Protein Of Unknown Function Research Articles

To Be or Not to Be an Ion Channel: Cryo-EM Structures Have a Say.

Ion channels are the second largest class of drug targets after G protein-coupled receptors. In addition to well-recognized ones like voltage-gated Na/K/Ca channels in the heart and neurons, novel ion channels are continuously discovered in both excitable and non-excitable cells and demonstrated to play important roles in many physiological processes and diseases such as developmental disorders, neurodegenerative diseases, and cancer. However, in the field of ion channel discovery, there are an unignorable number of published studies that are unsolid and misleading. Despite being the gold standard of a functional assay for ion channels, electrophysiological recordings are often accompanied by electrical noise, leak conductance, and background currents of the membrane system. These unwanted signals, if not treated properly, lead to the mischaracterization of proteins with seemingly unusual ion-conducting properties. In the recent ten years, the technical revolution of cryo-electron microscopy (cryo-EM) has greatly advanced our understanding of the structures and gating mechanisms of various ion channels and also raised concerns about the pore-forming ability of some previously identified channel proteins. In this review, we summarize cryo-EM findings on ion channels with molecular identities recognized or disputed in recent ten years and discuss current knowledge of proposed channel proteins awaiting cryo-EM analyses. We also present a classification of ion channels according to their architectures and evolutionary relationships and discuss the possibility and strategy of identifying more ion channels by analyzing structures of transmembrane proteins of unknown function. We propose that cross-validation by electrophysiological and structural analyses should be essentially required for determining molecular identities of novel ion channels.

Longitudinal map of transcriptome changes in the Lyme pathogen Borrelia burgdorferi during tick-borne transmission

Borrelia burgdorferi (Bb), the causative agent of Lyme disease, adapts to vastly different environments as it cycles between tick vector and vertebrate host. During a tick bloodmeal, Bb alters its gene expression to prepare for vertebrate infection; however, the full range of transcriptional changes that occur over several days inside of the tick are technically challenging to capture. We developed an experimental approach to enrich Bb cells to longitudinally define their global transcriptomic landscape inside nymphal Ixodes scapularis ticks during a transmitting bloodmeal. We identified 192 Bb genes that substantially change expression over the course of the bloodmeal from 1 to 4 days after host attachment. The majority of upregulated genes encode proteins found at the cell envelope or proteins of unknown function, including 45 outer surface lipoproteins embedded in the unusual protein-rich coat of Bb. As these proteins may facilitate Bb interactions with the host, we utilized mass spectrometry to identify candidate tick proteins that physically associate with Bb. The Bb enrichment methodology along with the ex vivo Bb transcriptomes and candidate tick interacting proteins presented here provide a resource to facilitate investigations into key determinants of Bb priming and transmission during the tick stage of its unique transmission cycle.

Longitudinal map of transcriptome changes in the Lyme pathogen Borrelia burgdorferi during tick-borne transmission.

Borrelia burgdorferi (Bb), the causative agent of Lyme disease, adapts to vastly different environments as it cycles between tick vector and vertebrate host. During a tick bloodmeal, Bb alters its gene expression to prepare for vertebrate infection; however, the full range of transcriptional changes that occur over several days inside of the tick are technically challenging to capture. We developed an experimental approach to enrich Bb cells to longitudinally define their global transcriptomic landscape inside nymphal Ixodes scapularis ticks during a transmitting bloodmeal. We identified 192 Bb genes that substantially change expression over the course of the bloodmeal from 1 to 4 days after host attachment. The majority of upregulated genes encode proteins found at the cell envelope or proteins of unknown function, including 45 outer surface lipoproteins embedded in the unusual protein-rich coat of Bb. As these proteins may facilitate Bb interactions with the host, we utilized mass spectrometry to identify candidate tick proteins that physically associate with Bb. The Bb enrichment methodology along with the ex vivo Bb transcriptomes and candidate tick interacting proteins presented here provide a resource to facilitate investigations into key determinants of Bb priming and transmission during the tick stage of its unique transmission cycle.

