Encapsulated Ferritin-like Proteins: A Structural Perspective.

Encapsulins are self-assembling nano-compartments that naturally occur in bacteria and archaea. These nano-compartments encapsulate cargo proteins that bind to the shell's interior through specific recognition sequences and perform various metabolic processes. Encapsulation enables organisms to perform chemical reactions without exposing the rest of the cell to potentially harmful substances while shielding cargo molecules from degradation and other adverse effects of the surrounding environment. One particular type of cargo protein, the ferritin-like protein (FLP), is the focus of this review. Encapsulated FLPs are members of the ferritin-like protein superfamily, and they play a crucial role in converting ferrous iron (Fe+2) to ferric iron (Fe+3), which is then stored inside the encapsulin in mineralized form. As such, FLPs regulate iron homeostasis and protect organisms against oxidative stress. Recent studies have demonstrated that FLPs have tremendous potential as biosensors and bioreactors because of their ability to catalyze the oxidation of ferrous iron with high specificity and efficiency. Moreover, they have been investigated as potential targets for therapeutic intervention in cancer drug development and bacterial pathogenesis. Further research will likely lead to new insights and applications for these remarkable proteins in biomedicine and biotechnology.

The ribosome-inactivating proteins MAP30 and Momordin inhibit SARS-CoV-2.

The continuing emergence of SARS-CoV-2 variants has highlighted the need to identify additional points for viral inhibition. Ribosome inactivating proteins (RIPs), such as MAP30 and Momordin which are derived from bitter melon (Momordica charantia), have been found to inhibit a broad range of viruses. MAP30 has been shown to potently inhibit HIV-1 with minimal cytotoxicity. Here we show that MAP30 and Momordin potently inhibit SARS-CoV-2 replication in A549 human lung cells (IC50 ~ 0.2 µM) with little concomitant cytotoxicity (CC50 ~ 2 µM). Both viral inhibition and cytotoxicity remain unaltered by appending a C-terminal Tat cell-penetration peptide to either protein. Mutation of tyrosine 70, a key residue in the active site of MAP30, to alanine completely abrogates both viral inhibition and cytotoxicity, indicating the involvement of its RNA N-glycosylase activity. Mutation of lysine 171 and lysine 215, residues corresponding to those in Ricin which when mutated prevented ribosome binding and inactivation, to alanine in MAP30 decreased cytotoxicity (CC50 ~ 10 µM) but also the viral inhibition (IC50 ~ 1 µM). Unlike with HIV-1, neither Dexamethasone nor Indomethacin exhibited synergy with MAP30 in the inhibition of SARS-CoV-2. From a structural comparison of the two proteins, one can explain their similar activities despite differences in both their active-sites and ribosome-binding regions. We also note points on the viral genome for potential inhibition by these proteins.

Variant STAT4 and Response to Ruxolitinib in an Autoinflammatory Syndrome.

BACKGROUND: Disabling pansclerotic morphea (DPM) is a rare systemic inflammatory disorder, characterized by poor wound healing, fibrosis, cytopenias, hypogammaglobulinemia, and squamous-cell carcinoma. The cause is unknown, and mortality is high. METHODS: We evaluated four patients from three unrelated families with an autosomal dominant pattern of inheritance of DPM. Genomic sequencing independently identified three heterozygous variants in a specific region of the gene that encodes signal transducer and activator of transcription 4 (STAT4). Primary skin fibroblast and cell-line assays were used to define the functional nature of the genetic defect. We also assayed gene expression using single-cell RNA sequencing of peripheral-blood mononuclear cells to identify inflammatory pathways that may be affected in DPM and that may respond to therapy. RESULTS: Genome sequencing revealed three novel heterozygous missense gain-of-function variants in STAT4. In vitro, primary skin fibroblasts showed enhanced interleukin-6 secretion, with impaired wound healing, contraction of the collagen matrix, and matrix secretion. Inhibition of Janus kinase (JAK)-STAT signaling with ruxolitinib led to improvement in the hyperinflammatory fibroblast phenotype in vitro and resolution of inflammatory markers and clinical symptoms in treated patients, without adverse effects. Single-cell RNA sequencing revealed expression patterns consistent with an immunodysregulatory phenotype that were appropriately modified through JAK inhibition. CONCLUSIONS: Gain-of-function variants in STAT4 caused DPM in the families that we studied. The JAK inhibitor ruxolitinib attenuated the dermatologic and inflammatory phenotype in vitro and in the affected family members. (Funded by the American Academy of Allergy, Asthma, and Immunology Foundation and others.).

