Structural characterisation of the interaction between RBBP6 and the multifunctional protein YB-1

Magister Scientiae - MScAs a means of further localising the interaction, truncated fragments derived from the C-terminal region of YB-1, were tested for their interaction with the RING finger domain of RBBP6 using three different assays: a directed yeast 2-hybrid assay, co-immunoprecipitation and NMR chemical shift perturbation analysis. Our results suggest that the entire 62 amino acid region at the C-terminal domain of YB-1 may be involved in the interaction with RBBP6. Using chemical shift perturbation analysis, this study provides an indication of where YB-1 binds to the RING finger. This represents the first step towards the design of therapeutics aimed at modulating the interaction between RBBP6 and YB-1 as a means of targeting the oncogenic effects of YB-1. In order to identify E2 enzymes involved in the ubiquitination of YB-1, we examined the efficiencies of selected E2s in an in vitro ubiquitination assay. UbcH5c and UbcH7 were both found to catalyse the ubiquitination of YB-1 in conjuction with RBBP6, whereas Ubc13 was not. Finally, we show using NMR that two single-point mutations of the RING finger-like domain are sufficient to abolish homodimerisation of the domain. These will be used in future studies to investigate the requirement for homodimerisation on the ubiquitination activity of RBBP6.South Afric

First COVID-19 case in Zambia — Comparative phylogenomic analyses of SARS-CoV-2 detected in African countries

Since its first discovery in December 2019 in Wuhan, China, COVID-19, caused by the novel coronavirus SARS-CoV-2, has spread rapidly worldwide. While African countries were relatively spared initially, the initial low incidence of COVID-19 cases was not sustained for long due to continuing travel links between China, Europe and Africa. In preparation, Zambia had applied a multisectoral national epidemic disease surveillance and response system resulting in the identification of the first case within 48 h of the individual entering the country by air travel from a trip to France. Contact tracing showed that SARS-CoV-2 infection was contained within the patient’s household, with no further spread to attending health care workers or community members. Phylogenomic analysis of the patient’s SARS-CoV-2 strain showed that it belonged to lineage B.1.1., sharing the last common ancestor with SARS-CoV-2 strains recovered from South Africa. At the African continental level, our analysis showed that B.1 and B.1.1 lineages appear to be predominant in Africa. Whole genome sequence analysis should be part of all surveillance and case detection activities in order to monitor the origin and evolution of SARS-CoV-2 lineages across Africa

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Mechanistic modulation of a bifunctional GC-linked receptor kinase, PSKR1

Receptor kinases possessing intrinsic guanylate cyclase (GC) activity constitute a family of catalytically active membrane-bound proteins that play crucial roles in a myriad of signal transduction processes. Currently known GC-linked receptor kinases have an extracellular ligand-binding domain and an intracellular portion that is composed of an inactive kinase homology domain and a functional GC catalytic centre. A novel class of GC-linked receptor kinases was unearthed using homologyguided bioinformatic data mining tools designed from annotated amino acid residues in the GC catalytic centres of lower eukaryotes. The GC catalytic centre in this new class of receptor kinases is encapsulated within an active kinase domain, thereby conferring a dual catalytic function to this class of proteins. This is contrary to currently known classical GC-linked receptor kinases, which are mono-functional. There are currently four members of this novel class of receptor kinases which have been demonstrated to possess intrinsic GC and kinase activity. However, there is a paucity of information as to how this dual catalysis is physiologically regulated. In an attempt to uncover the modulatory mechanisms regulating the dual catalysis of novel GC-linked receptor kinases, this thesis examines the elements influencing the dual catalysis of the phytosulfokine receptor, PSKR1. Most specifically, this study focuses on how receptor dimerisation, phosphorylation, and the effect of intracellular signalling molecules like calcium and ATP influence PSKR1’s intrinsic dual catalysis. Analytical ultracentrifugation and size exclusion chromatography have demonstrated that the recombinantly expressed cytoplasmic domain of PSKR1 is reversibly dimeric in solution. This observation probably represents a catalytically competent iv physiological arrangement of the receptor that may be a pre-requisite for PSKR1- mediated nucleotide cyclase and phospho-transferase activity. Mapping out of the phosphorylation pattern in the cytoplasmic domain of PSKR1 using tandem mass spectroscopy identified 14 phosphorylation sites; eight phosphoserines, three phosphothreonines and three phosphotyrosines. Further in vitro studies revealed that PSKR1 is capable of self-phosphorylating and that phosphorylation is essential for PSKR1-mediated dual catalysis. Apart from its obvious role in phosphorylation, ATP seemed to potentiate the nucleotide cyclase activity of PSKR1, possibly through allosteric modulation. Intriguingly, calcium had a direct influence in regulating PSKR1-mediated dual catalysis without the aid of calcium sensor proteins such as calmodulin. At physiologically high calcium concentrations, PSKR1’s nucleotide cyclase activity was enhanced while on the other hand the phospho-transferase activity of PSKR1 was significantly suppressed. This observation points to calcium acting as a molecular switch regulating the dual catalysis of PSKR1. Taken together, the findings obtained in this study informs a proposed model of how all these regulatory elements modulate PSKR1-mediated dual catalysis. In summation, the presence of catalytically relevant motifs within functional protein domains allows signalling proteins to self-regulate and fine-tune their signal output in concert with their immediate cellular environment. This necessitates the need for prospective studies to carry out similar bioinformatics search strategies in order to uncover hidden regulatory elements that facilitate the self-regulation of proteins

