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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.

Encapsulins are protein nanocompartments that regulate cellular metabolism in several bacteria and archaea. Myxococcus xanthus encapsulins protect the bacterial cells against oxidative stress by sequestering cytosolic iron. These encapsulins are formed by the shell protein EncA and three cargo proteins: EncB, EncC, and EncD. EncB and EncC form rotationally symmetric decamers with ferroxidase centers (FOCs) that oxidize Fe +2 to Fe +3 for iron storage in mineral form. However, the structure and function of the third cargo protein, EncD, have yet to be determined. Here, we report the x-ray crystal structure of EncD in complex with flavin mononucleotide. EncD forms an α-helical hairpin arranged as an antiparallel dimer, but unlike other flavin-binding proteins, it has no β-sheet, showing that EncD and its homologs represent a unique class of bacterial flavin-binding proteins. The cryo-EM structure of EncA-EncD encapsulins confirms that EncD binds to the interior of the EncA shell via its C-terminal targeting peptide. With only 100 amino acids, the EncD α-helical dimer forms the smallest flavin-binding domain observed to date. Unlike EncB and EncC, EncD lacks a FOC, and our biochemical results show that EncD instead is a NAD(P)H-dependent ferric reductase, indicating that the M. xanthus encapsulins act as an integrated system for iron homeostasis. Overall, this work contributes to our understanding of bacterial metabolism and could lead to the development of technologies for iron biomineralization and the production of iron-containing materials for the treatment of various diseases associated with oxidative stress., Competing Interests: Competing interests statement:The authors declare no competing interest.

HIV-1 Rev is an essential regulatory protein that transports unspliced and partially spliced viral mRNAs from the nucleus to the cytoplasm for the expression of viral structural proteins. During its nucleocytoplasmic shuttling, Rev interacts with several host proteins to use the cellular machinery for the advantage of the virus. Here, we report the 3.5 Å cryo-EM structure of a 4.8 MDa Rev-tubulin ring complex. Our structure shows that Rev's arginine-rich motif (ARM) binds to both the acidic surfaces and the C-terminal tails of α/β-tubulin. The Rev-tubulin interaction is functionally homologous to that of kinesin-13, potently destabilizing microtubules at sub-stoichiometric levels. Expression of Rev in astrocytes and HeLa cells shows that it can modulate the microtubule cytoskeleton within the cellular environment. These results show a previously undefined regulatory role of Rev., Competing Interests: Declaration of interests The authors declare that they have no conflicts of interest with the contents of this article., (Published by Elsevier Ltd.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

As ACE2 is the critical SARS-CoV-2 receptor, we hypothesized that aerosol administration of clinical grade soluble human recombinant ACE2 (APN01) will neutralize SARS-CoV-2 in the airways, limit spread of infection in the lung, and mitigate lung damage caused by deregulated signaling in the renin-angiotensin (RAS) and Kinin pathways. Here, after demonstrating in vitro neutralization of SARS-CoV-2 by APN01, and after obtaining preliminary evidence of its tolerability and preventive efficacy in a mouse model, we pursued development of an aerosol formulation. As a prerequisite to a clinical trial, we evaluated both virus binding activity and enzymatic activity for cleavage of Ang II following aerosolization. We report successful aerosolization for APN01, retaining viral binding as well as catalytic RAS activity. Dose range-finding and IND-enabling repeat-dose aerosol toxicology testing were conducted in dogs. Twice daily aerosol administration for two weeks at the maximum feasible concentration revealed no notable toxicities. Based on these results, a Phase I clinical trial in healthy volunteers has now been initiated (NCT05065645), with subsequent Phase II testing planned for individuals with SARS-CoV-2 infection., Competing Interests: Gerald Wirnsberger and Sonja Holler and Romana Gugensberger were employed by Apeiron Biologics A.G. Apeiron supplied the APN01 for study. Josef M. Pettinger was a founder of Apeiron, is a current shareholder and inventor of APN01. David L. McCormick is a Section Editor for PLOS One. Other authors declare no competing interests.

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., Competing Interests: Declaration of interests The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022. Published by Elsevier Ltd.)

Cryptophycin-52 (Cp-52) is potentially the most potent anticancer drug known, with IC 50 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 β-tubulin, particularly in helices H8 and H10, with distinct differences between α and β 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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Published by Elsevier Inc.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for coronavirus disease 2019 (COVID-19), encodes two proteases required for replication. The main protease (M pro ), encoded as part of two polyproteins, pp1a and pp1ab, is responsible for 11 different cleavages of these viral polyproteins to produce mature proteins required for viral replication. M pro is therefore an attractive target for therapeutic interventions. Certain proteins in cells under oxidative stress undergo modification of reactive cysteines. We show M pro is susceptible to glutathionylation, leading to inhibition of dimerization and activity. Activity of glutathionylated M pro could be restored with reducing agents or glutaredoxin. Analytical studies demonstrated that glutathionylated M pro primarily exists as a monomer and that modification of a single cysteine with glutathione is sufficient to block dimerization and inhibit its activity. Gel filtration studies as well as analytical ultracentrifugation confirmed that glutathionylated M pro exists as a monomer. Tryptic and chymotryptic digestions of M pro as well as experiments using a C300S M pro mutant revealed that Cys300, which is located at the dimer interface, is a primary target of glutathionylation. Moreover, Cys300 is required for inhibition of activity upon M pro glutathionylation. These findings indicate that M pro dimerization and activity can be regulated through reversible glutathionylation of a non-active site cysteine, Cys300, which itself is not required for M pro activity, and provides a novel target for the development of agents to block M pro dimerization and activity. This feature of M pro may have relevance to the pathophysiology of SARS-CoV-2 and related bat coronaviruses. IMPORTANCE SARS-CoV-2 is responsible for the devastating COVID-19 pandemic. Therefore, it is imperative that we learn as much as we can about the biochemistry of the coronavirus proteins to inform development of therapy. One attractive target is the main protease (M pro ), a dimeric enzyme necessary for viral replication. Most work thus far developing M pro inhibitors has focused on the active site. Our work has revealed a regulatory mechanism for M pro activity through glutathionylation of a cysteine (Cys300) at the dimer interface, which can occur in cells under oxidative stress. Cys300 glutathionylation inhibits M pro activity by blocking its dimerization. This provides a novel accessible and reactive target for drug development. Moreover, this process may have implications for disease pathophysiology in humans and bats. It may be a mechanism by which SARS-CoV-2 has evolved to limit replication and avoid killing host bats when they are under oxidative stress during flight.

