Immunotherapy targeting isoDGR-protein damage extends lifespan in a mouse model of protein deamidation.

Aging results from the accumulation of molecular damage that impairs normal biochemical processes. We previously reported that age-linked damage to amino acid sequence NGR (Asn-Gly-Arg) results in "gain-of-function" conformational switching to isoDGR (isoAsp-Gly-Arg). This integrin-binding motif activates leukocytes and promotes chronic inflammation, which are characteristic features of age-linked cardiovascular disorders. We now report that anti-isoDGR immunotherapy mitigates lifespan reduction of Pcmt1-/- mouse. We observed extensive accumulation of isoDGR and inflammatory cytokine expression in multiple tissues from Pcmt1-/- and naturally aged WT animals, which could also be induced via injection of isoDGR-modified plasma proteins or synthetic peptides into young WT animals. However, weekly injection of anti-isoDGR mAb (1 mg/kg) was sufficient to significantly reduce isoDGR-protein levels in body tissues, decreased pro-inflammatory cytokine concentrations in blood plasma, improved cognition/coordination metrics, and extended the average lifespan of Pcmt1-/- mice. Mechanistically, isoDGR-mAb mediated immune clearance of damaged isoDGR-proteins via antibody-dependent cellular phagocytosis (ADCP). These results indicate that immunotherapy targeting age-linked protein damage may represent an effective intervention strategy in a range of human degenerative disorders.

Gram-negative bacteria resist antimicrobial agents by a DzrR-mediated envelope stress response.

BACKGROUND: Envelope stress responses (ESRs) are critical for adaptive resistance of Gram-negative bacteria to envelope-targeting antimicrobial agents. However, ESRs are poorly defined in a large number of well-known plant and human pathogens. Dickeya oryzae can withstand a high level of self-produced envelope-targeting antimicrobial agents zeamines through a zeamine-stimulated RND efflux pump DesABC. Here, we unraveled the mechanism of D. oryzae response to zeamines and determined the distribution and function of this novel ESR in a variety of important plant and human pathogens. RESULTS: In this study, we documented that a two-component system regulator DzrR of D. oryzae EC1 mediates ESR in the presence of envelope-targeting antimicrobial agents. DzrR was found modulating bacterial response and resistance to zeamines through inducing the expression of RND efflux pump DesABC, which is likely independent on DzrR phosphorylation. In addition, DzrR could also mediate bacterial responses to structurally divergent envelope-targeting antimicrobial agents, including chlorhexidine and chlorpromazine. Significantly, the DzrR-mediated response was independent on the five canonical ESRs. We further presented evidence that the DzrR-mediated response is conserved in the bacterial species of Dickeya, Ralstonia, and Burkholderia, showing that a distantly located DzrR homolog is the previously undetermined regulator of RND-8 efflux pump for chlorhexidine resistance in B. cenocepacia. CONCLUSIONS: Taken together, the findings from this study depict a new widely distributed Gram-negative ESR mechanism and present a valid target and useful clues to combat antimicrobial resistance.

Endothelial Damage Arising From High Salt Hypertension Is Elucidated by Vascular Bed Systematic Profiling.

BACKGROUND: Considerable evidence links dietary salt intake with the development of hypertension, left ventricular hypertrophy, and increased risk of stroke and coronary heart disease. Despite extensive epidemiological and basic science interrogation of the relationship between high salt (HS) intake and blood pressure, it remains unclear how HS impacts endothelial cell (EC) and vascular structure in vivo. This study aims to elucidate HS-induced vascular pathology using a differential systemic decellularization in vivo approach. METHODS: We performed systematic molecular characterization of the endothelial glycocalyx and EC proteomes in mice with HS (8%) diet-induced hypertension versus healthy control animals. Isolation of eGC and EC compartments was achieved using differential systemic decellularization in vivo methodology. Altered protein expression in hypertensive compared to normal mice was characterized by liquid chromatography tandem mass spectrometry. Proteomic results were validated using functional assays, microscopic imaging, and histopathologic evaluation. RESULTS: Proteomic analysis revealed a significant downregulation of eGC and associated proteins in HS diet-induced hypertensive mice (among 1696 proteins identified in this group, 723 were markedly decreased in abundance, while only 168 were increased in abundance. Bioinformatic analysis indicated substantial derangement of the eGC layer, which was subsequently confirmed by fluorescent and electron microscopy assessment of vessel damage ex vivo. In the EC fraction, HS-induced hypertension significantly altered protein mediators of contractility, metabolism, mechanotransduction, renal function, and the coagulation cascade. In particular, we observed dysregulation of integrin subunits α2, α2b, and α5, which was associated with arterial wall inflammation and substantial infiltration of CD68+ monocyte-macrophages. Consequently, HS-induced hypertensive mice also displayed reduced vascular integrity of multiple organs including lungs, kidneys, and heart. CONCLUSIONS: These findings provide novel molecular insight into HS-induced structural changes in eGC and EC composition that may increase cardiovascular risk and potentially guide the development of new diagnostics and therapeutic interventions.