Identification of IQCH as a calmodulin-associated protein required for sperm motility in humans

Sperm fertilization ability mainly relies on proper sperm progression through the female genital tract and capacitation, which involves phosphorylation signaling pathways triggered by calcium and bicarbonate. We performed exome sequencing of an infertile asthenozoospermic patient and identified truncating variants in MAP7D3, encoding a microtubule-associated protein, and IQCH, encoding a protein of unknown function with enzymatic and signaling features. We demonstrate the deleterious impact of both variants on sperm transcripts and proteins from the patient. We show that, invitro, patient spermatozoa could not induce the phosphorylation cascades associated with capacitation. We also provide evidence for IQCH association with calmodulin, a well-established calcium-binding protein that regulates the calmodulin kinase. Notably, we describe IQCH spatial distribution around the sperm axoneme, supporting its function within flagella. Overall, our work highlights the cumulative pathological impact of gene mutations and identifies IQCH as a key protein required for sperm motility and capacitation.

Role of fourteen XRE-DUF397 pairs from Streptomyces coelicolor as regulators of antibiotic production and differentiation. New players in a complex regulatory network.

Bacteria of the genus Streptomyces have a plethora of transcriptional regulators, among which the xenobiotic response element (XRE) plays an important role. In this organism, XRE regulators are often followed downstream by small proteins of unknown function containing a DUF397 domain. It has been proposed that XRE/DUF397 pairs constitute type II toxin-antitoxin (TA) systems. However, previous work carried out by our group has shown that one of these systems is a strong activator of antibiotic production in S. coelicolor and other Streptomyces species. In this work, we have studied the overexpression of fourteen XRE/DUF397 pairs present in the S. coelicolor genome and found that none behave as a type II TA system. Instead, they act as pleiotropic regulators affecting, in a dependent manner, antibiotic production and morphological differentiation on different culture media. After deleting, individually, six XRE/DUF397 pairs (those systems producing more notable phenotypic changes when overexpressed: SCO2246/45, SCO2253/52, SCO4176/77, SCO4678/79, SCO6236/35, and SCO7615/16), the pair SCO7615/16 was identified as producing the most dramatic differences as compared to the wild-type strain. The SCO7615/16 mutant had a different phenotype on each of the media tested (R2YE, LB, NMMP, YEPD, and MSA). In particular, on R2YE and YEPD media, a bald phenotype was observed even after 7 days, with little or no actinorhodin (ACT) production. Lower ACT production was also observed on LB medium, but the bacteria were able to produce aerial mycelium. On NMMP medium, the mutant produced a larger amount of ACT as compared with the wild-type strain.

Molecular characterization of a novel ambiguivirus isolated from the phytopathogenic fungus Setosphaeria turcica.

In this study, a novel single-stranded RNA virus was isolated from the plant-pathogenic fungus Setosphaeria turcica strain TG2, and the virus was named "Setosphaeria turcica ambiguivirus 2" (StAV2). The complete nucleotide sequence of the StAV2 genome was determined using RT-PCR and RLM-RACE. The StAV2 genome comprises 3,000 nucleotides with a G+C content of 57.77%. StAV2 contains two in-frame open reading frames (ORFs) with the potential to produce an ORF1-ORF2 fusion protein via a stop codon readthrough mechanism. ORF1 encodes a hypothetical protein (HP) of unknown function. The ORF2-encoded protein shows a high degree of sequence similarity to the RNA-dependent RNA polymerases (RdRps) of ambiguiviruses. BLASTp searches showed that the StAV2 HP and RdRp share the highest amino acid sequence identity (46.38% and 69.23%, respectively) with the corresponding proteins of a virus identified as "Riboviria sp." isolated from a soil sample. Multiple sequence alignments and phylogenetic analysis based on the amino acid sequences of the RdRp revealed that StAV2 is a new member of the proposed family "Ambiguiviridae".

The enigmatic nucleus of the marine dinoflagellate Prorocentrum cordatum.