Hepatitis B virus (HBV) is a leading cause of liver disease. The capsid is an essential component of the virion and it is therefore of interest how it assembles and disassembles. The capsid protein is unusual both for its rare fold and that it polymerizes according to two different icosahedral symmetries, causing the polypeptide chain to exist in seven quasi-equivalent environments: A, B, and C in AB and CC dimers in T = 3 capsids, and A, B, C, and D in AB and CD dimers in T = 4 capsids. We have compared the two capsids by cryo-EM at 3.5 Å resolution. To ensure a valid comparison, the two capsids were prepared and imaged under identical conditions. We find that the chains have different conformations and potential energies, with the T = 3 C chain having the lowest. Three of the four quasi-equivalent dimers are asymmetric with respect to conformation and potential energy; however, the T = 3 CC dimer is symmetrical and has the lowest potential energy although its intra-dimer interface has the least free energy of formation. Of all the inter-dimer interfaces, the CB interface has the least area and free energy, in both capsids. From the calculated energies of higher-order groupings of dimers discernible in the lattices we predict early assembly intermediates, and indeed we observe such structures by negative stain EM of in vitro assembly reactions. By sequence analysis and computational alanine scanning we identify key residues and motifs involved in capsid assembly. Our results explain several previously reported observations on capsid assembly, disassembly, and dimorphism., Competing Interests: The authors have declared that no competing interests exist.

The hepatitis B virus e antigen, an alternative transcript of the core gene, is a secreted protein that maintains viral persistence. The physiological form has extended C termini relative to Cp(-10)149, the construct used in many studies. To examine the role of the C termini, we expressed the constructs Cp(-10)151 and Cp(-10)154, which have additional arginine residues. Both constructs when treated with reductant formed capsids more efficiently than Cp(-10)149. These capsids were also substantially more stable, as measured by thermal denaturation and resistance to urea dissociation. Mutagenesis suggests that electrostatic interactions between the additional arginine residues and glutamate residues on adjacent subunits play a role in the extra stabilization. These findings have implications for the physiological role and biotechnological potential of this protein., (Published 2019. This article is a U.S. Government work and is in the public domain in the USA.)

Pigmentation is the result of a complex process by which the biopolymer melanin is synthesized and packed into melanosomes of melanocytes. Various types of oculocutaneous albinism (OCA), a series of autosomal recessive disorders, are associated with reduced pigmentation in the skin, eyes, and hair due to genetic mutations of proteins involved in melanogenesis. Human tyrosinase (Tyr) and tyrosinase-related protein 1 (Tyrp1) drives the enzymatic process of pigment bio-polymerization. However, within the melanogenic pathway, Tyrp1 has catalytic functions not clearly defined and distinct from Tyr. Here, we characterize the biochemical and biophysical properties of recombinant human Tyrp1. For this purpose, we purified and analyzed the intra-melanosomal domain (Tyrp1tr) for protein stability and enzymatic function in conditions mimicking the environment within melanosomes and the endoplasmic reticulum. The study suggests that Tyrp1tr is a monomeric molecule at ambient temperatures and below (<25 °C). At higher temperatures, >31 °C, higher protein aggregates form with a concurrent decrease of monomers in solution. Also, Tyrp1tr diphenol oxidase activity at pH 5.5 rises as both the pre-incubation temperature and the higher molecular weight protein aggregates formation increases. The enhanced protein activity is consistent with the volume exclusion change caused by protein aggregates.

Tyrosinases are melanocyte-specific enzymes involved in melanin biosynthesis. Mutations in their genes cause oculocutaneous albinism associated with reduced or altered pigmentation of skin, hair, and eyes. Here, the recombinant human intra-melanosomal domains of tyrosinase, TYRtr (19-469), and tyrosinase-related protein 1, TYRP1tr (25-472), were studied in vitro to define their functional relationship. Proteins were expressed or coexpressed in whole Trichoplusia ni larvae and purified. Their associations were studied using gel filtration and sedimentation equilibrium methods. Protection of TYRtr was studied by measuring the kinetics of tyrosinase diphenol oxidase activity in the presence (1:1 and 1:20 molar ratios) or the absence of TYRP1tr for 10 hr under conditions mimicking melanosomal and ER pH values. Our data indicate that TYRtr incubation with excess TYRP1tr protects TYR, increasing its stability over time. However, this mechanism does not appear to involve the formation of stable hetero-oligomeric complexes to maintain the protective function., (Published 2019. This article is a U.S. Government work and is in the public domain in the USA.)

