Four-amino Acid Research Articles

Determinants of maturation of the Staphylococcus aureus autoinducing peptide.

The accessory gene regulatory (Agr) system is required for virulence factor gene expression and pathogenesis of Staphylococcus aureus. The Agr system is activated in response to the accumulation of a cyclic autoinducing peptide (AIP), which is matured and secreted by the bacterium. The precursor of AIP, AgrD, consists of the AIP flanked by an N-terminal [Formula: see text]-helical Leader and a charged C-terminal tail. AgrD is matured to AIP by the action of two proteases, AgrB and MroQ. AgrB cleaves the C-terminal tail and promotes the formation of a thiolactone ring, whereas MroQ cleaves the N-terminal Leader in a manner that depends on the four-amino acid linker immediately following a conserved IG helix breaker motif. However, the attributes of AgrD that dictate the sequence of events in peptide maturation are not fully defined. Here, we used engineered AgrD peptide intermediates to ascertain the sufficiency of MroQ for N-terminal peptide cleavage, peptide export, and generation of mature AIP. We found that MroQ promotes the removal of the N-terminal Leader peptide from both linear and cyclic peptide intermediates, while peptide cyclization remained essential for signaling. The expression of the Leader peptide in isolation was sufficient for MroQ-dependent cleavage proximal to the four-amino-acid linker. In addition, active site mutations within AgrB destabilized full-length AgrD and thiolactone-containing intermediates and prevented the release of the Leader peptide. Altogether, our data support a tandem peptide maturation event involving both MroQ and AgrB that appears to couple protease activity and export of bioactive AIP.IMPORTANCEThe accessory gene regulatory (Agr) system is important for S. aureus pathogenesis. Activation of the Agr system requires recognition of a cyclic peptide pheromone, which must be fully matured to exert its biological activity. The complete events in cyclic peptide maturation and export from the bacterial cell remain to be fully defined. We and others recently discovered that the membrane peptidase MroQ is required for pheromone maturation. This study builds off the identification of MroQ and considers the attributes of the pheromone pro-peptide that are required for MroQ-mediated processing as well as uncovers features important for peptide stability and export. Overall, the findings in this study have implications for understanding bacterial pheromone maturation and virulence.

The pathogenicity and potential risk evaluation of Zika virus to cause mysterious "Disease X": A brief report.

Disease X is included to represent an unknown pathogen that has the potential to spread globally and trigger significant epidemics. The World Health Organization (WHO) and the Global Alliance for Vaccine and Immunization (GAVI) recommend 10 potential disease organisms responsible for disease X. Among them, the Zika virus (ZIKV) is one of the potential organisms. The present work aimed to evaluate the current pathogenicity and potential risk of the ZIKV for public health emergencies. We performed a brief review of existing literature from available sources using Zika fever, Zika virus, and Flaviviridae as keywords and extracted relevant information for this review. The primary way that the ZIKV transmits to humans is by mosquito bites (Aedes aegypti and Aedes albopictus). Typically, Aedes mosquitoes bite during the day. As an RNA virus, the ZIKV is well-known for its rapid mutation rate. ZIKV strains or variations can arise due to genomic mutations, and they occur through various processes, including replication errors, recombination, and selection pressure. Nowadays, the ZIKV has also evolved into a global pandemic because the four-amino acid sequence fits the envelope protein's 154 glycosylation motif, which may be involved in virulence. There is no specific medicine or vaccine available for the prevention or treatment of ZIKV infection. Therefore, personal protective measures should be the way to prevent ZIKV disease. Therefore, avoiding exposure is the best way to prevent problems from ZIKV to mosquitoes.

