A genetic screen to isolate “Lariat” peptide inhibitors of protein function

Functional genomic analyses provide information that allows hypotheses to be formulated on protein function. These hypotheses, however, need to be validated using reverse genetic approaches, which are difficult to perform on a large scale and in diploid organisms. To address this problem, we developed a genetic screen to rapidly isolate “lariat” peptides that function as trans dominant inhibitors of protein function. We engineered intein proteins to genetically produce lariats. A lariat consists of a lactone peptide covalently attached to a linear peptide. Cyclizing peptides with a lactone bond imposes a constraint even within the reducing environment found inside of cells. The covalently attached linear peptide provides a site for fusing protein moieties. We fused a transcriptional activation domain to a combinatorial lactone peptide, which allowed combinatorial lariat libraries to be screened for protein interactions using the yeast two-hybrid assay. We confirmed that the intein processed in yeast using Western blot analysis. A chemoselective ring opening of the lactone bond with heavy water, followed by mass spectrometry analysis showed that ~ 44% of purified lariat contained an intact lactone bond. To improve the stability of the lactone bond, we introduced mutations into the engineered intein and analyzed their processing and stability by mass spectrometery. Several mutations were identified that increased the amount of intact lariat. Combinatorial libraries of lactone peptides were generated and screened using the yeast-two-hybrid interaction trap. Lactone cyclic peptides that bound to a number of different targets including LexA, Jak2, and Riz1 were isolated. A lactone cyclic peptide isolated against the bacterial repressor protein LexA was characterized. LexA regulates bacterial SOS response and LexA mutants that cannot undergo autoproteolyis make bacteria more sensitive to, and inhibit resistance against cytotoxic reagents. The anti-LexA lariat interacted with LexA with a dissociation constant of 37 µM by surface plasmon resonance. The lactone constraint was determined to be required for the interaction of the anti-LexA L2 lariat with LexA in the yeast-two-hybrid assay. Alanine scanning showed that only two amino acids (G8 and E9) in the anti-LexA L2 sequence (1-SRSWDLPGEY-10) were not required for the interaction with LexA. The interaction of the anti-LexA lariat with LexA in vivo was confirmed by chromatin precipitation of the lactone peptide-LexA-DNA complex. The anti-microbial properties of the anti-LexA lariat were also characterized. The anti-LexA lariat potentiated the activity of a DNA damaging agent mitomycin C and inhibited the cleavage of LexA, preventing the SOS response pathway from being activated. In summary, lariats possess desired traits for characterizing the function and therapeutic potential of proteins. The ability to genetically and chemically synthesize lariats allows the lariat transcription activation domain to be replaced by other peptide and chemical moieties such as affinity tags, fluorescent molecules, localization sequences, et cetera, which give them advantages over “head to tail” cyclized peptides, which have no free end to attach moieties

Biotechnologies for Plant Mutation Breeding: Protocols

Plant Breeding/Biotechnology; Agriculture; Genetic Engineering; Plant Genetics & Genomic

2006 Eighteenth Annual IMSA Presentation Day

We believe that our goal of creating decidedly-different learners is already being met and will make a profound impact on the future of humanity.https://digitalcommons.imsa.edu/archives_sir/1020/thumbnail.jp

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

Advances in Polyhydroxyalkanoate (PHA) Production, Volume 3

Nowadays, we are witnessing highly dynamic research activities related to the intriguing field of biodegradable materials with plastic-like properties. These activities are currently intensified by a strengthened public awareness of prevailing ecological issues connected to growing piles of plastic waste, microplastic formation, and increasing greenhouse gas emissions; this goes hand-in-hand with the ongoing depletion of fossil feedstocks, which are traditionally used to produce full carbon backbone polymers. To a steadily increasing extend, polyhydroxyalkanoate (PHA) biopolyesters, a family of plastic-like materials with versatile material properties, are considered a future-oriented solution for diminishing these concerns. PHA production is based on renewable resources, and occurs in a bio-mediated fashion by the action of living organisms. If accomplished in an optimized way, PHA production and the entire PHA lifecycle are embedded into nature´s closed cycles of carbon. Holistic improvement of PHA production, applicable on an industrially relevant scale, calls for inter alia: consolidated knowledge about the enzymatic and genetic particularities of PHA accumulating organisms, in-depth understanding of the kinetics of the bioprocess, the selection of appropriate inexpensive fermentation feedstocks, tailoring the composition of PHA on the level of the monomeric constituents, optimized biotechnological engineering, and novel strategies for PHA recovery from biomass characterized by minor energy and chemical requirement