Hepatitis B virus (HBV) is the leading cause of liver disease worldwide. While an adequate vaccine is available, current treatment options are limited, not highly effective, and associated with adverse effects, encouraging the development of alternative therapeutics. The HBV core gene encodes two different proteins: core, which forms the viral nucleocapsid, and pre-core, which serves as an immune modulator with multiple points of action. The two proteins mostly have the same sequence, although they differ at their N and C termini and in their dimeric arrangements. Previously, we engineered two human-framework antibody fragments (Fab/scFv) with nano- to picomolar affinities for both proteins. Here, by means of X-ray crystallography, analytical ultracentrifugation, and electron microscopy, we demonstrate that the antibodies have non-overlapping epitopes and effectively block biologically important assemblies of both proteins. These properties, together with the anticipated high tolerability and long half-lives of the antibodies, make them promising therapeutics., (Published by Elsevier Ltd.)

HIV-1 Rev protein mediates nuclear export of unspliced and partially spliced viral RNAs for production of viral genomes and structural proteins. Rev assembles on a 351-nt Rev response element (RRE) within viral transcripts and recruits host export machinery. Small (<40-nt) RNA aptamers that compete with the RRE for Rev binding inhibit HIV-1 viral replication. We determined the X-ray crystal structure of a potential anti-HIV-1 aptamer that binds Rev with high affinity (K d  = 5.9 nM). The aptamer is structurally similar to the RRE high-affinity site but forms additional contacts with Rev unique to its sequence. Exposed bases of the aptamer interleave with the guanidinium groups of two arginines of Rev, forming stacking interactions and hydrogen bonds. The aptamer also obstructs an oligomerization interface of Rev, blocking Rev self-assembly. We propose that this aptamer can inhibit HIV-1 replication by interfering with Rev-RRE, Rev-Rev, and possibly Rev-host protein interactions., (Published by Elsevier Ltd.)

HIV-1 Rev mediates the nuclear export of unspliced and partially-spliced viral transcripts for the production of progeny genomes and structural proteins. In this process, four (or more) copies of Rev assemble onto a highly-structured 351-nt region in such viral transcripts, the Rev response element (RRE). How this occurs is not known. The Rev assembly domain has a helical-hairpin structure which associates through three (A-A, B-B and C-C) interfaces. The RRE has the topology of an upper-case letter A, with the two known Rev binding sites mapping onto the legs of the A. We have determined a crystal structure for the Rev assembly domain at 2.25 Å resolution, without resort to either mutations or chaperones. It shows that B-B dimers adopt an arrangement reversed relative to that previously reported, and join through a C-C interface to form tetramers. The new subunit arrangement shows how four Rev molecules can assemble on the two sites on the RRE to form the specificity checkpoint, and how further copies add through A-A interactions. Residues at the C-C interface, specifically the Pro31-Trp45 axis, are a potential target for intervention., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Human tyrosinase (hTyr) is a Type 1 membrane bound glycoenzyme that catalyzes the initial and rate-limiting steps of melanin production in the melanosome. Mutations in the Tyr gene are linked to oculocutaneous albinism type 1 (OCA1), an autosomal recessive disorder. Currently, the application of enzyme replacement therapy for a treatment of OCA1 is hampered by the absence of pure hTyr. Here, full-length hTyr (residues 1-529) was overexpressed in Trichoplusia ni larvae infected with a baculovirus, solubilized with detergent and purified using chromatography. Michaelis-Menten kinetics, enzymatic specific activity, and analytical ultracentrifugation were used to compare the hTyr in detergent with the soluble recombinant intra-melanosomal domain, hTyrCtr (residues 19-469). Active hTyr is monomeric in detergent micelles suggesting no stable interactions between protein molecules. Both, hTyr and hTyrCtr, exhibited similar enzymatic activity and ligand affinity in L-DOPA and L-Tyrosine reactions. In addition, expression in larvae is a scalable process that will allow high yield protein production. Thus, larval production of enzymatically active human tyrosinase potentially could be a useful tool in developing a cure for OCA1., Competing Interests: The authors have declared that no competing interests exist.

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 ( K d ∼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.

Interleukin 35 (IL-35) is a heterodimeric cytokine composed of IL-12p35 and Ebi3 subunits. IL-35 suppresses autoimmune diseases while preventing host defense to infection and promoting tumor growth and metastasis by converting resting B and T cells into IL-10-producing and IL-35-producing regulatory B (Breg) and T (Treg) cells. Despite sharing the IL-12p35 subunit, IL-12 (IL-12p35/IL-12p40) promotes inflammatory responses whereas IL-35 (IL-12p35/Ebi3) induces regulatory responses, suggesting that IL-12p35 may have unknown intrinsic immune-regulatory functions regulated by its heterodimeric partner. Here we show that the IL-12p35 subunit has immunoregulatory functions hitherto attributed to IL-35. IL-12p35 suppresses lymphocyte proliferation, induces expansion of IL-10-expressing and IL-35-expressing B cells and ameliorates autoimmune uveitis in mice by antagonizing pathogenic Th17 responses. Recapitulation of essential immunosuppressive activities of IL-35 indicates that IL-12p35 may be utilized for in vivo expansion of Breg cells and autologous Breg cell immunotherapy. Furthermore, our uveitis data suggest that intrinsic immunoregulatory activities of other single chain IL-12 subunits might be exploited to treat other autoimmune diseases.IL-12p35 is common to IL-35 and IL-12, which have opposing effects on inflammation. Here the authors show that the IL-12p35 subunit induces regulatory B cells and can be used therapeutically to limit autoimmune uveitis in mice.

