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1. Profiling thimet oligopeptidase‐mediated proteolysis in Arabidopsis thaliana

   https://doi.org/10.1111/tpj.15165  

   Iannetta, Anthony A. ; Rogers, Holden T. ; Al‐Mohanna, Thualfeqar ; O’Brien, Juliana N. ; Wommack, Andrew J. ; Popescu, Sorina C. ; Hicks, Leslie M. ( February 2021 , The Plant Journal)

   Protein homeostasis (proteostasis) is crucial for proper cellular function, including the production of peptides with biological functions through controlled proteolysis. Proteostasis has roles in maintenance of cellular functions and plant interactions with the environment under physiological conditions. Plant stress continues to reduce agricultural yields causing substantial economic losses; thus, it is critical to understand how plants perceive stress signals to elicit responses for survival. As previously shown inArabidopsis thaliana, thimet oligopeptidases (TOPs) TOP1 (also referred to as organellar oligopeptidase) and TOP2 (also referred to as cytosolic oligopeptidase) are essential components in plant response to pathogens, but further characterization of TOPs and their peptide substrates is required to understand their contributions to stress perception and defense signaling. Herein, label‐free peptidomics via liquid chromatography‐tandem mass spectrometry was used to differentially quantify 1111 peptides, originating from 369 proteins, between the Arabidopsis Col‐0 wild type andtop1top2knock‐out mutant. This revealed 350 peptides as significantly more abundant in the mutant, representing accumulation of these potential TOP substrates. Ten direct substrates were validated usingin vitroenzyme assays with recombinant TOPs and synthetic candidate peptides. These TOP substrates are derived from proteins involved in photosynthesis, glycolysis, protein folding, biogenesis, and antioxidant defense, implicating TOP involvement in processes aside from defense signaling. Sequence motif analysis revealed TOP cleavage preference for non‐polar residues in the positions surrounding the cleavage site. Identification of these substrates provides a framework for TOP signaling networks, through which the interplay between proteolytic pathways and defense signaling can be further characterized.

    

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2. The maize ZmMIEL1 E3 ligase and ZmMYB83 transcription factor proteins interact and regulate the hypersensitive defence response

   https://doi.org/10.1111/mpp.13057  

   Karre, Shailesh ; Kim, Saet‐Byul ; Samira, Rozalynne ; Balint‐Kurti, Peter ( April 2021 , Molecular Plant Pathology)

   The plant hypersensitive response (HR), a rapid cell death at the point of pathogenesis, is mediated by nucleotide‐binding site, leucine‐rich repeat (NLR) resistance proteins (R‐proteins) that recognize the presence of specific pathogen‐derived proteins. Rp1‐D21 is an autoactive maize NLR R‐protein that triggers HR spontaneously. We previously mapped loci associated with variation in the strength of HR induced by Rp1‐D21. Here we identify the E3 ligaseZmMIEL1as the causal gene at a chromosome 10 modifier locus. TransientZmMIEL1expression inNicotiana benthamianareduced HR induced by Rp1‐D21, while suppression ofZmMIEL1expression in maize carryingRp1‐D21increased HR. ZmMIEL1 also suppressed HR induced by another autoactive NLR, theArabidopsisR‐protein RPM1D505V, inN. benthamiana. We demonstrated that ZmMIEL1 is a functional E3 ligase and that the effect of ZmMIEL1 was dependent on the proteasome but also that levels of Rp1‐D21 and RPM1D505V were not reduced when coexpressed with ZmMIEL1 in theN. benthamianasystem. By comparison to a similar system inArabidopsis, we identify ZmMYB83 as a potential target of ZmMIEL1. Suppression ofZmMYB83expression in maize lines carryingRp1‐D21suppressed HR. Suppression ofZmMIEL1expression caused an increase inZmMYB83transcript and protein levels inN. benthamianaand maize. Using coimmunoprecipitation and bimolecular fluorescence complementation assays, we demonstrated that ZmMIEL1 and ZmMYB83 physically interacted. Additionally,ZmMYB83andZmMIEL1regulated the expression of a set of maize very long chain fatty acid (VLCFA) biosynthetic genes that may be involved in regulating HR.