Structural characterization of the Myxococcus xanthus encapsulin and ferritin-like cargo system gives insight into its iron storage mechanism.

Encapsulins are bacterial organelle-like cages involved in various aspects of metabolism, especially protection from oxidative stress. They can serve as vehicles for a wide range of medical applications. Encapsulin shell proteins are structurally similar to HK97 bacteriophage capsid protein and their function depends on the encapsulated cargos. The Myxococcus xanthus encapsulin system comprises EncA and three cargos: EncB, EncC, and EncD. EncB and EncC are similar to bacterial ferritins that can oxidize Fe+2 to less toxic Fe+3. We analyzed EncA, EncB, and EncC by cryo-EM and X-ray crystallography. Cryo-EM shows that EncA cages can have T = 3 and T = 1 symmetry and that EncA T = 1 has a unique protomer arrangement. Also, we define EncB and EncC binding sites on EncA. X-ray crystallography of EncB and EncC reveals conformational changes at the ferroxidase center and additional metal binding sites, suggesting a mechanism for Fe oxidation and storage within the encapsulin shell.

Role of peripheral inflammatory biomarkers, transforming growth factor-beta and interleukin 6 in predicting peritoneal adhesions following repeat cesarean delivery.

BACKGROUND: Repeat cesarean deliveries (CDs) pose a risk in the development of intra-abdominal adhesions. AIM: We aimed to examine the incidence and severity of adhesions in repeat CDs using a specific scoring system and assess the predictive power of the pre-operative value of transforming growth factor (TGF)-ß and interleukin (IL)-6 with selected peripheral inflammatory biomarkers (PIBs) in the prediction of adhesion formation. METHODS: This prospective study enrolled 91 pregnant women at term, who had previously undergone at least one or more scheduled CDs. PIBs, namely C-reactive protein, white blood cell count, neutrophil-to-lymphocyte ratio, platelet-to-lymphocyte ratio, and systemic immune-inflammation index, TGF-ß, and IL-6 were studied according to the presence and location of adhesions. RESULTS: There was a significant difference only in the variables of the number of CDs, post-operative adhesion index (PAI) score, IL-6, and TGF-ß on the presence of adhesion (p < 0.05). The linear regression model revealed that the effect of the number of CDs, PAI score, and IL-6 values on TGF-ß was significant (p < 0.05). The effect of the PAI score on TGF-ß was higher than that of IL-6. As a reciprocal relationship, the effect of the TGF-ß value on the PAI score was also higher than that of IL-6. CONCLUSION: In patients with a history of repeat CDs, the preoperative determination of TGF-ß seems to be an important independent predictor of POA. The adverse events due to post-operative adhesion caused by repeat CDs can be overcome by detecting high-risk patients with a comprehensive assessment and individualized intervention integrated into overall patient management.

Conformational changes in tubulin upon binding cryptophycin-52 reveal its mechanism of action.

Cryptophycin-52 (Cp-52) is potentially the most potent anticancer drug known, with IC50 values in the low picomolar range, but its binding site on tubulin and mechanism of action are unknown. Here, we have determined the binding site of Cp-52, and its parent compound, cryptophycin-1, on HeLa tubulin, to a resolution of 3.3 Å and 3.4 Å, respectively, by cryo-EM and characterized this binding further by molecular dynamics simulations. The binding site was determined to be located at the tubulin interdimer interface and partially overlap that of maytansine, another cytotoxic tubulin inhibitor. Binding induces curvature both within and between tubulin dimers that is incompatible with the microtubule lattice. Conformational changes occur in both α-tubulin and ß-tubulin, particularly in helices H8 and H10, with distinct differences between α and ß monomers and between Cp-52-bound and cryptophycin-1-bound tubulin. From these results, we have determined: (i) the mechanism of action of inhibition of both microtubule polymerization and depolymerization, (ii) how the affinity of Cp-52 for tubulin may be enhanced, and (iii) where linkers for targeted delivery can be optimally attached to this molecule.