The purification of an enzyme from insect larvae infected with a baculovirus vector is described. The enzyme tyrosinase is of biomedical importance and catalyzes the first rate-limiting steps in melanin production. Tyrosinase mutations can result in oculocutaneous albinism type 1 (OCA1), an inherited eye disease associated with decreased melanin pigment production and vision defects. To simplify expression and subsequent purification, the extracellular domain is expressed in insect cells, produced in Trichoplusia ni larvae, and purified using affinity and size-exclusion chromatography. The purified recombinant human tyrosinase is a soluble monomeric glycoprotein with an activity that mirrors the tyrosinase in vivo function. © 2017 by John Wiley & Sons, Inc., (Copyright © 2017 John Wiley & Sons, Inc.)

The transcriptional programs that guide lymphocyte differentiation depend on the precise expression and timing of transcription factors (TFs). The TF BACH2 is essential for T and B lymphocytes and is associated with an archetypal super-enhancer (SE). Single-nucleotide variants in the BACH2 locus are associated with several autoimmune diseases, but BACH2 mutations that cause Mendelian monogenic primary immunodeficiency have not previously been identified. Here we describe a syndrome of BACH2-related immunodeficiency and autoimmunity (BRIDA) that results from BACH2 haploinsufficiency. Affected subjects had lymphocyte-maturation defects that caused immunoglobulin deficiency and intestinal inflammation. The mutations disrupted protein stability by interfering with homodimerization or by causing aggregation. We observed analogous lymphocyte defects in Bach2-heterozygous mice. More generally, we observed that genes that cause monogenic haploinsufficient diseases were substantially enriched for TFs and SE architecture. These findings reveal a previously unrecognized feature of SE architecture in Mendelian diseases of immunity: heterozygous mutations in SE-regulated genes identified by whole-exome/genome sequencing may have greater significance than previously recognized.

Z-disc-associated, alternatively spliced, PDZ motif-containing protein (ZASP) is a principal component of the sarcomere. The three prevalent isoforms of ZASP in skeletal muscle are generated by alternative splicing of exons 9 and 10. The long isoforms, either having (ZASP-L) or lacking exon 10 (ZASP-LΔex10), include an N-terminal PDZ domain, an actin-binding region (ABR) with a conserved motif (ZM), and three C-terminal LIM domains. The short isoform (ZASP-S) lacks the LIM domains. Mutations, A147T and A165V, within the ZM of ZASP-LΔex10 cause myofibrillar myopathy, but the mechanism is unknown. We have prepared these proteins, their ABR, and the respective mutant variants in recombinant form, characterized them biophysically, and analyzed their actin-binding properties by surface plasmon resonance and electron microscopy. All the proteins were physically homogeneous and monomeric and had circular dichroic spectra consistent with partially folded conformations. Comparison of the NMR HSQC spectra of ZASP-S and the PDZ domain showed that the ABR is unstructured. ZASP-S and its mutant variants and ZASP-LΔex10 all bound to immobilized G-actin with high affinity (K d ≈ 10 -8 to 10 -9 M). Constructs of the isolated actin-binding region missing exon 10 (ABRΔ10) bound with lower affinity (K d ≈ 10 -7 M), but those retaining exon 10 (ABR+10) did so only weakly (K d ≈ 10 -5 M). ZASP-S, and the ABRΔ10, also induced F-actin and array formation, even in conditions of low ionic strength and in the absence of KCl and Mg 2+ ions. Interestingly, the ZM mutations A147T and A165V did not affect any of the results described above.

Protein synthesis is initiated by methionine in eukaryotes and by formylmethionine in prokaryotes. N-terminal methionine can be co-translationally cleaved by the enzyme methionine aminopeptidase (MAP). When recombinant proteins are expressed in bacterial and mammalian expression systems, there is a simple universal rule that predicts whether the initiating methionine will be processed by MAP based on the size of the residue adjacent (penultimate) to the N-methionine. In general, if the side chains of the penultimate residues have a radius of gyration of 1.29 Å or less, methionine is cleaved. © 2017 by John Wiley & Sons, Inc., (Copyright © 2017 John Wiley & Sons, Inc.)

Oculocutaneous albinism type 1 (OCA1) is an autosomal recessive disorder caused by mutations in the tyrosinase gene. Two subtypes of OCA1 have been described: severe OCA1A with complete absence of tyrosinase activity and less severe OCA1B with residual tyrosinase activity. Here, we characterize the recombinant human tyrosinase intramelanosomal domain and mutant variants, which mimic genetic changes in both subtypes of OCA1 patients. Proteins were prepared using site-directed mutagenesis, expressed in insect larvae, purified by chromatography, and characterized by enzymatic activities, tryptophan fluorescence, and Gibbs free energy changes. The OCA1A mutants showed very low protein expression and protein yield and are enzymatically inactive. Mutants mimicking OCA1B were biochemically similar to the wild type, but exhibited lower specific activities and protein stabilities. The results are consistent with clinical data, which indicates that OCA1A mutations inactivate tyrosinase and result in severe phenotype, while OCA1B mutations partially inactivate tyrosinase and result in OCA1B albinism., (Published 2017. This article is a U.S. Government work and is in the public domain in the USA.)