Molecular condensation and mechanoregulation of plant class I formin, an integrin-like actin nucleator.

The actin cytoskeleton (AC) undergoes rapid remodelling to coordinate cellular processes during signal transduction, including changes in actin nucleation, crosslinking, and depolymerization in a time- and space-dependent manner. Switching the initial actin nucleation often provides timely control of the entire actin network formation. Located at the cell surface, the plant class I formin family is a major class of actin nucleators that rapidly respond to exterior chemical and environmental cues. Plant class I formins are structurally integrated within the plant cell wall-plasma membrane-actin cytoskeleton (CW-PM-AC) continuum, sharing similar biophysical properties to mammalian integrins that are embedded within the extracellular matrix-PM-AC continuum. In plants, perturbation of structural components of the CW-PM-AC continuum changes the biophysical properties of two dimensional-scaffolding structures, which results in uncontrolled molecular diffusion and interactions of class I formins, as well as their clustering and activities in the nucleation of the AC. Emerging studies have shown that the PM-integrated formins are highly responsive to the mechanical perturbation of CW and AC integrity changes that tune the oligomerization and condensation of formin on the cell surface. However, during diverse signalling transductions, the molecular mechanisms that spatiotemporally underlie the mechanosensing and mechanoregulation of formin for remodelling actin remain unclear. Here, the emphasis will be placed on recent developments in understanding how the molecular condensation of class I formin regulates the biochemical activities in tuning actin polymerization during plant immune signalling, as well as how the plant structural components of the CW-PM-AC continuum control formin condensation at a nanometre scale.

Identification of atypical T4SS effector proteins mediating bacterial defense.

To remain competitive, proteobacteria use various contact-dependent weapon systems to defend against microbial competitors. The bacterial-killing type IV secretion system (T4SS) is one such powerful weapon. It commonly controls the killing/competition between species by secreting the lethal T4SS effector (T4E) proteins carrying conserved XVIPCD domains into competing cells. In this study, we sought knowledge to understand whether the bacterial-killing T4SS-producing bacteria encode T4E-like proteins and further explore their biological functions. To achieve this, we designed a T4E-guided approach to discover T4E-like proteins that are designated as atypical T4Es. Initially, this approach required scientists to perform simple BlastP search to identify T4E homologs that lack the XVIPCD domain in the genomes of T4SS-producing bacteria. These homologous genes were then screened in Escherichia coli to identify antibacterial candidates (atypical T4Es) and their neighboring detoxification proteins, followed by testing their gene cotranscription and validating their physical interactions. Using this approach, we did discover two atypical T4E proteins from the plant-beneficial Lysobacter enzymogenes and the phytopathogen Xanthomonas citri. We also provided substantial evidence to show that the atypical T4E protein Le1637-mediated bacterial defense in interspecies interactions between L. enzymogenes and its competitors. Therefore, the newly designed T4E-guided approach holds promise for detecting functional atypical T4E proteins in bacterial cells.

Quantitative Proteomics of Medium-Sized Extracellular Vesicle-Enriched Plasma of Lacunar Infarction for the Discovery of Prognostic Biomarkers.