Roles of the N-terminal motif in improving the activity and soluble expression of phenylalanine ammonia lyases in Escherichia coli

Phenylalanine ammonia-lyase (PAL) has various applications in fine chemical manufacturing and the pharmaceutical industry. In particular, PAL derived from Anabaena variabilis (AvPAL) is used as a therapeutic agent to the treat phenylketonuria in clinical settings. In this study, we aligned the amino acid sequences of AvPAL and PAL derived from Nostoc punctiforme (NpPAL) to obtain several mutants with enhanced activity, expression yield, and thermal stability via amino acid substitution and saturation mutagenesis at the N-terminal position. Enzyme kinetic experiments revealed that the kcat values of NpPAL-N2K, NpPAL-I3T, and NpPAL-T4L mutants were increased to 3.2-, 2.8-, and 3.3-fold that of the wild-type, respectively. Saturation mutagenesis of the fourth amino acid in AvPAL revealed that the kcat values of AvPAL-L4N, AvPAL-L4P, AvPAL-L4Q and AvPAL-L4S increased to 4.0-, 3.7-, 3.6-, and 3.2-fold, respectively. Additionally, the soluble protein yield of AvPAL-L4K increased to approximately 14 mg/L, which is approximately 3.5-fold that of AvPAL. Molecular dynamics studies further revealed that maintaining the attacking state of the reaction and N-terminal structure increased the rate of catalytic reaction and improved the solubility of proteins. These findings provide new insights for the rational design of PAL in the future.

FgSdhC Paralog Confers Natural Resistance toward SDHI Fungicides in Fusarium graminearum.

Fusarium graminearum exhibited natural resistance to a majority of succinate dehydrogenase inhibitor fungicides (SDHIs) and the molecular mechanisms responsible for the natural resistance were still unknown. Succinate dehydrogenase subunit C (SdhC) is an essential gene for maintaining succinate-ubiquinone oxidoreductase (SQR) function in fungi. In F. graminearum, a paralog of FgSdhC named as FgSdhC1 was identified. Based on RNA-Seq and qRT-PCR assay, we found that the expression level of FgSdhC1 was very low but upregulated by SDHIs treatment. Based on reverse genetics, we demonstrated that FgSdhC1 was an inessential gene in normal growth but was sufficient for maintaining SQR function and conferred natural resistance or reduced sensitivity toward SDHIs. Additionally, we found that the standard F. graminearum isolate PH-1 had high sensitivity to a majority of SDHIs. A single nucleotide variation (C to T) in the FgSdhC1 of isolate PH-1, resulting in a premature termination codon (TAA) replacing the fourth amino acid glutamine (Q), led to the failure of FgSdhC1 to perform functions of conferring nature resistance. These results established that a dispensable paralogous gene determined SDHIs resistance in natural populations of F. graminearum.

Can fosfomycin be an alternative therapy for infections caused by E. coli harbouring dual resistance: NDM and four-amino acid insertion in PBP3?

NDM-expressing Escherichia coli infections are challenging to treat, due to limited treatment options. E. coli with four-amino acid inserts (YRIN/YRIK) are also common in India and it has been reported to reduce the susceptibility to aztreonam/avibactam and the clinically used triple combination ceftazidime/avibactam with aztreonam. Thus, there is a severe dearth of antibiotics to treat infections of NDM + PBP3 insert E. coli. In this study, we determined the susceptibility of E. coli with NDM and PBP3 insert to fosfomycin as an alternative option to treat serious infections. Non-duplicate well-characterized NDM-expressing (without or with co-expression of OXA-48-like) E. coli isolates (n = 213) subsequently carrying four-amino acid inserts in PBP3 were included in this study. MICs of fosfomycin were determined by the agar dilution method with glucose-6-phosphate supplementation, while for other comparators the broth microdilution method was used. Collectively, 98% of NDM-expressing E. coli isolates with PBP3 insert were susceptible to fosfomycin at the MIC of ≤32 mg/L. Resistance to aztreonam was noticed in 38% of the tested isolates. Putting together fosfomycin's in vitro activity, clinical efficacy and safety in randomized controlled trials, we conclude that fosfomycin could be considered as an alternative option to treat infections caused by E. coli harbouring NDM and PBP3 insert resistance mechanisms.

Discovery of a Tricyclic β-Lactam as a Potent Antimicrobial Agent against Carbapenem-Resistant Enterobacterales, Including Strains with Reduced Membrane Permeability and Four-Amino Acid Insertion into Penicillin-Binding Protein 3: Structure-Activity-Relationships and In Vitro and In Vivo Activities.

