COPII collar defines the boundary between ER and ER exit site and does not coat cargo containers

COPII and COPI mediate the formation of membrane vesicles translocating in opposite directions within the secretory pathway. Live-cell and electron microscopy revealed a novel mode of function for COPII during cargo export from the ER. COPII is recruited to membranes defining the boundary between the ER and ER exit sites, facilitating selective cargo concentration. Using direct observation of living cells, we monitored cargo selection processes, accumulation, and fission of COPII-free ERES membranes. CRISPR/Cas12a tagging, the RUSH system, and pharmaceutical and genetic perturbations of ER-Golgi transport demonstrated that the COPII coat remains bound to the ER–ERES boundary during protein export. Manipulation of the cargo-binding domain in COPII Sec24B prohibits cargo accumulation in ERES. These findings suggest a role for COPII in selecting and concentrating exported cargo rather than coating Golgi-bound carriers. These findings transform our understanding of coat proteins’ role in ER-to-Golgi transport.     

Predicting and controlling the reactivity of immune cell populations against cancer

Heterogeneous cell populations form an interconnected network that determine their collective output. One example of such a heterogeneous immune population is tumor‐infiltrating lymphocytes (TILs), whose output can be measured in terms of its reactivity against tumors. While the degree of reactivity varies considerably between different TILs, ranging from null to a potent response, the underlying network that governs the reactivity is poorly understood. Here, we asked whether one can predict and even control this reactivity. To address this we measured the subpopulation compositions of 91 TILs surgically removed from 27 metastatic melanoma patients. Despite the large number of subpopulations compositions, we were able to computationally extract a simple set of subpopulation‐based rules that accurately predict the degree of reactivity. This raised the conjecture of whether one could control reactivity of TILs by manipulating their subpopulation composition. Remarkably, by rationally enriching and depleting selected subsets of subpopulations, we were able to restore anti‐tumor reactivity to nonreactive TILs. Altogether, this work describes a general framework for predicting and controlling the output of a cell mixture.     

Matching fusion protein systems for affinity analysis of two interacting families of proteins: the cohesin-dockerin interaction

Cellulosomes are multi-enzyme complexes that orchestrate the efficient degradation of cellulose and related plant cell wall polysaccharides. The complex is maintained by the high-affinity protein-protein interaction between two complementary modules: the cohesin and the dockerin. In order to characterize the interaction between different cohesins and dockerins, we have developed matching fusion-protein systems, which harbor either the cohesin or the dockerin component. For this purpose, corresponding plasmid cassettes were designed, which encoded for the following carrier proteins: (i) a thermostable xylanase with an appended His-tag; and (ii) a highly stable cellulose-binding module (CBM). The resultant xylanase-dockerin and CBM-cohesin fusion products exhibited high expression levels of soluble protein. The expressed, affinity-purified proteins were extremely stable, and the functionality of the cohesin or dockerin component was retained. The fusion protein system was used to establish a sensitive and reliable, semi-quantitative enzyme-linked affinity assay for determining multiple samples of cohesin-dockerin interactions in microtiter plates. A variety of cohesin-dockerin systems, which had been examined previously using other methodologies, were revisited applying the affinity-based enzyme assay, the results of which served to verify the validity of the approach.     

Systemic dysregulation of CEACAM1 in melanoma patients

It was previously shown that CEACAM1 on melanoma cells strongly predicts poor outcome. Here, we show a statistically significant increase of serum CEACAM1 in 64 active melanoma patients, as compared to 48 patients with no evidence of disease and 37 healthy donors. Among active patients, higher serum CEACAM1 correlated with LDH values and with decreased survival. Multivariate analysis with neutralization of LDH showed that increased serum CEACAM1 carries a hazard ratio of 2.40. In vitro, soluble CEACAM1 was derived from CEACAM1(+), but neither from CEACAM1(?) melanoma cells nor from CEACAM1(+) lymphocytes, and directly correlated with the number of CEACAM1(+) melanoma cells. Production of soluble CEACAM1 depended on intact de novo protein synthesis and secretion machineries, but not on metalloproteinase function. An unusually high percentage of CEACAM1(+) circulating NK and T lymphocytes was demonstrated in melanoma patients. CEACAM1 inhibited killing activity in functional assays. CEACAM1 expression could not be induced on lymphocytes by serum from patients with high CEACAM1 expression. Further, expression of other NK receptors was impaired, which collectively indicate on a general abnormality. In conclusion, the systemic dysregulation of CEACAM1 in melanoma patients further denotes the role of CEACAM1 in melanoma and may provide a basis for new tumor monitoring and prognostic platforms.     