Lacunar infarction (LACI), a subtype of acute ischemic stroke, has poor mid- to long-term prognosis due to recurrent vascular events or incident dementia which is difficult to predict using existing clinical data. Herein, we aim to discover blood-based biomarkers for LACI as a complementary prognostic tool. Convalescent plasma was collected from forty-five patients following a non-disabling LACI along with seventeen matched control subjects. The patients were followed up prospectively for up to five years to record an occurrence of adverse outcome and grouped accordingly (i.e., LACI-no adverse outcome, LACI-recurrent vascular event, and LACI-cognitive decline without any recurrence of vascular events). Medium-sized extracellular vesicles (MEVs), isolated from the pooled plasma of four groups, were analyzed by stable isotope labeling and 2D-LC-MS/MS. Out of 573 (FDR < 1%) quantified proteins, 146 showed significant changes in at least one LACI group when compared to matched healthy control. A systems analysis revealed that major elements (~85%) of the MEV proteome are different from the proteome of small-sized extracellular vesicles obtained from the same pooled plasma. The altered MEV proteins in LACI patients are mostly reduced in abundance. The majority of the shortlisted MEV proteins are not linked to commonly studied biological processes such as coagulation, fibrinolysis, or inflammation. Instead, they are linked to oxygen-glucose deprivation, endo-lysosomal trafficking, glucose transport, and iron homeostasis. The dataset is provided as a web-based data resource to facilitate meta-analysis, data integration, and targeted large-scale validation.

Molecular basis of the key regulator WRINKLED1 in plant oil biosynthesis.

Vegetable oils are not only major components of human diet but also vital for industrial applications. WRINKLED1 (WRI1) is a pivotal transcription factor governing plant oil biosynthesis, but the underlying DNA-binding mechanism remains incompletely understood. Here, we resolved the structure of Arabidopsis WRI1 (AtWRI1) with its cognate double-stranded DNA (dsDNA), revealing two antiparallel ß sheets in the tandem AP2 domains that intercalate into the adjacent major grooves of dsDNA to determine the sequence recognition specificity. We showed that AtWRI1 represented a previously unidentified structural fold and DNA-binding mode. Mutations of the key residues interacting with DNA element affected its binding affinity and oil biosynthesis when these variants were transiently expressed in tobacco leaves. Seed oil content was enhanced in stable transgenic wri1-1 expressing an AtWRI1 variant (W74R). Together, our findings offer a structural basis explaining WRI1 recognition and binding of DNA and suggest an alternative strategy to increase oil yield in crops through WRI1 bioengineering.

Cryo-EM structure of the entire FtsH-HflKC AAA protease complex.

The membrane-bound AAA protease FtsH is the key player controlling protein quality in bacteria. Two single-pass membrane proteins, HflK and HflC, interact with FtsH to modulate its proteolytic activity. Here, we present structure of the entire FtsH-HflKC complex, comprising 12 copies of both HflK and HflC, all of which interact reciprocally to form a cage, as well as four FtsH hexamers with periplasmic domains and transmembrane helices enclosed inside the cage and cytoplasmic domains situated at the base of the cage. FtsH K61/D62/S63 in the ß2-ß3 loop in the periplasmic domain directly interact with HflK, contributing to complex formation. Pull-down and in vivo enzymatic activity assays validate the importance of the interacting interface for FtsH-HflKC complex formation. Structural comparison with the substrate-bound human m-AAA protease AFG3L2 offers implications for the HflKC cage in modulating substrate access to FtsH. Together, our findings provide a better understanding of FtsH-type AAA protease holoenzyme assembly and regulation.

Structural analyses of the AAA+ ATPase domain of the transcriptional regulator GtrR in the BDSF quorum-sensing system in Burkholderia cenocepacia.

Global transcriptional regulator downstream RpfR (GtrR) is a key downstream regulator for quorum-sensing signaling molecule cis-2-dodecenoic acid (BDSF). As a bacterial enhancer-binding protein (bEBP), GtrR is composed of an N-terminal receiver domain, a central ATPases associated with diverse cellular activities (AAA+) ATPase σ54 -interaction domain, and a C-terminal helix-turn-helix DNA-binding domain. In this work, we solved its AAA+ ATPase domain in both apo and GTP-bound forms. The structure revealed how GtrR specifically recognizes GTP. In addition, we also revealed that GtrR has moderate GTPase activity in vitro in the absence of its activation signal. Finally, we found the residues K170, D236, R311, and R357 in GtrR that are crucial to its biological function, any single mutation leading to completely abolishing GtrR activity.