The marine, bloom-forming dinoflagellate Prorocentrum cordatum CCMP 1329 (formerly P. minimum) has a genome atypical of eukaryotes, with a large size of ~4.15 Gbp, organized in plentiful, highly condensed chromosomes and packed in a dinoflagellate-specific nucleus (dinokaryon). Here, we apply microscopic and proteogenomic approaches to obtain new insights into this enigmatic nucleus of axenic P. cordatum. High-resolution focused ion beam/scanning electron microscopy analysis of the flattened nucleus revealed highest density of nuclear pores in the vicinity of the nucleolus, a total of 62 tightly packed chromosomes (~0.4-6.7 µm3), and interaction of several chromosomes with the nucleolus and other nuclear structures. A specific procedure for enriching intact nuclei was developed to enable proteomic analyses of soluble and membrane protein-enriched fractions. These were analyzed with geLC and shotgun approaches employing ion-trap and timsTOF (trapped-ion-mobility-spectrometry time-of-flight) mass spectrometers, respectively. This allowed identification of 4,052 proteins (39% of unknown function), out of which 418 were predicted to serve specific nuclear functions; additional 531 proteins of unknown function could be allocated to the nucleus. Compaction of DNA despite very low histone abundance could be accomplished by highly abundant major basic nuclear proteins (HCc2-like). Several nuclear processes including DNA replication/repair and RNA processing/splicing can be fairly well explained on the proteogenomic level. By contrast, transcription and composition of the nuclear pore complex remain largely elusive. One may speculate that the large group of potential nuclear proteins with currently unknown functions may serve yet to be explored functions in nuclear processes differing from those of typical eukaryotic cells. IMPORTANCE Dinoflagellates form a highly diverse group of unicellular microalgae. They provide keystone species for the marine ecosystem and stand out among others by their very large, unusually organized genomes embedded in the nuclei markedly different from other eukaryotic cells. Functional insights into nuclear and other cell biological structures and processes of dinoflagellates have long been hampered by the paucity of available genomic sequences. The here studied cosmopolitan P. cordatum belongs to the harmful algal bloom-forming, marine dinoflagellates and has a recently de novo assembled genome. We present a detailed 3D reconstruction of the P. cordatum nucleus together with comprehensive proteogenomic insights into the protein equipment mastering the broad spectrum of nuclear processes. This study significantly advances our understanding of mechanisms and evolution of the conspicuous dinoflagellate cell biology.

Four distinct isolates of a novel polymycovirus identified in Setosphaeria turcica.

Isolation and analysis of double-stranded RNA (dsRNA) from the phytopathogenic fungus Setosphaeria turcica f. sp. zeae revealed the presence of a new double-stranded RNA (dsRNA) virus, tentatively named "Setosphaeria turcica polymycovirus 2" (StPmV2). The genome of StPmV2 consists of five segments (dsRNA1-5), ranging in size from 965 bp to 2462 bp. Each dsRNA contains oneopen reading frame (ORF) flanked by 5' and 3' untranslated regions (UTRs) with conserved terminal sequences.The putative protein encoded by dsRNA1 shows 64.52% amino acid sequence identity to the RNA-dependent RNA polymerase (RdRp) of the most closely related virus, Cladosporium cladosporioides virus 1, which belongs to the family Polymycoviridae. dsRNAs 2-4 encode the putative coat protein, methyltransferase (MTR), and proline-alanine-serine-rich protein (PASrp), respectively, and dsRNA5 encodes a protein of unknown function. Phylogenetic analysis based on the RdRp protein indicated that StPmV2 clustered with members of the family Polymycoviridae and is therefore a new mycovirus belonging to the genus Polymycovirus in the family Polymycoviridae. In addition, three other distinct isolates of StPmV2 were identified: one isolated from S. turcica f. sp. zeae and two from S. turcica f. sp. sorghi. To our knowledge, this is the first report of a polymycovirus infecting both S. turcica f. sp. zeae and S. turcica f. sp. sorghi.

Global Proteome Profiling Revealed the Adaptive Reprogramming of Barley Flag Leaf to Drought and Elevated Temperature.