A Case of Smith-Lemli-Opitz Syndrome Diagnosed with Hypertrophic Pyloric Stenosis.

Smith-Lemli-Opitz syndrome (SLOS) is caused by a deficiency in the enzyme 7-dehydrocholesterol reductase (DHCR7) that results in an abnormality in cholesterol metabolism. SLOS is inherited as an autosomal recessive genetic disorder. In this case, we describe a 34-day-old patient with postnatal progressive projectile vomiting, diagnosed with hypertrophic pyloric stenosis, who was suspected to have SLOS during treatment clinical and biochemical profile. A 34-day-old patient with progressively worsening vomiting and abdominal distention, diagnosed as hypertrophic pyloric stenosis, was operated by pediatric surgery department. After operation, the patient required pediatric intensive care unit admission due to respiratory distress, anemia, hypoalbuminemia, and generalized edema. Physical examination of our patient revealed dysmorphic facial features, finger anomalies, sacral dimple, and ambiguous genitalia, with chromosomal determination as XY. Molecular genetic testing was performed, and mutations in the DHCR7 gene of homozygous c.1342G>A/p.Glu448Lys (rs80338864) were detected. Infants with progressive projectile vomiting, feeding problems, and multiple anomalies with dysmorphic facial anomalies may be suspected to have SLOS and their families should be advised to have genetic testing and genetic counseling.

The effect of tension-free vaginal tape on coital incontinence concomitant with stress urinary incontinence.

OBJECTIVES: The aim of this study was to evaluate the impact of tension-free vaginal tape (TVT) on coital incontinence concomitant with stress urinary incontinence. METHODS: TVT was performed on sexually active women diagnosed with urodynamic stress incontinence (USI) who also experienced coital incontinence with penetration and/or orgasm. The patient-reported success rate was assessed by the Patient's Global Impression of Improvement (PGI-I) scale. The sexual function of the women was evaluated by the fulfilled Female Sexual Function Index (FSFI) before and after the operations. RESULTS: Eighty-two women underwent the TVT procedure with epidural anesthesia and 80 of them (97%) answered pre-operative and post-operative FSFI questionnaires. In the pre-operative clinical assessment, 48 women (58%) stated they experienced urinary incontinence during penetration, 13 (15%) during orgasm, and 21 (25%) identified it for both. The patient-reported success rate was 86% (71 of 82 patients) according to the PGI-I results: 44 of 48 women (91%) during penetration, nine of 13 (69%) during orgasm, and 18 of 21 (85%) for both. The FSFI scores for sexual desire, lubrication, and sexual arousal domains increased in 57 (71%), 49 (61%), and 44 (55%) patients, respectively, whereas they remained unchanged in 23 (28%), 31 (38%), and 36 (45%) patients. For the orgasm, satisfaction, and pain domains, the results were similar. The mean total FSFI score before the operations was 23.63 ± 6.84 and it significantly increased after surgery to 29.47 ± 4.28 (P < .05). CONCLUSIONS: The TVT procedure may offer treatment for coital incontinence accompanying USI. It also provides significant improvement in the sexual lives of women.

Irgm2 and Gate-16 cooperatively dampen Gram-negative bacteria-induced caspase-11 response.

Inflammatory caspase-11 (rodent) and caspases-4/5 (humans) detect the Gram-negative bacterial component LPS within the host cell cytosol, promoting activation of the non-canonical inflammasome. Although non-canonical inflammasome-induced pyroptosis and IL-1-related cytokine release are crucial to mount an efficient immune response against various bacteria, their unrestrained activation drives sepsis. This suggests that cellular components tightly control the threshold level of the non-canonical inflammasome in order to ensure efficient but non-deleterious inflammatory responses. Here, we show that the IFN-inducible protein Irgm2 and the ATG8 family member Gate-16 cooperatively counteract Gram-negative bacteria-induced non-canonical inflammasome activation, both in cultured macrophages and in vivo. Specifically, the Irgm2/Gate-16 axis dampens caspase-11 targeting to intracellular bacteria, which lowers caspase-11-mediated pyroptosis and cytokine release. Deficiency in Irgm2 or Gate16 induces both guanylate binding protein (GBP)-dependent and GBP-independent routes for caspase-11 targeting to intracellular bacteria. Our findings identify molecular effectors that fine-tune bacteria-activated non-canonical inflammasome responses and shed light on the understanding of the immune pathways they control.