Translational GTPase BipA Is Involved in the Maturation of a Large Subunit of Bacterial Ribosome at Suboptimal Temperature.

BPI-inducible protein A (BipA), a highly conserved paralog of the well-known translational GTPases LepA and EF-G, has been implicated in bacterial motility, cold shock, stress response, biofilm formation, and virulence. BipA binds to the aminoacyl-(A) site of the bacterial ribosome and establishes contacts with the functionally important regions of both subunits, implying a specific role relevant to the ribosome, such as functioning in ribosome biogenesis and/or conditional protein translation. When cultured at suboptimal temperatures, the Escherichia coli bipA genomic deletion strain (ΔbipA) exhibits defects in growth, swimming motility, and ribosome assembly, which can be complemented by a plasmid-borne bipA supplementation or suppressed by the genomic rluC deletion. Based on the growth curve, soft agar swimming assay, and sucrose gradient sedimentation analysis, mutation of the catalytic residue His78 rendered plasmid-borne bipA unable to complement its deletion phenotypes. Interestingly, truncation of the C-terminal loop of BipA exacerbates the aforementioned phenotypes, demonstrating the involvement of BipA in ribosome assembly or its function. Furthermore, tandem mass tag-mass spectrometry analysis of the ΔbipA strain proteome revealed upregulations of a number of proteins (e.g., DeaD, RNase R, CspA, RpoS, and ObgE) implicated in ribosome biogenesis and RNA metabolism, and these proteins were restored to wild-type levels by plasmid-borne bipA supplementation or the genomic rluC deletion, implying BipA involvement in RNA metabolism and ribosome biogenesis. We have also determined that BipA interacts with ribosome 50S precursor (pre-50S), suggesting its role in 50S maturation and ribosome biogenesis. Taken together, BipA demonstrates the characteristics of a bona fide 50S assembly factor in ribosome biogenesis.

Structural Basis and Function of the N Terminus of SARS-CoV-2 Nonstructural Protein 1.

Nonstructural protein 1 (Nsp1) of severe acute respiratory syndrome coronaviruses (SARS-CoVs) is an important pathogenic factor that inhibits host protein translation by means of its C terminus. However, its N-terminal function remains elusive. Here, we determined the crystal structure of the N terminus (amino acids [aa] 11 to 125) of SARS-CoV-2 Nsp1 at a 1.25-Å resolution. Further functional assays showed that the N terminus of SARS-CoVs Nsp1 alone loses the ability to colocalize with ribosomes and inhibit protein translation. The C terminus of Nsp1 can colocalize with ribosomes, but its protein translation inhibition ability is significantly weakened. Interestingly, fusing the C terminus of Nsp1 with enhanced green fluorescent protein (EGFP) or other proteins in place of its N terminus restored the protein translation inhibitory ability to a level equivalent to that of full-length Nsp1. Thus, our results suggest that the N terminus of Nsp1 is able to stabilize the binding of the Nsp1 C terminus to ribosomes and act as a nonspecific barrier to block the mRNA channel, thus abrogating host mRNA translation.

Ribosome Protection Proteins-"New" Players in the Global Arms Race with Antibiotic-Resistant Pathogens.

Bacteria have evolved an array of mechanisms enabling them to resist the inhibitory effect of antibiotics, a significant proportion of which target the ribosome. Indeed, resistance mechanisms have been identified for nearly every antibiotic that is currently used in clinical practice. With the ever-increasing list of multi-drug-resistant pathogens and very few novel antibiotics in the pharmaceutical pipeline, treatable infections are likely to become life-threatening once again. Most of the prevalent resistance mechanisms are well understood and their clinical significance is recognized. In contrast, ribosome protection protein-mediated resistance has flown under the radar for a long time and has been considered a minor factor in the clinical setting. Not until the recent discovery of the ATP-binding cassette family F protein-mediated resistance in an extensive list of human pathogens has the significance of ribosome protection proteins been truly appreciated. Understanding the underlying resistance mechanism has the potential to guide the development of novel therapeutic approaches to evade or overcome the resistance. In this review, we discuss the latest developments regarding ribosome protection proteins focusing on the current antimicrobial arsenal and pharmaceutical pipeline as well as potential implications for the future of fighting bacterial infections in the time of "superbugs."