The current worldwide emergence of carbapenem-resistant enterobacterales (CREs) constitutes an important growing clinical and public health threat. Acquired carbapenemases are the most important determinants of resistance to carbapenems. In the development of the previously reported tricyclic β-lactam skeleton which exhibits potent antibacterial activities against several problematic β-lactamase-producing CREs without a β-lactamase inhibitor, we found that these activities were reduced against clinical isolates with resistance mechanisms other than β-lactamase production. These mechanisms were the reduction of outer membrane permeability with the production of β-lactamases and the insertion of four amino acids into penicillin-binding protein 3. Here, we report the discovery of a potent compound that overcomes these resistance mechanisms by the conversion of the alkoxyimino moiety of the aminothiazole side chain in which a hydrophilic functional group is introduced and the carboxylic acid of the alkoxyimino moiety is converted to reduce the negative charge of the whole molecule from 2 to 1. This potent tricyclic β-lactam is a promising drug candidate for infectious diseases caused by CREs due to its potent therapeutic efficacy in the neutropenic mouse lung infection model and low frequency of producing spontaneously resistant mutants.

SARS-CoV-2 spike and its adaptable furin cleavage site

SARS-CoV-2 spike and its adaptable furin cleavage site

Tryptophanemia is controlled by a tryptophan-sensing mechanism ubiquitinating tryptophan 2,3-dioxygenase

Maintaining stable tryptophan levels is required to control neuronal and immune activity. We report that tryptophan homeostasis is largely controlled by the stability of tryptophan 2,3-dioxygenase (TDO), the hepatic enzyme responsible for tryptophan catabolism. High tryptophan levels stabilize the active tetrameric conformation of TDO through binding noncatalytic exosites, resulting in rapid catabolism of tryptophan. In low tryptophan, the lack of tryptophan binding in the exosites destabilizes the tetramer into inactive monomers and dimers and unmasks a four-amino acid degron that triggers TDO polyubiquitination by SKP1-CUL1-F-box complexes, resulting in proteasome-mediated degradation of TDO and rapid interruption of tryptophan catabolism. The nonmetabolizable analog alpha-methyl-tryptophan stabilizes tetrameric TDO and thereby stably reduces tryptophanemia. Our results uncover a mechanism allowing a rapid adaptation of tryptophan catabolism to ensure quick degradation of excess tryptophan while preventing further catabolism below physiological levels. This ensures a tight control of tryptophanemia as required for both neurological and immune homeostasis.

In silico approach on sequential and structural variability in oryzacystatin and its interaction with cysteine protease enzymes of insect

Phytoprotease inhibitors (PI) are important defence compounds produced by plants against microbes and insect herbivory. Oryzacystatins (OCs) are a group of protease inhibitors from Oryza sativa L. that are specific against cysteine protease enzymes. This study revealed the evolutionary relationship of eleven different oryzacystatins from rice and their interaction with cysteine protease enzymes from brown planthopper (BPH) and striped stem borer (SSB). Three-dimensional structure of eleven different oryzacystatins and six cysteine protease enzymes were homologically modelled, and their interaction was analysed to explore the sequence heterogeneity, structural variability and functional significance. OC XI and OC V showed higher docking score and hydrogen bond interaction with all the six tested cysteine protease enzymes. N terminal glycine residue, central conserved QVVXG, C terminal AVVXXXPW regions are involved in interacting with the active site residue of protease enzymes. Substitution of N terminal glycine by any other residue in OC VI significantly reduced the interaction efficiency with cysteine proteases. In OC XI, glutamine in the fourth position of QVVXG showed higher interaction efficiency with all cysteine proteases than serine at the fourth amino acid position. N terminal glycine plays a vital role in OC XI for interacting with active sites of cysteine protease enzymes whereas, in the OC V central conserved region QVVSG and C terminal PW region plays a major part in the interaction. However, either N terminal or C terminal region along with the central conserved region of oryzacystatin is involved in mediating an efficient interaction with the active site residues of cysteine proteases. Molecular dynamic (MD) simulation study revealed the stability of the OC X1 - cathepsin O2 like and OC V - cathepsin F like complexes during a simulation for 20 ns. Insilico results of the present study predict the potential of oryzacystatins interaction with cysteine protease enzymes of insects.