HIV-1 Rev protein mediates the nuclear export of viral RNA genomes. To do so, Rev oligomerizes cooperatively onto an RNA motif, the Rev response element (RRE), forming a complex that engages with the host nuclear export machinery. To better understand Rev oligomerization, we determined four crystal structures of Rev N-terminal domain dimers, which show that they can pivot about their dyad axis, giving crossing angles of 90° to 140°. In parallel, we performed cryoelectron microscopy of helical Rev filaments. Filaments vary from 11 to 15 nm in width, reflecting variations in dimer crossing angle. These structures contain additional density, indicating that C-terminal domains become partially ordered in the context of filaments. This conformational variability may be exploited in the assembly of RRE/Rev complexes. Our data also revealed a third interface between Revs, which offers an explanation for how the arrangement of Rev subunits adapts to the "A"-shaped architecture of the RRE in export-active complexes., (Published by Elsevier Ltd.)

When the first version of this unit was written in 1995, protein purification of recombinant proteins was based on a variety of standard chromatographic methods and approaches, many of which were described and mentioned throughout Current Protocols in Protein Science. In the interim, there has been a shift toward an almost universal usage of the affinity or fusion tag. This may not be the case for biotechnology manufacture where affinity tags can complicate producing proteins under regulatory conditions. Regardless of the protein expression system, questions are asked as to which and how many affinity tags to use, where to attach them in the protein, and whether to engineer a self-cleavage system or simply leave them on. We will briefly address some of these issues. Also, although this overview focuses on E.coli, protein expression and purification, other commonly used expression systems are mentioned and, apart from cell-breakage methods, protein purification methods and strategies are essentially the same., (Copyright © 2015 John Wiley & Sons, Inc.)

Purification of human IL-1β is used in this unit as an example of the preparation of a soluble protein from E. coli. Bacteria containing IL-1β are lysed, and IL-1 β in the resulting supernatant is purified by anion-exchange chromatography, salt precipitation, and cation-exchange chromatography, and then concentrated. Finally, the IL-1 β protein is applied to a gel-filtration column to separate it from remaining higher- and lower-molecular-weight contaminants, the purified protein is stored frozen or is lyophilized. The purification protocol described is typical for a protein that is expressed in fairly high abundance (i.e., >5% total protein) and accumulates in a soluble state. In addition, the purification procedure serves as an example of how to use classical protein purifications methods, which may also be used in conjunction with the affinity-based methods now more commonly used., (Copyright © 2014 John Wiley & Sons, Inc.)

Heterologous expression of recombinant proteins in E. coli often results in the formation of insoluble and inactive protein aggregates, commonly referred to as inclusion bodies. To obtain the native (i.e., correctly folded) and hence active form of the protein from such aggregates, four steps are usually followed: (1) the cells are lysed, (2) the cell wall and outer membrane components are removed, (3) the aggregates are solubilized (or extracted) with strong protein denaturants, and (4) the solubilized, denatured proteins are folded with concomitant oxidation of reduced cysteine residues into the correct disulfide bonds to obtain the native protein. This unit features three different approaches to the final step of protein folding and purification. In the first, guanidine·HCl is used as the denaturant, after which the solubilized protein is folded (before purification) in an "oxido-shuffling" buffer system to increase the rate of protein oxidation. In the second, acetic acid is used to solubilize the protein, which is then partially purified by gel filtration before folding; the protein is then folded and oxidized by simple dialysis against water. Thirdly, folding and purification of a fusion protein using metal-chelate affinity chromatography are described., (Copyright © 2014 John Wiley & Sons, Inc.)