Plants, as sessile organisms, have developed sophisticated mechanisms to survive in changing environments. Recent advances in omics approaches have facilitated the exploration of plant genomes; however, the molecular mechanisms underlying the responses of barley and other cereals to multiple abiotic stresses remain largely unclear. Exposure to stress stimuli affects many proteins with regulatory and protective functions. In the present study, we employed liquid chromatography coupled with high-resolution mass spectrometry to identify stress-responsive proteins on the genome-wide scale of barley flag leaves exposed to drought, heat, or both. Profound alterations in the proteome of genotypes with different flag leaf sizes were found. The role of stress-inducible proteins was discussed and candidates underlying the universal stress response were proposed, including dehydrins. Moreover, the putative functions of several unknown proteins that can mediate responses to stress stimuli were explored using Pfam annotation, including calmodulin-like proteins. Finally, the confrontation of protein and mRNA abundances was performed. A correlation network between transcripts and proteins performance revealed several components of the stress-adaptive pathways in barley flag leaf. Taking the findings together, promising candidates for improving the tolerance of barley and other cereals to multivariate stresses were uncovered. The presented proteomic landscape and its relationship to transcriptomic remodeling provide novel insights for understanding the molecular responses of plants to environmental cues.

Proximity-dependent biotin identification links cholesterol catabolism with branched-chain amino acid degradation in Mycobacterium smegmatis.

Cholesterol is a crucial component in Mycobacterium tuberculosis virulence as it is required for phagocytosis of mycobacteria by macrophages. In addition, the tubercle bacilli can grow using cholesterol as the sole carbon source. Thus, cholesterol catabolism represents a valuable target for the development of new antitubercular drugs. However, the molecular partners of cholesterol catabolism remain elusive in mycobacteria. Here, we focused on HsaC and HsaD, enzymes involved in two consecutive steps of cholesterol ring degradation and identified putative partners, using a BirA-based proximity-dependent biotin identification (BioID) approach in Mycobacterium smegmatis. In rich medium, the fusion protein BirA-HsaD was able to fish the endogenous cognate HsaC, thus validating this approach to study protein-protein interactions and to infer metabolic channeling of cholesterol ring degradation. In chemically defined medium, both HsaC and HsaD interacted with four proteins, BkdA, BkdB, BkdC, and MSMEG_1634. BkdA, BkdB, and BkdC are enzymes that participate in the degradation of branched-chain amino acids. As cholesterol and branched-chain amino acid catabolism both generate propionyl-CoA, which is a toxic metabolite for mycobacteria, this interconnection suggests a compartmentalization to avoid dissemination of propionyl-CoA into the mycobacterial cytosol. Moreover, the BioID approach allowed us to decipher the interactome of MSMEG_1634 and MSMEG_6518, two proteins of unknown function, which are proximal to the enzymes involved in cholesterol and branched-chain amino acid catabolism. In conclusion, BioID is a powerful tool to characterize protein-protein interactions and to decipher the interconnections between different metabolic pathways, thereby facilitating the identification of new mycobacterial targets.

Molecular characterization of a novel fungal alphaflexivirus reveals potential inter-species horizontal gene transfer

Sclerotinia sclerotiorum is a notorious phytopathogenic fungus that harbors diverse mycoviruses. A novel positive-sense single-stranded RNA virus, Sclerotinia sclerotiorum alphaflexivirus 2 (SsAFV2), was isolated from the hypovirulent strain 32–9 of S. sclerotiorum, and its complete genome was determined. The SsAFV2 genome contains 7,162 nucleotides (nt), excluding the poly (A) structure, and is composed of four open reading frames (ORF1–4). ORF1 encodes a polyprotein that contains three conserved domains: methyltransferase, helicase, and RNA-dependent RNA polymerase (RdRp). The ORF3 putative encodes coat proteins (CP), with ORF2 and ORF4 encoding hypothetical proteins of unknown functions. Phylogenetic analysis revealed that SsAFV2 clustered with Botrytis virus X (BVX) based on multiple alignments of helicase, RdRp, and CP, but the methyltransferase of SsAFV2 was most closely related to Sclerotinia sclerotiorum alphaflexivirus 1, suggesting that SsAFV2 is a new member of the Botrexvirus genus within the Alphaflexiviridae family, and also revealed the occurrence of potential inter-species horizontal gene transfer events within the Botrexvirus genus during the evolutionary process. Our results contribute to the current knowledge regarding the evolution and divergence of Botrexviruses.

TurboID reveals the proxiomes of Chlamydomonas proteins involved in thylakoid biogenesis and stress response.