Emerging evidence for kindlin oligomerization and its role in regulating kindlin function.

Integrin-mediated cell-extracellular matrix (ECM) interactions play crucial roles in a broad range of physiological and pathological processes. Kindlins are important positive regulators of integrin activation. The FERM-domain-containing kindlin family comprises three members, kindlin-1, kindlin-2 and kindlin-3 (also known as FERMT1, FERMT2 and FERMT3), which share high sequence similarity (identity >50%), as well as domain organization, but exhibit diverse tissue-specific expression patterns and cellular functions. Given the significance of kindlins, analysis of their atomic structures has been an attractive field for decades. Recently, the structures of kindlin and its ß-integrin-bound form have been obtained, which greatly advance our understanding of the molecular functions that involve kindlins. In particular, emerging evidence indicates that oligomerization of kindlins might affect their integrin binding and focal adhesion localization, positively or negatively. In this Review, we presented an update on the recent progress of obtaining kindlin structures, and discuss the implication for integrin activation based on kindlin oligomerization, as well as the possible regulation of this process.

Structure of Arabidopsis CESA3 catalytic domain with its substrate UDP-glucose provides insight into the mechanism of cellulose synthesis.

Cellulose is synthesized by cellulose synthases (CESAs) from the glycosyltransferase GT-2 family. In plants, the CESAs form a six-lobed rosette-shaped CESA complex (CSC). Here we report crystal structures of the catalytic domain of Arabidopsis thaliana CESA3 (AtCESA3CatD) in both apo and uridine diphosphate (UDP)-glucose (UDP-Glc)-bound forms. AtCESA3CatD has an overall GT-A fold core domain sandwiched between a plant-conserved region (P-CR) and a class-specific region (C-SR). By superimposing the structure of AtCESA3CatD onto the bacterial cellulose synthase BcsA, we found that the coordination of the UDP-Glc differs, indicating different substrate coordination during cellulose synthesis in plants and bacteria. Moreover, structural analyses revealed that AtCESA3CatD can form a homodimer mainly via interactions between specific beta strands. We confirmed the importance of specific amino acids on these strands for homodimerization through yeast and in planta assays using point-mutated full-length AtCESA3. Our work provides molecular insights into how the substrate UDP-Glc is coordinated in the CESAs and how the CESAs might dimerize to eventually assemble into CSCs in plants.

An engineered CRISPR-Cas12a variant and DNA-RNA hybrid guides enable robust and rapid COVID-19 testing.

Extensive testing is essential to break the transmission of SARS-CoV-2, which causes the ongoing COVID-19 pandemic. Here, we present a CRISPR-based diagnostic assay that is robust to viral genome mutations and temperature, produces results fast, can be applied directly on nasopharyngeal (NP) specimens without RNA purification, and incorporates a human internal control within the same reaction. Specifically, we show that the use of an engineered AsCas12a enzyme enables detection of wildtype and mutated SARS-CoV-2 and allows us to perform the detection step with loop-mediated isothermal amplification (LAMP) at 60-65 °C. We also find that the use of hybrid DNA-RNA guides increases the rate of reaction, enabling our test to be completed within 30 minutes. Utilizing clinical samples from 72 patients with COVID-19 infection and 57 healthy individuals, we demonstrate that our test exhibits a specificity and positive predictive value of 100% with a sensitivity of 50 and 1000 copies per reaction (or 2 and 40 copies per microliter) for purified RNA samples and unpurified NP specimens respectively.

Structural basis of a novel repressor, SghR, controlling Agrobacterium infection by cross-talking to plants.

Agrobacterium tumefaciens infects various plants and causes crown gall diseases involving temporal expression of virulence factors. SghA is a newly identified virulence factor enzymatically releasing salicylic acid from its glucoside conjugate and controlling plant tumor development. Here, we report the structural basis of SghR, a LacI-type transcription factor highly conserved in Rhizobiaceae family, regulating the expression of SghA and involved in tumorigenesis. We identified and characterized the binding site of SghR on the promoter region of sghA and then determined the crystal structures of apo-SghR, SghR complexed with its operator DNA, and ligand sucrose, respectively. These results provide detailed insights into how SghR recognizes its cognate DNA and shed a mechanistic light on how sucrose attenuates the affinity of SghR with DNA to modulate the expression of SghA. Given the important role of SghR in mediating the signaling cross-talk during Agrobacterium infection, our results pave the way for structure-based inducer analog design, which has potential applications for agricultural industry.