Human immunodeficiency viral (HIV-1) fusion is mediated by the viral envelope gp120/gp41 complex (ENVelope glycoprotein). After gp120 shedding, gp41 is exposed and elicits membrane fusion via a cascade of conformational changes. In contrast to prefusion and postfusion conformation, little is known about any intermediate conformation. We report on a solution NMR investigation of homotrimeric HIV-1 gp41(27-194), comprising the transmembrane region and reconstituted in dodecyl phosphocholine (DPC) micelles. The protein is mainly α-helical, but experiences internal dynamics on the nanosecond and micro to millisecond time scale and transient α-helical behavior for certain residues in the N-terminal heptad repeat (NHR). Strong lipid interactions are observed, in particular for C-terminal residues of the NHR and imunodominant loop region connecting NHR and C-terminal heptad repeat (CHR). Our data indicate an extended conformation with features anticipated for a prefusion intermediate, presumably in exchange with a lowly populated postfusion six-helical bundle conformation., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: The study of autoinflammatory diseases has uncovered mechanisms underlying cytokine dysregulation and inflammation., Methods: We analyzed the DNA of an index patient with early-onset systemic inflammation, cutaneous vasculopathy, and pulmonary inflammation. We sequenced a candidate gene, TMEM173, encoding the stimulator of interferon genes (STING), in this patient and in five unrelated children with similar clinical phenotypes. Four children were evaluated clinically and immunologically. With the STING ligand cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), we stimulated peripheral-blood mononuclear cells and fibroblasts from patients and controls, as well as commercially obtained endothelial cells, and then assayed transcription of IFNB1, the gene encoding interferon-β, in the stimulated cells. We analyzed IFNB1 reporter levels in HEK293T cells cotransfected with mutant or nonmutant STING constructs. Mutant STING leads to increased phosphorylation of signal transducer and activator of transcription 1 (STAT1), so we tested the effect of Janus kinase (JAK) inhibitors on STAT1 phosphorylation in lymphocytes from the affected children and controls., Results: We identified three mutations in exon 5 of TMEM173 in the six patients. Elevated transcription of IFNB1 and other gene targets of STING in peripheral-blood mononuclear cells from the patients indicated constitutive activation of the pathway that cannot be further up-regulated with stimulation. On stimulation with cGAMP, fibroblasts from the patients showed increased transcription of IFNB1 but not of the genes encoding interleukin-1 (IL1), interleukin-6 (IL6), or tumor necrosis factor (TNF). HEK293T cells transfected with mutant constructs show elevated IFNB1 reporter levels. STING is expressed in endothelial cells, and exposure of these cells to cGAMP resulted in endothelial activation and apoptosis. Constitutive up-regulation of phosphorylated STAT1 in patients' lymphocytes was reduced by JAK inhibitors., Conclusions: STING-associated vasculopathy with onset in infancy (SAVI) is an autoinflammatory disease caused by gain-of-function mutations in TMEM173. (Funded by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases; ClinicalTrials.gov number, NCT00059748.).

Proteins self-associate to form dimers and tetramers. Purified proteins are used to study the thermodynamics of protein interactions using the analytical ultracentrifuge. In this approach, monomer-dimer equilibrium constants are directly measured at various temperatures. Data analysis is used to derive thermodynamic parameters, such as Gibbs free energy, enthalpy, and entropy, which can predict which major forces are involved in protein association., (Copyright © 2014 John Wiley & Sons, Inc.)

The HIV-1 protein Rev oligomerizes on viral transcripts and directs their nuclear export. Previously, a Fab against Rev generated by phage display was used to crystallize and solve the structure of the Rev oligomerization domain. Here we have investigated the capability of this Fab to block Rev oligomerization and inhibit HIV-1 replication. The Fab itself did not have antiviral activity, but when a Tat-derived cell-penetrating peptide was appended, the resulting molecule (FabRev1-Tat) was strongly inhibitory of three different CCR5-tropic HIV-1 isolates (IC50 = 0.09-0.44 μg/ml), as assessed by suppression of reverse transcriptase activity in infected peripheral blood mononuclear cells, and had low cell toxicity (TC50 > 100 μg/ml). FabRev1-Tat was taken up by both peripheral blood mononuclear and HEK293T cells, appearing in both the cytoplasm and nucleus, as shown by immunofluorescence confocal laser scanning microscopy. Computational alanine scanning was used to identify key residues in the complementarity-determining regions to guide mutagenesis experiments. Residues in the light chain CDR3 (LCDR3) were assessed to be important. Residues in LCDR3 were mutated, and LCDR3-Tyr(92) was found to be critical for binding to Rev, as judged by surface plasmon resonance and electron microscopy. Peptides corresponding to all six CDR regions were synthesized and tested for Rev binding. None of the linear peptides had significant affinity for Rev, but four of the amide-cyclic forms did. Especially cyclic-LCDR3 (LGGYPAASYRTA) had high affinity for Rev and was able to effectively depolymerize Rev filaments, as shown by both surface plasmon resonance and electron microscopy., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

In the present study, GRL008, a novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI), and darunavir (DRV), both of which contain a P2-bis-tetrahydrofuranyl urethane (bis-THF) moiety, were found to exert potent antiviral activity (50% effective concentrations [EC50s], 0.029 and 0.002 μM, respectively) against a multidrug-resistant clinical isolate of HIV-1 (HIVA02) compared to ritonavir (RTV; EC50, >1.0 μM) and tipranavir (TPV; EC50, 0.364 μM). Additionally, GRL008 showed potent antiviral activity against an HIV-1 variant selected in the presence of DRV over 20 passages (HIVDRV(R)P20), with a 2.6-fold increase in its EC50 (0.097 μM) compared to its corresponding EC50 (0.038 μM) against wild-type HIV-1NL4-3 (HIVWT). Based on X-ray crystallographic analysis, both GRL008 and DRV showed strong hydrogen bonds (H-bonds) with the backbone-amide nitrogen/carbonyl oxygen atoms of conserved active-site amino acids G27, D29, D30, and D30' of HIVA02 protease (PRA02) and wild-type PR in their corresponding crystal structures, while TPV lacked H-bonds with G27 and D30' due to an absence of polar groups. The P2' thiazolyl moiety of RTV showed two conformations in the crystal structure of the PRA02-RTV complex, one of which showed loss of contacts in the S2' binding pocket of PRA02, supporting RTV's compromised antiviral activity (EC50, >1 μM). Thus, the conserved H-bonding network of P2-bis-THF-containing GRL008 with the backbone of G27, D29, D30, and D30' most likely contributes to its persistently greater antiviral activity against HIVWT, HIVA02, and HIVDRV(R)P20., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Hepatitis B virus core-antigen (capsid protein) and e-antigen (an immune regulator) have almost complete sequence identity, yet the dimeric proteins (termed Cp149d and Cp(-10)149d , respectively) adopt quite distinct quaternary structures. Here we use hydrogen deuterium exchange-mass spectrometry (HDX-MS) to study their structural properties. We detect many regions that differ substantially in their HDX dynamics. Significantly, whilst all regions in Cp(-10)149d exchange by EX2-type kinetics, a number of regions in Cp149d were shown to exhibit a mixture of EX2- and EX1-type kinetics, hinting at conformational heterogeneity in these regions. Comparison of the HDX of the free Cp149d with that in assembled capsids (Cp149c ) indicated increased resistance to exchange at the C-terminus where the inter-dimer contacts occur. Furthermore, evidence of mixed exchange kinetics were not observed in Cp149c , implying a reduction in flexibility upon capsid formation. Cp(-10)149d undergoes a drastic structural change when the intermolecular disulphide bridge is reduced, adopting a Cp149d -like structure, as evidenced by the detected HDX dynamics being more consistent with Cp149d in many, albeit not all, regions. These results demonstrate the highly dynamic nature of these similar proteins. To probe the effect of these structural differences on the resulting antigenicity, we investigated binding of the antibody fragment (Fab E1) that is known to bind a conformational epitope on the four-helix bundle. Whilst Fab E1 binds to Cp149c and Cp149d , it does not bind non-reduced and reduced Cp(-10)149d , despite unhindered access to the epitope. These results imply a remarkable sensitivity of this epitope to its structural context., (© 2014 The Protein Society.)