Feleucin-K3 Analogue with an α-(4-Pentenyl)-Ala Substitution at the Key Site Has More Potent Antimicrobial and Antibiofilm Activities in Vitro and in Vivo.

The development of antimicrobial compounds is now regarded as an urgent problem. Antimicrobial peptides (AMPs) have great potential to become novel antimicrobial drugs. Feleucin-K3 is an α-helical cationic AMP isolated from the skin secretion of the Asian bombinid toad species Bombina orientalis and has antimicrobial activity. In our previous studies, amino acid scanning of Feleucin-K3 was performed to determine the key site affecting its activity. In this study, we investigated and synthesized a series of analogues that have either a natural or an unnatural hydrophobic amino acid substitution at the fourth amino acid residue of Feleucin-K3. Among these analogues, Feleucin-K59 (K59), which has an α-(4-pentenyl)-Ala substitution, was shown to have increased antimicrobial activity against both standard and drug-resistant strains of clinical common bacteria, improved stability, no hemolytic activity at antimicrobial concentrations, and no resistance. In addition, K59 has potent antibiofilm activity in vitro. More importantly, K59 showed better antimicrobial and antibiofilm activities against drug-resistant bacteria in in vivo experiments in mice than traditional antibiotics. In this preliminary study of the mechanism of action, we found that K59 could rapidly kill bacteria by a dual-action mechanism of disrupting the cell membrane and binding to intracellular DNA, thus making it difficult for bacteria to develop resistance.

Crystal Structure of Geranylgeranyl Pyrophosphate Synthase (CrtE) Involved in Cyanobacterial Terpenoid Biosynthesis.

Cyanobacteria are photosynthetic prokaryotes that perform oxygenic photosynthesis. Due to their ability to use the photon energy of sunlight to fix carbon dioxide into biomass, cyanobacteria are promising hosts for the sustainable production of terpenoids, also known as isoprenoids, a diverse class of natural products with potential as advanced biofuels and high-value chemicals. However, the cyanobacterial enzymes involved in the biosynthesis of the terpene precursors needed to make more complicated terpenoids are poorly characterized. Here we show that the predicted type II prenyltransferase CrtE encoded by the model cyanobacterium Synechococcus sp. PCC 7002 is homodimeric and able to synthesize C20-geranylgeranyl pyrophosphate (GGPP) from C5-isopentenyl pyrophosphate (IPP) and C5-dimethylallyl pyrophosphate (DMAPP). The crystal structure of CrtE solved to a resolution of 2.7 Å revealed a strong structural similarity to the large subunit of the heterodimeric geranylgeranyl pyrophosphate synthase 1 from Arabidopsis thaliana with each subunit containing 14 helices. Using mutagenesis, we confirmed that the fourth and fifth amino acids (Met-87 and Ser-88) before the first conserved aspartate-rich motif (FARM) play important roles in controlling chain elongation. While the WT enzyme specifically produced GGPP, variants M87F and S88Y could only generate C15-farnesyl pyrophosphate (FPP), indicating that residues with large side chains obstruct product elongation. In contrast, replacement of M87 with the smaller Ala residue allowed the formation of the longer C25-geranylfarnesyl pyrophosphate (GFPP) product. Overall, our results provide new structural and functional information on the cyanobacterial CrtE enzyme that could lead to the development of improved cyanobacterial platforms for terpenoid production.

In vitro activity of the novel β-lactamase inhibitor taniborbactam (VNRX-5133), in combination with cefepime or meropenem, against MDR Gram-negative bacterial isolates from China.