In Chlamydomonas (Chlamydomonas reinhardtii), the VESICLE-INDUCING PROTEIN IN PLASTIDS 1 and 2 (VIPP1 and VIPP2) play roles in the sensing and coping with membrane stress and in thylakoid membrane biogenesis. To gain more insight into these processes, we aimed to identify proteins interacting with VIPP1/2 in the chloroplast and chose proximity labeling (PL) for this purpose. We used the transient interaction between the nucleotide exchange factor CHLOROPLAST GRPE HOMOLOG 1 (CGE1) and the stromal HEAT SHOCK PROTEIN 70B (HSP70B) as test system. While PL with APEX2 and BioID proved to be inefficient, TurboID resulted in substantial biotinylation in vivo. TurboID-mediated PL with VIPP1/2 as baits under ambient and H2O2 stress conditions confirmed known interactions of VIPP1 with VIPP2, HSP70B, and the CHLOROPLAST DNAJ HOMOLOG 2 (CDJ2). Proteins identified in the VIPP1/2 proxiomes can be grouped into proteins involved in the biogenesis of thylakoid membrane complexes and the regulation of photosynthetic electron transport, including PROTON GRADIENT REGULATION 5-LIKE 1 (PGRL1). A third group comprises 11 proteins of unknown function whose genes are upregulated under chloroplast stress conditions. We named them VIPP PROXIMITY LABELING (VPL). In reciprocal experiments, we confirmed VIPP1 in the proxiomes of VPL2 and PGRL1. Our results demonstrate the robustness of TurboID-mediated PL for studying protein interaction networks in the chloroplast of Chlamydomonas and pave the way for analyzing functions of VIPPs in thylakoid biogenesis and stress responses.

Novel protein CcmS is required for stabilization of the assembly of β-carboxysome in Synechocystis sp. strain PCC 6803.

The carboxysome plays an essential role in the carbon concentration mechanism in cyanobacteria. Although significant progress has been made in the structural analysis of the carboxysome, little is still known about its biosynthesis. We identified slr1911, a gene encoding a protein of unknown function in cyanobacterium Synechocystis sp. Strain PCC 6803 (Syn6803), which we termed ccmS by screening a low CO2 -sensitive mutant. CcmS interacts with CcmK1 and CcmM. The former is a shell protein of the β-carboxysome and the latter is a crucial component of the β-carboxysome, which is responsible for aggregating RuBisCO and recruiting shell proteins. The deletion of ccmS lowers the accumulation and assembly of CcmK1, resulting in aberrant carboxysomes, suppressed photosynthetic capacities, and leads to a slow growth phenotype, especially under CO2 -limited conditions. These observations suggest that CcmS stabilizes the assembly of the β-carboxysome shell and likely connects the carboxysome core with the shell. Our results provide a molecular view of the role played by CcmS in the formation of the β-carboxysome and its function in Syn6803.

Analysis of infections and parameters of humoral immunity in patients (pts) with relapsed/refractory multiple myeloma (RRMM) treated with talquetamab (tal) monotherapy in MonumenTAL-1.