Chimeric rabbit/human Fab antibodies against the hepatitis Be-antigen and their potential applications in assays, characterization, and therapy.

Hepatitis B virus (HBV) infection afflicts millions worldwide, causing cirrhosis and liver cancer. HBV e-antigen (HBeAg), a clinical marker for disease severity, is a soluble variant of the viral capsid protein. HBeAg is not required for viral replication but is implicated in establishing immune tolerance and chronic infection. The structure of recombinant e-antigen (rHBeAg) was recently determined, yet to date, the exact nature and quantitation of HBeAg still remain uncertain. Here, to further characterize HBeAg, we used phage display to produce a panel of chimeric rabbit/human monoclonal antibody fragments (both Fab and scFv) against rHBeAg. Several of the Fab/scFv, expressed in Escherichia coli, had unprecedentedly high binding affinities (Kd ∼10-12 m) and high specificity. We used Fab/scFv in the context of an enzyme-linked immunosorbent assay (ELISA) for HBeAg quantification, which we compared with commercially available kits and verified with seroconversion panels, the WHO HBeAg standard, rHBeAg, and patient plasma samples. We found that the specificity and sensitivity are superior to those of existing commercial assays. To identify potential fine differences between rHBeAg and HBeAg, we used these Fabs in microscale immunoaffinity chromatography to purify HBeAg from individual patient plasmas. Western blotting and MS results indicated that rHBeAg and HBeAg are essentially structurally identical, although HBeAg from different patients exhibits minor carboxyl-terminal heterogeneity. We discuss several potential applications for the humanized Fab/scFv.

Novel NLRP3/cryopyrin mutations and pro-inflammatory cytokine profiles in Behçet's syndrome patients.

Behçet's syndrome (BS) is a systemic inflammatory disorder with unknown etiology. Features of both innate and adaptive immunity have been claimed in the pathogenesis of BS. To test the possible dysregulation of the NLRP3/cryopyrin (Nod-like receptor with a pyrin domain 3) inflammasome, as a result of mutation(s), we performed single-strand conformation polymorphism analyses and/or sequencing of all the coding regions and intron-exon boundaries of NLRP3/cryopyrin and ASC (apoptosis-associated speck-like protein containing CARD) genes from Turkish BS patients and healthy controls. At the same time, we determined pro-inflammatory cytokine secretion profiles of peripheral blood cells in response to LPS treatment using ELISA. BS patients with vascular involvement showed significantly increased levels of TNF-α release at 2-, 4- and 8-h post-treatment and significantly increased IL-1ß levels were detected at 2h (P = 0.005) and 4h (P = 0.025) (n = 10). We identified four mutations in the NLRP3/cryopyrin gene, V200M (n = 3/104) and T195M (n = 1/104), in BS patients but none in control samples. No mutations were detected in the ASC gene. The effect of these NLRP3/cryopyrin mutants on ASC speck assembly and IL-1ß secretion was tested and the V200M mutant was shown to induce IL-1ß secretion. Thus, it is likely that certain mutations in NLRP3/cryopyrin in combination with yet unknown other factors may contribute to the pro-inflammatory cytokine profiles in BS patients.

Structural basis for activation of an integral membrane protease by lipopolysaccharide.

Omptins constitute a unique family of outer membrane proteases that are widespread in Enterobacteriaceae. The plasminogen activator (Pla) of Yersinia pestis is an omptin family member that is very important for development of both bubonic and pneumonic plague. The physiological function of Pla is to cleave (activate) human plasminogen to form the plasma protease plasmin. Uniquely, lipopolysaccharide (LPS) is essential for the catalytic activity of all omptins, including Pla. Why omptins require LPS for enzymatic activity is unknown. Here, we report the co-crystal structure of LPS-free Pla in complex with the activation loop peptide of human plasminogen, its natural substrate. The structure shows that in the absence of LPS, the peptide substrate binds deep within the active site groove and displaces the nucleophilic water molecule, providing an explanation for the dependence of omptins on LPS for enzymatic activity.

Structure of the two transmembrane Cu+ transport sites of the Cu+ -ATPases.