To evaluate in vitro activity of the novel β-lactamase boronate inhibitor taniborbactam (VNRX-5133) combined with cefepime or meropenem against 500 urinary Gram-negative bacilli. Cefepime/taniborbactam and 14 comparators were tested by broth microdilution or agar dilution methods. A total of 450 Enterobacteriaceae and 50 Pseudomonas aeruginosa were selected from 2017 to 2019 based on different β-lactamase-producing or resistance phenotypes. For carbapenem-non-susceptible isolates, the modified carbapenem inactivation method (mCIM), EDTA-CIM (eCIM) and amplification of carbapenemase genes were performed. For NDM-producing isolates and those with cefepime/taniborbactam MICs >8 mg/L, the MICs of meropenem/taniborbactam and/or mutations in PBP3 were investigated. Taniborbactam improved cefepime activity with the same efficiency as avibactam improved ceftazidime activity against 66 KPC-2 producers, 30 non-carbapenemase-producing carbapenem-non-susceptible Enterobacteriaceae and 28 meropenem-susceptible P. aeruginosa. However, cefepime/taniborbactam exhibited more potent activity than ceftazidime/avibactam against 56 ESBL-producing, 61 AmpC-producing, 32 ESBL and AmpC co-producing, 87 NDM-producing and 21 MBL-producing Enterobacteriaceae predicted by phenotypic mCIM and eCIM, 82 Enterobacteriaceae that were susceptible to all tested β-lactams and 22 carbapenem-non-susceptible P. aeruginosa. A four-amino acid 'INYR' or 'YRIN' insertion, with or without a one/two-amino acid mutation in PBP3, may have caused cefepime/taniborbactam MICs >8 mg/L among 96.6% (28/29) of the NDM-5-producing Escherichia coli, which accounted for the majority of isolates with cefepime/taniborbactam MICs >8 mg/L (76.1%, 35/46). Taniborbactam's superior breadth of activity, when paired with cefepime or meropenem, suggests these β-lactam/β-lactamase inhibitor combinations could be promising candidates for treating urinary tract infections caused by ESBL and/or AmpC, KPC or NDM-producing Enterobacteriaceae or P. aeruginosa.

Protein Farnesyltransferase Catalyzes Unanticipated Farnesylation and Geranylgeranylation of Shortened Target Sequences.

Protein prenylation is a posttranslational modification involving the attachment of a C15 or C20 isoprenoid group to a cysteine residue near the C-terminus of the target substrate by protein farnesyltransferase (FTase) or protein geranylgeranyltransferase type I (GGTase-I), respectively. Both of these protein prenyltransferases recognize a C-terminal "CaaX" sequence in their protein substrates, but recent studies in yeast- and mammalian-based systems have demonstrated FTase can also accept sequences that diverge in length from the canonical four-amino acid motif, such as the recently reported five-amino acid C(x)3X motif. In this work, we further expand the substrate scope of FTase by demonstrating sequence-dependent farnesylation of shorter three-amino acid "Cxx" C-terminal sequences using both genetic and biochemical assays. Strikingly, biochemical assays utilizing purified mammalian FTase and Cxx substrates reveal prenyl donor promiscuity leading to both farnesylation and geranylgeranylation of these sequences. These findings expand the substrate pool of sequences that can be potentially prenylated, further refine our understanding of substrate recognition by FTase and GGTase-I, and suggest the possibility of a new class of prenylated proteins within proteomes.

Engineering a Proximity-Directed O-GlcNAc Transferase for Selective Protein O-GlcNAcylation in Cells.

O-Linked β-N-acetylglucosamine (O-GlcNAc) is a monosaccharide that plays an essential role in cellular signaling throughout the nucleocytoplasmic proteome of eukaryotic cells. Strategies for selectively increasing O-GlcNAc levels on a target protein in cells would accelerate studies of this essential modification. Here, we report a generalizable strategy for introducing O-GlcNAc into selected target proteins in cells using a nanobody as a proximity-directing agent fused to O-GlcNAc transferase (OGT). Fusion of a nanobody that recognizes GFP (nGFP) or a nanobody that recognizes the four-amino acid sequence EPEA (nEPEA) to OGT yielded nanobody-OGT constructs that selectively delivered O-GlcNAc to a series of tagged target proteins (e.g., JunB, cJun, and Nup62). Truncation of the tetratricopeptide repeat domain as in OGT(4) increased selectivity for the target protein through the nanobody by reducing global elevation of O-GlcNAc levels in the cell. Quantitative chemical proteomics confirmed the increase in O-GlcNAc to the target protein by nanobody-OGT(4). Glycoproteomics revealed that nanobody-OGT(4) or full-length OGT produced a similar glycosite profile on the target protein JunB and Nup62. Finally, we demonstrate the ability to selectively target endogenous α-synuclein for O-GlcNAcylation in HEK293T cells. These first proximity-directed OGT constructs provide a flexible strategy for targeting additional proteins and a template for further engineering of OGT and the O-GlcNAc proteome in the future. The use of a nanobody to redirect OGT substrate selection for glycosylation of desired proteins in cells may further constitute a generalizable strategy for controlling a broader array of post-translational modifications in cells.