8020 Background: Infection, a key complication of MM, may be due to patient, disease, or treatment-related factors. Immunotherapies that impact normal immune cells may increase risk of infection. Tal, a bispecific antibody with clinical benefit in pts with RRMM, targets G protein–coupled receptor family C group 5 member D (GPRC5D), a protein of unknown function with significantly higher expression on malignant versus normal plasma cells. We report the infection profile and immune function with tal in the phase (ph) 1/2 MonumenTAL-1 study (NCT03399799/NCT04634552). Methods: Pts had RRMM, were intolerant to/progressed on established therapies (ph 1), or had ≥3 prior lines of therapy (ph 2; ≥1 proteasome inhibitor/immunomodulatory drug/anti-CD38 antibody). Subcutaneous tal was given at 0.4 mg/kg QW or 0.8 mg/kg Q2W. Infections (graded by CTCAE v4.03) were treated per local guidelines. B-cell subpopulations and IgG levels were assessed from whole blood and serum samples, respectively. Results: We evaluated 339 pts on tal QW or Q2W, of whom 51 had prior T-cell redirection therapy (pTCRT); infection rates are shown in the table (median follow-up, 15.9, 10.1, and 13.1 mo, respectively). New-onset infections were most prevalent during cycles 1–2. Grade (gr) 3/4 infections observed in >2 pts were pneumonia (3.5%) and UTI (2.1%) on tal QW; pneumonia (2.1%) and COVID-19 (2.1%) on tal Q2W; and pneumonia (5.9%) with pTCRT. Opportunistic infections were observed in 3.5%, 4.1%, and 5.9% of pts, respectively. Less than 1.5% of pts died from infections: COVID-19 pneumonia (n=2) and one each due to septic shock, fungal sepsis, and unknown etiology. Hypogammaglobulinemia rates by IgG values were 64.3% (tal QW), 65.5% (tal Q2W), and 70.6% (pTCRT); IVIg use was 14.7%, 12.4%, and 15.7%, respectively. CD19+ B-cell levels were stable, and there was a trend toward increased non-clonal IgG over time. Conclusions: Roughly 20% of pts had gr 3/4 infections on tal (most frequently cycles 1–2), with low rates of opportunistic infections, discontinuation, and death. Infection rates, particularly rates of fatal infections, appear lower with tal than with BCMA-targeted T-cell–based therapies. A trend toward increased non-clonal IgG suggests potential recovery of humoral immunity accompanies rapid, deep, and durable responses to tal. These results distinguish tal as an important emerging therapy for RRMM. Clinical trial information: NCT03399799 , NCT04634552 . [Table: see text]

Glutamine Metabolism, Sensing andSignaling inPlants.

Glutamine (Gln) is the first amino acid synthesized in nitrogen (N) assimilation in plants. Gln synthetase (GS), converting glutamate (Glu) and NH4+ into Gln at the expense of ATP, is one of the oldest enzymes in all life domains. Plants have multiple GS isoenzymes that work individually or cooperatively to ensure that the Gln supply is sufficient for plant growth and development under various conditions. Gln is a building block for protein synthesis and an N-donor for the biosynthesis of amino acids, nucleic acids, amino sugars and vitamin B coenzymes. Most reactions using Gln as an N-donor are catalyzed by Gln amidotransferase (GAT) that hydrolyzes Gln to Glu and transfers the amido group of Gln to an acceptor substrate. Several GAT domain-containing proteins of unknown function in the reference plant Arabidopsis thaliana suggest that some metabolic fates of Gln have yet to be identified in plants. In addition to metabolism, Gln signaling has emerged in recent years. The N regulatory protein PII senses Gln to regulate arginine biosynthesis in plants. Gln promotes somatic embryogenesis and shoot organogenesis with unknown mechanisms. Exogenous Gln has been implicated in activating stress and defense responses in plants. Likely, Gln signaling is responsible for some of the new Gln functions in plants.

The Rvv two-component regulatory system regulates biofilm formation and colonization in Vibrio cholerae.

The facultative human pathogen, Vibrio cholerae, employs two-component signal transduction systems (TCS) to sense and respond to environmental signals encountered during its infection cycle. TCSs consist of a sensor histidine kinase (HK) and a response regulator (RR); the V. cholerae genome encodes 43 HKs and 49 RRs, of which 25 are predicted to be cognate pairs. Using deletion mutants of each HK gene, we analyzed the transcription of vpsL, a biofilm gene required for Vibrio polysaccharide and biofilm formation. We found that a V. cholerae TCS that had not been studied before, now termed Rvv, controls biofilm gene transcription. The Rvv TCS is part of a three-gene operon that is present in 30% of Vibrionales species. The rvv operon encodes RvvA, the HK; RvvB, the cognate RR; and RvvC, a protein of unknown function. Deletion of rvvA increased transcription of biofilm genes and altered biofilm formation, while deletion of rvvB or rvvC lead to no changes in biofilm gene transcription. The phenotypes observed in ΔrvvA depend on RvvB. Mutating RvvB to mimic constitutively active and inactive versions of the RR only impacted phenotypes in the ΔrvvA genetic background. Mutating the conserved residue required for kinase activity in RvvA did not affect phenotypes, whereas mutation of the conserved residue required for phosphatase activity mimicked the phenotype of the rvvA mutant. Furthermore, ΔrvvA displayed a significant colonization defect which was dependent on RvvB and RvvB phosphorylation state, but not on VPS production. We found that RvvA's phosphatase activity regulates biofilm gene transcription, biofilm formation, and colonization phenotypes. This is the first systematic analysis of the role of V. cholerae HKs in biofilm gene transcription and resulted in the identification of a new regulator of biofilm formation and virulence, advancing our understanding of the role TCSs play in regulating these critical cellular processes in V. cholerae.