A novel regulatory mechanism of the plant TIR immune signaling

Plant defense against microbial pathogens is mainly realized by pattern-triggered immunity (PTI) mediated by pattern recognition receptors (PRRs) at the cell surface, and effectortriggered immunity (ETI) mediated by nucleotide-binding leucine-rich repeat (NLR) immune receptors inside cells. Based on their N-terminal domains, plant NLRs can be divided into two categories: CC-NLRs (CNLs) with a coiled-coil (CC) domain and TIR-NLRs (TNLs) with a toll/interleukin 1 receptor (TIR) domain. Specific recognition of pathogen effectors induces oligomerization of NLRs, termed resistosomes, to transduce plant immune signaling. CNLs are able to form pentameric resistosomes upon activation and function as calcium (Ca2+)-permeable channels in the plasma membrane. Whether TNLs form resistosomes in response to pathogen infection remained an open question, although the TIR domain in TNLs has NADase activity that is required for TNL-mediated immunity. NADase activity, although essential, is not sufficient for TIR-triggered immune responses in plants, suggesting that other components may be required for TIR-mediated signaling. In my dissertation, I employed multiple approaches including biochemistry and structural biology to address these questions. The thesis contains three parts: In the first part, I present multiple lines of evidence showing that the Arabidopsis TNL RPP1 (for recognition of Peronospora parasitica 1) forms a tetrameric resistosome upon recognition of the cognate Hyaloperonospora arabidopsidis effector ATR1. Biochemical and structural data are summarized revealing the mechanism underlying the requirement of the RPP1 resistosome formation for NADase activity. The data from this study define the mechanism of direct effector recognition by a TNL, and demonstrate that the assembly of RPP1 resistosomes is required for TIR-encoded NADase activity and RPP1 function. In the second part, I describe biochemical evidence that TIR domain proteins also exhibit 2′,3′-cAMP/cGMP synthetase activity with RNA and probably DNA (RNA/DNA) as substrates. Then I present functional data supporting the physiological relevance of the synthetase activity in TIR-mediated immune responses. Structural data on a TIR domain protein bound by its dsDNA substrate are described, and the mechanisms of how TIR domain proteins encode both NADase and synthetase activities and how the two activities may act together to mediate TIR signaling are discussed. The data presented in this part reveal a novel enzymatic activity of plant TIR domain proteins and establish a role of 2′,3′-cAMP/cGMP in plant immunity. In the last section of my thesis, I describe experiments testing whether the RNase-like effector proteins associated with haustoria (RALPH effectors) have RNase activity

Gene Regulatory Network Inference Using Machine Learning Techniques

Systems Biology is a field that models complex biological systems in order to better understand the working of cells and organisms. One of the systems modeled is the gene regulatory network that plays the critical role of controlling an organism's response to changes in its environment. Ideally, we would like a model of the complete gene regulatory network. In recent years, several advances in technology have permitted the collection of an unprecedented amount and variety of data such as genomes, gene expression data, time-series data, and perturbation data. This has stimulated research into computational methods that reconstruct, or infer, models of the gene regulatory network from the data. Many solutions have been proposed, yet there remain open challenges in utilising the range of available data as it is inherently noisy, and must be integrated by the inference techniques. The thesis seeks to contribute to this discourse by investigating challenges of performance, scale, and data integration. We propose a new algorithm BENIN that views network inference as feature selection to address issues of scale, that uses elastic net regression for improved performance, and adapts elastic net to integrate different types of biological data. The BENIN algorithm is benchmarked on a synthetic dataset from the DREAM4 challenge, and on real expression data for the human HeLa cell cycle. On the DREAM4 dataset BENIN out-performed all DREAM4 competitors on the size 100 subchallenge, and is also competitive with more recent state-of-the-art methods. Moreover, on the HeLa cell cycle data, BENIN could infer known regulatory interactions and propose new interactions that warrant further experimental investigation. Keys words: gene regulatory network, network inference, feature selection, elastic net regression