Interleukin-10 (IL-10)-producing regulatory B (Breg) cells suppress autoimmune disease, and increased numbers of Breg cells prevent host defense to infection and promote tumor growth and metastasis by converting resting CD4(+) T cells to regulatory T (Treg) cells. The mechanisms mediating the induction and development of Breg cells remain unclear. Here we show that IL-35 induces Breg cells and promotes their conversion to a Breg subset that produces IL-35 as well as IL-10. Treatment of mice with IL-35 conferred protection from experimental autoimmune uveitis (EAU), and mice lacking IL-35 (p35 knockout (KO) mice) or defective in IL-35 signaling (IL-12Rβ2 KO mice) produced less Breg cells endogenously or after treatment with IL-35 and developed severe uveitis. Adoptive transfer of Breg cells induced by recombinant IL-35 suppressed EAU when transferred to mice with established disease, inhibiting pathogenic T helper type 17 (TH17) and TH1 cells while promoting Treg cell expansion. In B cells, IL-35 activates STAT1 and STAT3 through the IL-35 receptor comprising the IL-12Rβ2 and IL-27Rα subunits. As IL-35 also induced the conversion of human B cells into Breg cells, these findings suggest that IL-35 may be used to induce autologous Breg and IL-35(+) Breg cells and treat autoimmune and inflammatory disease.

The core of skeletal muscle Z-discs consists of actin filaments from adjacent sarcomeres that are cross-linked by α-actinin homodimers. Z-disc-associated, alternatively spliced, PDZ motif-containing protein (ZASP)/Cypher interacts with α-actinin, myotilin, and other Z-disc proteins via the PDZ domain. However, these interactions are not sufficient to maintain the Z-disc structure. We show that ZASP directly interacts with skeletal actin filaments. The actin-binding domain is between the modular PDZ and LIM domains. This ZASP region is alternatively spliced so that each isoform has unique actin-binding domains. All ZASP isoforms contain the exon 6-encoded ZASP-like motif that is mutated in zaspopathy, a myofibrillar myopathy (MFM), whereas the exon 8-11 junction-encoded peptide is exclusive to the postnatal long ZASP isoform (ZASP-LΔex10). MFM is characterized by disruption of skeletal muscle Z-discs and accumulation of myofibrillar degradation products. Wild-type and mutant ZASP interact with α-actin, α-actinin, and myotilin. Expression of mutant, but not wild-type, ZASP leads to Z-disc disruption and F-actin accumulation in mouse skeletal muscle, as in MFM. Mutations in the actin-binding domain of ZASP-LΔex10, but not other isoforms, cause disruption of the actin cytoskeleton in muscle cells. These isoform-specific mutation effects highlight the essential role of the ZASP-LΔex10 isoform in F-actin organization. Our results show that MFM-associated ZASP mutations in the actin-binding domain have deleterious effects on the core structure of the Z-discs in skeletal muscle.

The capsid of hepatitis B virus (HBV) is a major viral antigen and important diagnostic indicator. HBV capsids have prominent protrusions ('spikes') on their surface and are unique in having either T = 3 or T = 4 icosahedral symmetry. Mouse monoclonal and also human polyclonal antibodies bind either near the spike apices (historically the 'α-determinant') or in the 'floor' regions between them (the 'β-determinant'). Native mass spectrometry (MS) and gas-phase electrophoretic mobility molecular analysis (GEMMA) were used to monitor the titration of HBV capsids with the antigen-binding domain (Fab) of mAb 3120, which has long defined the β-determinant. Both methods readily distinguished Fab binding to the two capsid morphologies and could provide accurate masses and dimensions for these large immune complexes, which range up to ~8 MDa. As such, native MS and GEMMA provide valuable alternatives to a more time-consuming cryo-electron microscopy analysis for preliminary characterisation of virus-antibody complexes.