Sensory plastid-associated PsbP DOMAIN-CONTAINING PROTEIN 3 triggers plant growth- and defense-related epigenetic responses.

Sensory plastids are important in plant responses to environmental changes. Previous studies show that MutS HOMOLOG 1 (MSH1) perturbation in sensory plastids induces heritable epigenetic phenotype adjustment. Previously, the PsbP homolog DOMAIN-CONTAINING PROTEIN 3 (PPD3), a protein of unknown function, was postulated to be an interactor with MSH1. This study investigates the relationship of PPD3 with MSH1 and with plant environmental sensing. The ppd3 mutant displays a whole-plant phenotype variably altered in growth rate, flowering time, reactive oxygen species (ROS) modulation and response to salt, with effects on meristem growth. Present in both chloroplasts and sensory plastids, PPD3 colocalized with MSH1 in root tips but not in leaf tissues. The suppression or overexpression of PPD3 affected the plant growth rate and stress tolerance, and led to a heritable, heterogenous 'memory' state with both dwarfed and vigorous growth phenotypes. Gene expression and DNA methylome data sets from PPD3-OX and derived memory states showed enrichment in growth versus defense networks and meristem effects. Our results support a model of sensory plastid influence on nuclear epigenetic behavior and ppd3 as a second trigger, functioning within meristem plastids to recalibrate growth plasticity.

The Cornified Envelope: A Versatile Contributor to the Epidermal Barrier

The Cornified Envelope: A Versatile Contributor to the Epidermal Barrier

Differential domains and endoproteolytic processing in dominant surface proteins of unknown function from Mycoplasma hyopneumoniae and Mycoplasma flocculare

Mycoplasma hyopneumoniae causes porcine enzootic pneumonia (PEP), a chronic respiratory disease that leads to severe economic losses in the pig industry. Swine infection and PEP development depend on the adhesion of the pathogen to the swine respiratory tract and the host immune response, but these and other disease determinants are not fully understood. For instance, M. hyopneumoniae has a large repertoire of proteins of unknown function (PUFs) and some of them are abundant in the cell surface, where they likely mediate so far unknown pathogen-host interactions. Moreover, these surface PUFs may undergo endoproteolytic processing to generate larger repertoires of proteoforms to further complicate this scenario. Here, we investigated the five PUFs more represented on the surface of M. hyopneumoniae pathogenic strain 7448 in comparison with their orthologs from the nonpathogenic M. hyopneumoniae J strain and the closely related commensal species Mycoplasma flocculare. Comparative in silico analyses of deduced amino acid sequences and proteomic data identified differential domains, disordered regions and repeated motifs. We also provide evidence of differential endoproteolytic processing and antigenicity. Phylogenetic analyses were also performed with ortholog sequences, showing higher conservation of three of the assessed PUFs among Mycoplasma species related to respiratory diseases. Overall, our data point out to M. hyopneumoniae surface-dominant PUFs likely associated with pathogenicity.

Identifying the core genome of the nucleus-forming bacteriophage family and characterization of Erwinia phage RAY

We recently discovered that some bacteriophages establish a nucleus-like replication compartment (phage nucleus), but the core genes that define nucleus-based phage replication and their phylogenetic distribution were still to be determined. Here, we show that phages encoding the major phage nucleus protein chimallin share 72 conserved genes encoded within seven gene blocks. Of these, 21 core genes are unique to nucleus-forming phage, and all but one of these genes encode proteins of unknown function. We propose that these phages comprise a novel viral family we term Chimalliviridae. Fluorescence microscopy and cryoelectron tomography studies of Erwinia phage vB_EamM_RAY confirm that many of the key steps of nucleus-based replication are conserved among diverse chimalliviruses and reveal variations on this replication mechanism. This work expands our understanding of phage nucleus and PhuZ spindle diversity and function, providing a roadmap for identifying key mechanisms underlying nucleus-based phage replication.