Cu(+)-ATPases drive metal efflux from the cell cytoplasm. Paramount to this function is the binding of Cu(+) within the transmembrane region and its coupled translocation across the permeability barrier. Here, we describe the two transmembrane Cu(+) transport sites present in Archaeoglobus fulgidus CopA. Both sites can be independently loaded with Cu(+). However, their simultaneous occupation is associated with enzyme turnover. Site I is constituted by two Cys in transmembrane segment (TM) 6 and a Tyr in TM7. An Asn in TM7 and Met and Ser in TM8 form Site II. Single site x-ray spectroscopic analysis indicates a trigonal coordination in both sites. This architecture is distinct from that observed in Cu(+)-trafficking chaperones and classical cuproproteins. The high affinity of these sites for Cu(+) (Site I K(a)=1.3 fM(-1), Site II K(a)=1.1 fM(-1)), in conjunction with reversible direct Cu(+) transfer from chaperones, points to a transport mechanism where backward release of free Cu(+) to the cytoplasm is largely prevented.

Novel Zn2+ coordination by the regulatory N-terminus metal binding domain of Arabidopsis thaliana Zn(2+)-ATPase HMA2.

Arabidopsis thaliana HMA2 is a Zn2+ transporting P1B-type ATPase required for maintaining plant metal homeostasis. HMA2 and all eukaryote Zn2+-ATPases have unique conserved N- and C-terminal sequences that differentiate them from other P1B-type ATPases. Homology modeling and structural comparison by circular dichroism indicate that the 75 amino acid long HMA2 N-terminus shares the betaalphabetabetaalpha folding present in most P1B-type ATPase N-terminal metal binding domains (N-MBDs). However, the characteristic metal binding sequence CysXXCys is replaced by Cys17CysXXGlu21, a sequence present in all plant Zn2+-ATPases. The isolated HMA2 N-MBD fragment binds a single Zn2+ (Kd 0.18 microM), Cd2+ (Kd 0.27 microM), or, with less affinity, Cu+ (Kd 13 microM). Mutagenesis studies indicate that Cys17, Cys18, and Glu21 participate in Zn2+ and Cd2+ coordination, while Cys17 and Glu21, but not Cys18, are required for Cu+ binding. Interestingly, the Glu21Cys mutation that generates a CysCysXXCys site is unable to bind Zn2+ or Cd2+ but it binds Cu+ with affinity (Kd 1 microM) higher than wild type N-MBD. Truncated HMA2 lacking the N-MBD showed reduced ATPase activity without significant changes in metal binding to transmembrane metal binding sites. Likewise, ATPase activity of HMA2 carrying mutations Cys17Ala, Cys18Ala, and Glu21Ala/Cys was also reduced but showed a metal dependence similar to the wild type enzyme. These observations suggest that plant Zn2+-ATPase N-MBDs have a folding and function similar to Cu+-ATPase N-MBDs. However, the unique Zn2+ coordination via two thiols and a carboxyl group provides selective binding of the activating metals to these regulatory domains. Metal binding through these side chains, although found in different sequences, appears as a common feature of both bacterial and eukaryotic Zn2+-ATPase N-MBDs.

The structure and function of heavy metal transport P1B-ATPases.

P(1B)-type ATPases transport heavy metals (Cu+, Cu2+, Zn2+, Co2+, Cd2+, Pb2+) across membranes. Present in most organisms, they are key elements for metal homeostasis. P(1B)-type ATPases contain 6-8 transmembrane fragments carrying signature sequences in segments flanking the large ATP binding cytoplasmic loop. These sequences made possible the differentiation of at least four P(1B)-ATPase subgroups with distinct metal selectivity: P(1B-1): Cu+, P(1B-2): Zn2+, P(1B-3): Cu2+, P(1B-4): Co2+. Mutagenesis of the invariant transmembrane Cys in H6, Asn and Tyr in H7 and Met and Ser in H8 of the Archaeoglobus fulgidus Cu+-ATPase has revealed that their side chains likely coordinate the metals during transport and constitute a central unique component of these enzymes. The structure of various cytoplasmic domains has been solved. The overall structure of those involved in enzyme phosphorylation (P-domain), nucleotide binding (N-domain) and energy transduction (A-domain), appears similar to those described for the SERCA Ca2+-ATPase. However, they show different features likely associated with singular functions of these proteins. Many P(1B)-type ATPases, but not all of them, also contain a diverse arrangement of cytoplasmic metal binding domains (MBDs). In spite of their structural differences, all N- and C-terminal MBDs appear to control the enzyme turnover rate without affecting metal binding to transmembrane transport sites. In addition, eukaryotic Cu+-ATPases have multiple N-MBD regions that participate in the metal dependent targeting and localization of these proteins. The current knowledge of structure-function relationships among the different P(1B)-ATPases allows for a description of selectivity, regulation and transport mechanisms. Moreover, it provides a framework to understand mutations in human Cu+-ATPases (ATP7A and ATP7B) that lead to Menkes and Wilson diseases.

