintaining a healthy cellular proteome by performing protein quality control. Heat shock protein 70s (Hsp70s) are a diverse class of evolutionarily conserved chaperones that interact with short hydrophobic sequences presented in unfolded proteins, promoting productive folding, and preventing proteins from aggregation. Most of the extensive research on chaperone examines mechanism, substrate promiscuity, and engagement with many co-chaperones. Only recently were chaperones recognized to be frequent targets of post-translational modifications (PTMs). Despite the recent rise in PTMs identified, the impact of these modifications on chaperone function, whether singular or in concert with other modifications, remains elusive. To investigate the impact of PTMs on chaperone function, we chose to characterize two sites of phosphorylation on the linker of HspA1, the stress inducible human Hsp70. To mimic these phosphoserines, we used aspartate as a phosphomimetic substitution for all experiments. Interdomain allostery ties together chaperone structure and function. Therefore, the impact of phosphorylation on interdomain allostery is probed using biophysical and biochemical techniques. Altogether, data suggest that phosphorylation of the linker and SBD destabilizes the chaperone, while shifting the population towards the docked state. This result alludes to a previously described region of the protein that uncouples domain docking from conformational changes in the substrate-binding domain. The cross-communication between these phosphorylation sites reveals a novel, synergistic effect on chaperone structure and function.
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