Functional Roles of RNA-Binding Proteins in Plant Signaling

RNA-binding proteins (RBPs) are typical proteins that bind RNA through single or multiple RNA-binding domains (RBDs). These proteins have a functional role in determining the fate or function of the bound RNAs. A few hundred RBPs were known through in silico prediction based on computational assignment informed by structural similarity and the presence of classical RBDs. However, RBPs lacking such conventional RBDs were omitted. Owing to the recent mRNA interactome capture technology based on UV-crosslinking and fixing proteins to their mRNA targets followed by affinity capture purification and identification of RBPs by tandem mass spectrometry, several hundreds of RBPs have recently been discovered. These proteome-wide studies have colossally increased the number of proteins implicated in RNA binding and unearthed hundreds of novel RBPs lacking classical RBDs, such as proteins involved in intermediary metabolism. These discoveries provide wide insights into the post-transcriptional gene regulation players and their role in plant signaling, such as environmental stress conditions. In this review, novel discoveries of RBPs are explored, particularly on the evolving knowledge of their role in stress responses. The molecular functions of these RBPs, particularly focusing on those that do not have classical RBDs, are also elucidated at the systems level

Mechanistic modulation of a bifunctional GC-linked receptor kinase, PSKR1

Receptor kinases possessing intrinsic guanylate cyclase (GC) activity constitute a family of catalytically active membrane-bound proteins that play crucial roles in a myriad of signal transduction processes. Currently known GC-linked receptor kinases have an extracellular ligand-binding domain and an intracellular portion that is composed of an inactive kinase homology domain and a functional GC catalytic centre. A novel class of GC-linked receptor kinases was unearthed using homology-guided bioinformatic data mining tools designed from annotated amino acid residues in the GC catalytic centres of lower eukaryotes. The GC catalytic centre in this new class of receptor kinases is encapsulated within an active kinase domain, thereby conferring a dual catalytic function to this class of proteins. This is contrary to currently known classical GC-linked receptor kinases, which are mono-functional. There are currently four members of this novel class of receptor kinases which have been demonstrated to possess intrinsic GC and kinase activity. However, there is a paucity of information as to how this dual catalysis is physiologically regulated. In an attempt to uncover the modulatory mechanisms regulating the dual catalysis of novel GC-linked receptor kinases, this thesis examines the elements influencing the dual catalysis of the phytosulfokine receptor, PSKR1. Most specifically, this study focuses on how receptor dimerisation, phosphorylation, and the effect of intracellular signalling molecules like calcium and ATP influence PSKR1’s intrinsic dual catalysis. Analytical ultracentrifugation and size exclusion chromatography have demonstrated that the recombinantly expressed cytoplasmic domain of PSKR1 is reversibly dimeric in solution. This observation probably represents a catalytically competent physiological arrangement of the receptor that may be a pre-requisite for PSKR1-mediated nucleotide cyclase and phospho-transferase activity. Mapping out of the phosphorylation pattern in the cytoplasmic domain of PSKR1 using tandem mass spectroscopy identified 14 phosphorylation sites; eight phosphoserines, three phosphothreonines and three phosphotyrosines. Further in vitro studies revealed that PSKR1 is capable of self-phosphorylating and that phosphorylation is essential for PSKR1-mediated dual catalysis. Apart from its obvious role in phosphorylation, ATP seemed to potentiate the nucleotide cyclase activity of PSKR1, possibly through allosteric modulation. Intriguingly, calcium had a direct influence in regulating PSKR1-mediated dual catalysis without the aid of calcium sensor proteins such as calmodulin. At physiologically high calcium concentrations, PSKR1’s nucleotide cyclase activity was enhanced while on the other hand the phospho-transferase activity of PSKR1 was significantly suppressed. This observation points to calcium acting as a molecular switch regulating the dual catalysis of PSKR1. Taken together, the findings obtained in this study informs a proposed model of how all these regulatory elements modulate PSKR1-mediated dual catalysis. In summation, the presence of catalytically relevant motifs within functional protein domains allows signalling proteins to self-regulate and fine-tune their signal output in concert with their immediate cellular environment. This necessitates the need for prospective studies to carry out similar bioinformatics search strategies in order to uncover hidden regulatory elements that facilitate the self-regulation of proteins