Identification of three linear B cell epitopes against non-structural protein 3ABC of FMDV using monoclonal antibodies.

Foot-and-mouth disease virus (FMDV) has led to serious losses in the farming industry worldwide, particularly in cattle and swine. In developing countries, the control and eradication of FMD rely upon vaccination, in which the inactivated vaccine is predominant. In the preparation of inactivated vaccine, a series of purification methods were used to remove non-structural proteins (NSPs). It is necessary to develop a quantitative detection method of residual NSP and confirm a threshold value for the evaluation of the vaccine. Meanwhile, it is also important to develop a sensitive and rapid diagnostic method to distinguish infected animals from vaccinated animals (DIVA). In this study, three monoclonal antibodies (MAbs) against NSP 3ABC, designated 2G5, 9E2, and 1E10, were used. Subsequently, a series of overlapping peptides were expressed using a prokaryotic expression system to determine the minimal epitopes identified by the MAbs. Three linear B cell epitopes (BCEs), "92EYIEKA97" "23EGPYAGPLE31" and "209EPHH212", were identified by MAbs 2G5, 9E2, and 1E10, respectively. Alanine-scanning mutagenesis analysis confirmed the critical amino acid in these epitopes. The epitope "92EYIEKA97" is located in 3A, which is deleted in some natural deletion mutants that result in a change in virus tropism. MAb 9E2 that identified the epitope "23EGPYAGPLE31" reacted with 3B1 and 3B2, but did not react with 3B3. In combination with sequence alignment analysis, the epitope "23EGPYAGPLE31" is highly conserved among different FMDV isolates. Preliminary screening using the known positive and negative sera indicated the MAb 9E2 has the potential for the development of a diagnostic method for DIVA. The residual NSP in inactivated vaccines can be detected using 9E2-HRP, which indicated the MAb 9E2 is able to evaluate inactivated vaccines. The four-amino acid epitope is the first reported to date that is recognized by 1E10. These results provide valuable insight into the diagnosis of DIVA and the NSP residual evaluation in inactivated vaccines.

The expanded specificity and physiological role of a widespread N-degron recognin

All cells use proteases to maintain protein homeostasis. The proteolytic systems known as the N-degron pathways recognize signals at the N terminus of proteins and bring about the degradation of these proteins. The ClpS protein enforces the N-degron pathway in bacteria and bacteria-derived organelles by targeting proteins harboring leucine, phenylalanine, tryptophan, or tyrosine at the N terminus for degradation by the protease ClpAP. We now report that ClpS binds, and ClpSAP degrades, proteins still harboring the N-terminal methionine. We determine that ClpS recognizes a type of degron in intact proteins based on the identity of the fourth amino acid from the N terminus, showing a strong preference for large hydrophobic amino acids. We uncover natural ClpS substrates in the bacterium Salmonella enterica, including SpoT, the essential synthase/hydrolase of the alarmone (p)ppGpp. Our findings expand both the specificity and physiological role of the widespread N-degron recognin ClpS.

A 25-Residue Peptide From Botrytis cinerea Xylanase BcXyn11A Elicits Plant Defenses.

Plants activate defense responses against a possible pathogen once pattern-recognition receptors (PRRs) perceive the presence of pathogen-associated molecular patterns (PAMPs). Glycosyl hydrolase family 11 (GH11) endoxylanases from Trichoderma, Fusarium and Botrytis species have been described as being able to induce the defense response in plants, in a way that is independent of its enzymatic activity. However, until now, it has not been possible to establish with certainty which regions of these enzymes are recognized by plants as PAMPs. We show here for the first time that a short 25-residue peptide (named Xyn25) from the Botrytis cinerea xylanase BcXyn11A can reproduce by itself all the effects observed for the treatment of plants with the whole BcXyn11A protein. These include necrosis on leaves, seedling growth inhibition, induction of a ROS burst, electrolyte leakage, cytoplasm shrinkage, autofluorescence, cell death, and induction of defense genes. Two highly conserved four-amino acid regions within Xyn25 were shown to be necessary for the elicitation activity by substituting them with tracts of four alanine residues.