A novel regulatory metal binding domain is present in the C terminus of Arabidopsis Zn2+-ATPase HMA2.

HMA2 is a Zn2+-ATPase from Arabidopsis thaliana. It contributes to the maintenance of metal homeostasis in cells by driving Zn2+ efflux. Distinct from P1B-type ATPases, plant Zn2+-ATPases have long C-terminal sequences rich in Cys and His. Removal of the 244 amino acid C terminus of HMA2 leads to a 43% reduction in enzyme turnover without significant effect on the Zn2+ K(1/2) for enzyme activation. Characterization of the isolated HMA2 C terminus showed that this fragment binds three Zn2+ with high affinity (Kd = 16 +/- 3 nM). Circular dichroism spectral analysis indicated the presence of 8% alpha-helix, 45% beta-sheet, and 48% random coil in the C-terminal peptide with noticeable structural changes upon metal binding (8% alpha-helix, 39% beta-sheet, and 52% random coil). Zn K-edge XAS of Zn-C-MBD in the presence of one equivalent of Zn2+ shows that the average zinc complex formed is composed of three His and one Cys residues. Upon the addition of two extra Zn2+ ions per C-MBD, these appear coordinated primarily by His residues thus, suggesting that the three Zn2+ binding domains might not be identical. Modification of His residues with diethyl pyrocarbonate completely inhibited Zn2+ binding to the C terminus, pointing out the importance of His residues in Zn2+ coordination. In contrast, alkylation of Cys with iodoacetic acid did not prevent Zn2+ binding to the HMA2 C terminus. Zn K-edge XAS of the Cys-alkylated protein was consistent with (N/O)4 coordination of the zinc site, with three of those ligands fitting for His residues. In summary, plant Zn2+-ATPases contain novel metal binding domains in their cytoplasmic C terminus. Structurally distinct from the well characterized N-terminal metal binding domains present in most P1B-type ATPases, they also appear to regulate enzyme turnover rate.

Arabidopsis HMA2, a divalent heavy metal-transporting P(IB)-type ATPase, is involved in cytoplasmic Zn2+ homeostasis.

PIB-type ATPases transport heavy metal ions (Cu+, Cu2+, Zn2+, Cd2+, Co2+, etc.) across biological membranes. Several members of this subfamily are present in plants. Higher plants are the only eukaryotes where putative Zn(2+)-ATPases have been identified. We have cloned HMA2, a PIB-ATPase present in Arabidopsis (Arabidopsis thaliana), and functionally characterized this enzyme after heterologous expression in yeast (Saccharomyces cerevisiae). HMA2 is a Zn(2+)-dependent ATPase that is also activated by Cd2+ and, to a lesser extent, by other divalent heavy metals (Pb2+, Ni2+, Cu2+, and Co2+). The enzyme forms an acid-stable phosphorylated intermediate and is inhibited by vanadate. HMA2 interacts with Zn2+ and Cd2+ with high affinity (Zn2+ K(1/2) = 0.11 +/- 0.03 microm and Cd2+ K(1/2) = 0.031 +/- 0.007 microm). However, its activity is dependent on millimolar concentrations of Cys in the assay media. Zn2+ transport determinations indicate that the enzyme drives the outward transport of metals from the cell cytoplasm. Analysis of HMA2 mRNA suggests that the enzyme is present in all plant organs and transcript levels do not change in plants exposed to various metals. Removal of HMA2 full-length transcript results in Zn2+ accumulation in plant tissues. hma2 mutant plants also accumulate Cd2+ when exposed to this metal. These results suggest that HMA2 is responsible for Zn2+ efflux from the cells and therefore is required for maintaining low cytoplasmic Zn2+ levels and normal Zn2+ homeostasis.