Mechanistic modulation of a bifunctional GC-linked receptor kinase, PSKR1

Receptor kinases possessing intrinsic guanylate cyclase (GC) activity constitute a family of catalytically active membrane-bound proteins that play crucial roles in a myriad of signal transduction processes. Currently known GC-linked receptor kinases have an extracellular ligand-binding domain and an intracellular portion that is composed of an inactive kinase homology domain and a functional GC catalytic centre. A novel class of GC-linked receptor kinases was unearthed using homology-guided bioinformatic data mining tools designed from annotated amino acid residues in the GC catalytic centres of lower eukaryotes. The GC catalytic centre in this new class of receptor kinases is encapsulated within an active kinase domain, thereby conferring a dual catalytic function to this class of proteins. This is contrary to currently known classical GC-linked receptor kinases, which are mono-functional. There are currently four members of this novel class of receptor kinases which have been demonstrated to possess intrinsic GC and kinase activity. However, there is a paucity of information as to how this dual catalysis is physiologically regulated. In an attempt to uncover the modulatory mechanisms regulating the dual catalysis of novel GC-linked receptor kinases, this thesis examines the elements influencing the dual catalysis of the phytosulfokine receptor, PSKR1. Most specifically, this study focuses on how receptor dimerisation, phosphorylation, and the effect of intracellular signalling molecules like calcium and ATP influence PSKR1’s intrinsic dual catalysis. Analytical ultracentrifugation and size exclusion chromatography have demonstrated that the recombinantly expressed cytoplasmic domain of PSKR1 is reversibly dimeric in solution. This observation probably represents a catalytically competent physiological arrangement of the receptor that may be a pre-requisite for PSKR1-mediated nucleotide cyclase and phospho-transferase activity. Mapping out of the phosphorylation pattern in the cytoplasmic domain of PSKR1 using tandem mass spectroscopy identified 14 phosphorylation sites; eight phosphoserines, three phosphothreonines and three phosphotyrosines. Further in vitro studies revealed that PSKR1 is capable of self-phosphorylating and that phosphorylation is essential for PSKR1-mediated dual catalysis. Apart from its obvious role in phosphorylation, ATP seemed to potentiate the nucleotide cyclase activity of PSKR1, possibly through allosteric modulation. Intriguingly, calcium had a direct influence in regulating PSKR1-mediated dual catalysis without the aid of calcium sensor proteins such as calmodulin. At physiologically high calcium concentrations, PSKR1’s nucleotide cyclase activity was enhanced while on the other hand the phospho-transferase activity of PSKR1 was significantly suppressed. This observation points to calcium acting as a molecular switch regulating the dual catalysis of PSKR1. Taken together, the findings obtained in this study informs a proposed model of how all these regulatory elements modulate PSKR1-mediated dual catalysis. In summation, the presence of catalytically relevant motifs within functional protein domains allows signalling proteins to self-regulate and fine-tune their signal output in concert with their immediate cellular environment. This necessitates the need for prospective studies to carry out similar bioinformatics search strategies in order to uncover hidden regulatory elements that facilitate the self-regulation of proteins

Application of recombinant technology in protein investigations

Book ChapterA major breakthrough of modern biology is the development of recombinant technology which allows specific genes to be cloned and expressed in recombinant or chimeric forms in order to isolate or detect the defined products. Such technology is particularly useful in protein investigation. It not only helps to isolate target proteins from their natural complicated context and identify function, but also provides distinct and detectable means to track the protein activity in vivo. The current chapter covers a number of methodologies to isolate or detect gene products of interest, including recombinant protein purification and analysis, enzymatic reporter assays and fluorescent tag detection. The chapter is formalized with various research aspects, concrete experiments, step-by-step procedures, and notes dealing with possible problems. It is expected that this informative and practical manual will be applicable as a basic guide for readers

(De)Activation (Ir)Reversibly or Degradation : Dynamics of Post-Translational Protein Modifications in Plants

The increasing dynamic functions of post-translational modifications (PTMs) within protein molecules present outstanding challenges for plant biology even at this present day. Protein PTMs are among the first and fastest plant responses to changes in the environment, indicating that the mechanisms and dynamics of PTMs are an essential area of plant biology. Besides being key players in signaling, PTMs play vital roles in gene expression, gene, and protein localization, protein stability and interactions, as well as enzyme kinetics. In this review, we take a broader but concise approach to capture the current state of events in the field of plant PTMs. We discuss protein modifications including citrullination, glycosylation, phosphorylation, oxidation and disulfide bridges, N-terminal, SUMOylation, and ubiquitination. Further, we outline the complexity of studying PTMs in relation to compartmentalization and function. We conclude by challenging the proteomics community to engage in holistic approaches towards identification and characterizing multiple PTMs on the same protein, their interaction, and mechanism of regulation to bring a deeper understanding of protein function and regulation in plants