    

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3. A Quantitative Arabidopsis IRE1a Ribonuclease-Dependent in vitro mRNA Cleavage Assay for Functional Studies of Substrate Splicing and Decay Activities

   https://doi.org/10.3389/fpls.2021.707378  

   Diwan, Danish ; Liu, Xiaoyu ; Andrews, Caroline F. ; Pajerowska-Mukhtar, Karolina M. ( July 2021 , Frontiers in Plant Science)

   The unfolded protein response (UPR) is an adaptive eukaryotic reaction that controls the protein folding capacities of the endoplasmic reticulum (ER). The most ancient and well-conserved component of the UPR is Inositol-Requiring Enzyme 1 (IRE1). Arabidopsis IRE1a (AtIRE1) is a transmembrane sensor of ER stress equipped with dual protein kinase and ribonuclease (RNase) activities, encoded by its C-terminal domain. In response to both physiological stresses and pathological perturbations, AtIRE1a directly cleaves bZIP60 ( basic leucine zipper 60 ) mRNA. Here, we developed a quantitative in vitro cleavage assay that combines recombinant AtIRE1a protein that is expressed in Nicotiana benthamiana and total RNA isolated from Arabidopsis leaves. Wild-type AtIRE1a as well as its variants containing point mutations in the kinase or RNase domains that modify its cleavage activity were employed to demonstrate their contributions to cleavage activity levels. We show that, when exposed to total RNA in vitro , the AtIRE1a protein cleaves bZIP60 mRNA. Depletion of the bZIP60 transcript in the reaction mixture can be precisely quantified by a qRT-PCR-mediated assay. This method facilitates the functional studies of novel plant IRE1 variants by allowing to quickly and precisely assess the effects of protein mutations on the substrate mRNA cleavage activity before advancing to more laborious, stable transgenic approaches in planta . Moreover, this method is readily adaptable to other plant IRE1 paralogs and orthologs, and can also be employed to test additional novel mRNA substrates of plant IRE1, such as transcripts undergoing degradation through the process of regulated IRE1-dependent decay (RIDD). Finally, this method can also be modified and expanded to functional testing of IRE1 interactors and inhibitors, as well as for studies on the molecular evolution of IRE1 and its substrates, providing additional insights into the mechanistic underpinnings of IRE1-mediated ER stress homeostasis in plant tissues. 

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4. CPProtectides: Rapid uptake of well‐folded β‐hairpin peptides with enhanced resistance to intracellular degradation

   https://doi.org/10.1002/pep2.24092  

   Safa, Nora ; Anderson, Jeffery C. ; Vaithiyanathan, Manibarathi ; Pettigrew, Jacob H. ; Pappas, Gavin A. ; Liu, Dong ; Gauthier, Ted J. ; Melvin, Adam T. ( September 2018 , Peptide Science)

   Cell penetrating peptides (CPPs) have emerged as powerful tools for delivering bioactive cargoes, such as biosensors or drugs to intact cells. One limitation of CPPs is their rapid degradation by intracellular proteases. β‐hairpin “protectides” have previously been demonstrated to be long‐lived under cytosolic conditions due to their secondary structure. The goal of this work was to demonstrate that arginine‐rich β‐hairpin peptides function as both protectides and as CPPs. Peptides exhibiting a β‐hairpin motif were found to be rapidly internalized into cells with their uptake efficiency dependent on the number of arginine residues in the sequence. Cellular internalization of the β‐hairpin peptides was compared to unstructured, scrambled sequences and to commercially available, arginine‐rich CPPs. The unstructured peptides displayed greater uptake kinetics compared to the structured β‐hairpin sequences; however, intracellular stability studies revealed that the β‐hairpin peptides exhibited superior stability under cytosolic conditions with a 16‐fold increase in peptide half‐life. This study identifies a new class of long‐lived CPPs that can overcome the stability limitations of peptide‐based reporters or bioactive delivery mechanisms in intact cells.

    

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5. Cell Penetrating Peptides, Novel Vectors for Gene Therapy

   https://doi.org/10.3390/pharmaceutics12030225  

   Taylor, Rebecca E. ; Zahid, Maliha ( March 2020 , Pharmaceutics)

   null (Ed.)

   Cell penetrating peptides (CPPs), also known as protein transduction domains (PTDs), first identified ~25 years ago, are small, 6–30 amino acid long, synthetic, or naturally occurring peptides, able to carry variety of cargoes across the cellular membranes in an intact, functional form. Since their initial description and characterization, the field of cell penetrating peptides as vectors has exploded. The cargoes they can deliver range from other small peptides, full-length proteins, nucleic acids including RNA and DNA, liposomes, nanoparticles, and viral particles as well as radioisotopes and other fluorescent probes for imaging purposes. In this review, we will focus briefly on their history, classification system, and mechanism of transduction followed by a summary of the existing literature on use of CPPs as gene delivery vectors either in the form of modified viruses, plasmid DNA, small interfering RNA, oligonucleotides, full-length genes, DNA origami or peptide nucleic acids. 

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