Identification of key amino acid residues determining product specificity of 2,3-oxidosqualene cyclase in Oryza species.

Triterpene synthases, also known as 2,3-oxidosqualene cyclases (OSCs), synthesize diverse triterpene skeletons that form the basis of an array of functionally divergent steroids and triterpenoids. Tetracyclic and pentacyclic triterpene skeletons are synthesized via protosteryl and dammarenyl cations, respectively. The mechanism of conversion between two scaffolds is not well understood. Here, we report a promiscuous OSC from rice (Oryza sativa) (OsOS) that synthesizes a novel pentacyclic triterpene orysatinol as its main product. The OsOS gene is widely distributed in indica subspecies of cultivated rice and in wild rice accessions. Previously, we have characterized a different OSC, OsPS, a tetracyclic parkeol synthase found in japonica subspecies. Phylogenetic and protein structural analyses identified three key amino acid residues (#732, #365, #124) amongst 46 polymorphic sites that determine functional conversion between OsPS and OsOS, specifically, the chair-semi(chair)-chair and chair-boat-chair interconversions. The different orientation of a fourth amino acid residue Y257 was shown to be important for functional conversion The discovery of orysatinol unlocks a new path to triterpene diversity in nature. Our findings also reveal mechanistic insights into the cyclization of oxidosqualene into tetra- and pentacyclic skeletons, and provide a new strategy to identify key residues determining OSC specificity.

Efficient farnesylation of an extended C-terminal C(x)3X sequence motif expands the scope of the prenylated proteome

Protein prenylation is a post-translational modification that has been most commonly associated with enabling protein trafficking to and interaction with cellular membranes. In this process, an isoprenoid group is attached to a cysteine near the C terminus of a substrate protein by protein farnesyltransferase (FTase) or protein geranylgeranyltransferase type I or II (GGTase-I and GGTase-II). FTase and GGTase-I have long been proposed to specifically recognize a four-amino acid CAAX C-terminal sequence within their substrates. Surprisingly, genetic screening reveals that yeast FTase can modify sequences longer than the canonical CAAX sequence, specifically C(x)3X sequences with four amino acids downstream of the cysteine. Biochemical and cell-based studies using both peptide and protein substrates reveal that mammalian FTase orthologs can also prenylate C(x)3X sequences. As the search to identify physiologically relevant C(x)3X proteins begins, this new prenylation motif nearly doubles the number of proteins within the yeast and human proteomes that can be explored as potential FTase substrates. This work expands our understanding of prenylation's impact within the proteome, establishes the biologically relevant reactivity possible with this new motif, and opens new frontiers in determining the impact of non-canonically prenylated proteins on cell function.

C-reactive protein and pentraxin-3 binding of factor H-like protein 1 differs from complement factor H: implications for retinal inflammation

Retinal inflammation plays a key role in the progression of age-related macular degeneration (AMD), a condition that leads to loss of central vision. The deposition of the acute phase pentraxin C-reactive protein (CRP) in the macula activates the complement system, thereby contributing to dysregulated inflammation. The complement protein factor H (FH) can bind CRP and down-regulate an inflammatory response. However, it is not known whether a truncated form of FH, called factor H-like protein 1 (FHL-1), which plays a significant regulatory role in the eye, also interacts with CRP. Here, we compare the binding properties of FHL-1 and FH to both CRP and the related protein pentraxin-3 (PTX3). We find that, unlike FH, FHL-1 can bind pro-inflammatory monomeric CRP (mCRP) as well as the circulating pentameric form. Furthermore, the four-amino acid C-terminal tail of FHL-1 (not present in FH) plays a role in mediating its binding to mCRP. PTX3 was found to be present in the macula of donor eyes and the AMD-associated Y402H polymorphism altered the binding of FHL-1 to PTX3. Our findings reveal that the binding characteristics of FHL-1 differ from those of FH, likely underpinning independent immune regulatory functions in the context of the human retina.