Background: Tyrosinase (TYR) catalyzes the rate-limiting, first step in melanin production and its gene (TYR) is mutated in many cases of oculocutaneous albinism (OCA1), an autosomal recessive cause of childhood blindness. Patients with reduced TYR activity are classified as OCA1B; some OCA1B mutations are temperature-sensitive. Therapeutic research for OCA1 has been hampered, in part, by the absence of purified, active, recombinant wild-type and mutant human enzymes., Methodology/principal Findings: The intra-melanosomal domain of human tyrosinase (residues 19-469) and two OCA1B related temperature-sensitive mutants, R422Q and R422W were expressed in insect cells and produced in T. ni larvae. The short trans-membrane fragment was deleted to avoid potential protein insolubility, while preserving all other functional features of the enzymes. Purified tyrosinase was obtained with a yield of >1 mg per 10 g of larval biomass. The protein was a monomeric glycoenzyme with maximum enzyme activity at 37°C and neutral pH. The two purified mutants when compared to the wild-type protein were less active and temperature sensitive. These differences are associated with conformational perturbations in secondary structure., Conclusions/significance: The intramelanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their in vivo function. This advance allows for the structure - function analyses of different mutant TYR proteins and correlation with their corresponding human phenotypes; it also provides an important tool to discover drugs that may improve tyrosinase activity and treat OCA1.

With the rapid technological advances in all aspects of macromolecular X-ray crystallography the preparation of diffraction quality crystals has become the rate-limiting step. Crystallization chaperones have proven effective for overcoming this barrier. Here we describe the usage of a Fab chaperone for the crystallization of HIV-1 Rev, a protein that has long resisted all attempts at elucidating its complete atomic structure.

Nuclear export of unspliced and singly spliced viral mRNA is a critical step in the HIV life cycle. The structural basis by which the virus selects its own mRNA among more abundant host cellular RNAs for export has been a mystery for more than 25 years. Here, we describe an unusual topological structure that the virus uses to recognize its own mRNA. The viral Rev response element (RRE) adopts an "A"-like structure in which the two legs constitute two tracks of binding sites for the viral Rev protein and position the two primary known Rev-binding sites ~55 Å apart, matching the distance between the two RNA-binding motifs in the Rev dimer. Both the legs of the "A" and the separation between them are required for optimal RRE function. This structure accounts for the specificity of Rev for the RRE and thus the specific recognition of the viral RNA., (Copyright © 2013 Elsevier Inc. All rights reserved.)

GRL007 and GRL008, two structurally related nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) containing 3(R),3a(S),6a(R)-bis-tetrahydrofuranylurethane (bis-THF) as the P2 moiety and a sulfonamide isostere consisting of benzene carboxylic acid and benzene carboxamide as the P2' moiety, respectively, were evaluated for their antiviral activity and interactions with wild-type protease (PR(WT)). Both GRL007 (Ki of 12.7 pM with PR(WT)) and GRL008 (Ki of 8.9 pM) inhibited PR(WT) with high potency in vitro. X-ray crystallographic analysis of PR(WT) in complex with GRL007 or GRL008 showed that the bis-THF moiety of both compounds has three direct polar contacts with the backbone amide nitrogen atoms of Asp29 and Asp30 of PR(WT). The P2' moiety of both compounds showed one direct contact with the backbone of Asp30' and a bridging polar contact with Gly48' through a water molecule. Cell-based antiviral assays showed that GRL007 was inactive (50% effective concentration [EC50] of >1 μM) while GRL008 was highly active (EC50 of 0.04 μM) against wild-type HIV-1. High-performance liquid chromatography (HPLC)/mass spectrometry-based cellular uptake assays showed 8.1- and 84-fold higher intracellular concentrations of GRL008 than GRL007 in human MT-2 and MT-4 cell extracts, respectively. Thus, GRL007, in spite of its favorable enzyme-inhibitory activity and protease binding profile, exhibited a lack of antiviral activity in cell-based assays, most likely due to its compromised cellular uptake associated with its P2' benzene carboxylic acid moiety. The anti-HIV-1 potency, favorable toxicity, and binding profile of GRL008 suggest that further optimization of the P2' moiety may improve its antiretroviral features.

Infection of humans by hepatitis B virus (HBV) induces the copious production of antibodies directed against the capsid protein (Cp). A large variety of anticapsid antibodies have been identified that differ in their epitopes. These data, and the status of the capsid as a major clinical antigen, motivate studies to achieve a more detailed understanding of their interactions. In this study, we focused on the Fab fragments of two monoclonal antibodies, E1 and 3120. E1 has been shown to bind to the side of outward-protruding spikes whereas 3120 binds to the "floor" region of the capsid, between spikes. We used hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) to investigate the effects on HBV capsids of binding these antibodies. Conventionally, capsids loaded with saturating amounts of Fabs would be too massive to be readily amenable to HDX-MS. However, by focusing on the Cp protein, we were able to acquire deuterium uptake profiles covering the entire 149-residue sequence and reveal, in localized detail, changes in H/D exchange rates accompanying antibody binding. We find increased protection of the known E1 and 3120 epitopes on the capsid upon binding and show that regions distant from the epitopes are also affected. In particular, the α2a helix (residues 24-34) and the mobile C-terminus (residues 141-149) become substantially less solvent-exposed. Our data indicate that even at substoichiometric antibody binding an overall increase in the rigidity of the capsid is elicited, as well as a general dampening of its breathing motions.