Structural basis of human full-length kindlin-3 homotrimer in an auto-inhibited state.

Kindlin-1, -2, and -3 directly bind integrin ß cytoplasmic tails to regulate integrin activation and signaling. Despite their functional significance and links to several diseases, structural information on full-length kindlin proteins remains unknown. Here, we report the crystal structure of human full-length kindlin-3, which reveals a novel homotrimer state. Unlike kindlin-3 monomer, which is the major population in insect and mammalian cell expression systems, kindlin-3 trimer does not bind integrin ß cytoplasmic tail as the integrin-binding pocket in the F3 subdomain of 1 protomer is occluded by the pleckstrin homology (PH) domain of another protomer, suggesting that kindlin-3 is auto-inhibited upon trimer formation. This is also supported by functional assays in which kindlin-3 knockout K562 erythroleukemia cells reconstituted with the mutant kindlin-3 containing trimer-disrupting mutations exhibited an increase in integrin-mediated adhesion and spreading on fibronectin compared with those reconstituted with wild-type kindlin-3. Taken together, our findings reveal a novel mechanism of kindlin auto-inhibition that involves its homotrimer formation.

Structural and biochemical characterization of novel carbonic anhydrases from Phaeodactylum tricornutum.

Carbonic anhydrases (CAs) are a well characterized family of metalloenzymes that are highly efficient in facilitating the interconversion between carbon dioxide and bicarbonate. Recently, CA activity has been associated with the LCIB (limiting CO2-inducible protein B) protein family, which has been an interesting target in aquatic photosynthetic microorganisms. To gain further insight into the catalytic mechanism of this new group of CAs, the X-ray structure of a highly active LCIB homolog (PtLCIB3) from the diatom Phaeodactylum tricornutum was determined. The CA activities of PtLCIB3, its paralog PtLCIB4 and a variety of their mutants were also measured. It was discovered that PtLCIB3 has a classic ß-CA fold and its overall structure is highly similar to that of its homolog PtLCIB4. Subtle structural alterations between PtLCIB3 and PtLCIB4 indicate that an alternative proton-shuttle cavity could perhaps be one reason for their remarkable difference in CA activity. A potential alternative proton-shuttle route in the LCIB protein family is suggested based on these results.

Insights into the mechanism and regulation of the CbbQO-type Rubisco activase, a MoxR AAA+ ATPase.

The vast majority of biological carbon dioxide fixation relies on the function of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). In most cases the enzyme exhibits a tendency to become inhibited by its substrate RuBP and other sugar phosphates. The inhibition is counteracted by diverse molecular chaperones known as Rubisco activases (Rcas). In some chemoautotrophic bacteria, the CbbQO-type Rca Q2O2 repairs inhibited active sites of hexameric form II Rubisco. The 2.2-Å crystal structure of the MoxR AAA+ protein CbbQ2 from Acidithiobacillus ferrooxidans reveals the helix 2 insert (H2I) that is critical for Rca function and forms the axial pore of the CbbQ hexamer. Negative-stain electron microscopy shows that the essential CbbO adaptor protein binds to the conserved, concave side of the CbbQ2 hexamer. Site-directed mutagenesis supports a model in which adenosine 5'-triphosphate (ATP)-powered movements of the H2I are transmitted to CbbO via the concave residue L85. The basal ATPase activity of Q2O2 Rca is repressed but strongly stimulated by inhibited Rubisco. The characterization of multiple variants where this repression is released indicates that binding of inhibited Rubisco to the C-terminal CbbO VWA domain initiates a signal toward the CbbQ active site that is propagated via elements that include the CbbQ α4-ß4 loop, pore loop 1, and the presensor 1-ß hairpin (PS1-ßH). Detailed mechanistic insights into the enzyme repair chaperones of the highly diverse CO2 fixation machinery of Proteobacteria will facilitate their successful implementation in synthetic biology ventures.