Isolation and Chimerization of a Highly Neutralizing Antibody Conferring Passive Protection against Lethal Bacillus anthracis Infection

Several studies have demonstrated that the passive transfer of protective antigen (PA)-neutralizing antibodies can protect animals against Bacillus anthracis infection. The standard protocol for the isolation of PA-neutralizing monoclonal antibodies is based upon a primary selection of the highest PA-binders by ELISA, and usually yields only few candidates antibodies. We demonstrated that by applying a PA-neutralization functionality-based screen as the primary criterion for positive clones, it was possible to isolate more than 100 PA-neutralizing antibodies, some of which exhibited no measurable anti-PA titers in ELISA. Among the large panel of neutralizing antibodies identified, mAb 29 demonstrated the most potent activity, and was therefore chimerized. The variable region genes of the mAb 29 were fused to human constant region genes, to form the chimeric 29 antibody (cAb 29). Guinea pigs were fully protected against infection by 40LD₅₀ B. anthracis spores following two separate administrations with 10 mg/kg of cAb 29: the first administration was given before the challenge, and a second dose was administered on day 4 following exposure. Moreover, animals that survived the challenge and developed endogenous PA-neutralizing antibodies with neutralizing titers above 100 were fully protected against repeat challenges with 40LD₅₀ of B. anthracis spores. The data presented here emphasize the importance of toxin neutralization-based screens for the efficient isolation of protective antibodies that were probably overlooked in the standard screening protocol. The protective activity of the chimeric cAb 29 demonstrated in this study suggest that it may serve as an effective immunotherapeutic agent against anthrax.     

CaV1.2 I-II linker structure and Timothy syndrome

Ca_V channels are multi-subunit protein complexes that enable inward cellular Ca²⁺ currents in response to membrane depolarization. We recently described structure-function studies of the intracellular α1 subunit domain I-II linker, directly downstream of domain IS6. The results show the extent of the linker’s helical structure to be subfamily dependent, as dictated by highly conserved primary sequence differences. Moreover, the difference in structure confers different biophysical properties, particularly the extent and kinetics of voltage and calcium-dependent inactivation. Timothy syndrome is a human genetic disorder due to mutations in the Ca_V1.2 gene. Here, we explored whether perturbation of the I-II linker helical structure might provide a mechanistic explanation for a Timothy syndrome mutant’s (human Ca_V1.2 G406R equivalent) biophysical effects on inactivation and activation. The results are equivocal, suggesting that a full mechanistic explanation for this Timothy syndrome mutation requires further investigation.     

DOC2B,C2 Domains,and Calcium: A Tale of Intricate Interactions

Ca⁺²-dependent exocytosis involves vesicle docking, priming, fusion, and recycling. This process is performed and regulated by a vast number of synaptic proteins and depends on proper protein–protein and protein–lipid interactions. Double C2 domain (DOC2) is a protein family of three isoforms found while screening DNA libraries with a C2 probe. DOC2 has three domains: the Munc13-interacting domain and tandem C2s (designated C2A and C2B) connected by a short polar linker. The C2 domain binds phospholipids in a Ca²⁺-dependent manner. This review focuses on the ubiquitously expressed isoform DOC2B. Sequence alignment of the tandem C2 protein family in mouse revealed high homology (81%) between rabphilin-3A and DOC2B proteins. We created a structural model of DOC2B's C2A based on the crystal structure of rabphilin-3A with and without calcium and found that the calcium-binding loops of DOC2B move upon calcium binding, enabling efficient plasma membrane penetration of its C2A. Here, we discuss the potential relation between the DOC2B bioinformatical model and its function and suggest a possible working model for its interaction with other proteins of the exocytotic machinery, including Munc13, Munc18, and syntaxin.     

Staphylococcus aureus NrdH Redoxin Is a Reductant of the Class Ib Ribonucleotide Reductase

Staphylococci contain a class Ib NrdEF ribonucleotide reductase (RNR) that is responsible, under aerobic conditions, for the synthesis of deoxyribonucleotide precursors for DNA synthesis and repair. The genes encoding that RNR are contained in an operon consisting of three genes, nrdIEF, whereas many other class Ib RNR operons contain a fourth gene, nrdH, that determines a thiol redoxin protein, NrdH. We identified a 77-amino-acid open reading frame in Staphylococcus aureus that resembles NrdH proteins. However, S. aureus NrdH differs significantly from the canonical NrdH both in its redox-active site, C-P-P-C instead of C-M/V-Q-C, and in the absence of the C-terminal [WF]SGFRP[DE] structural motif. We show that S. aureus NrdH is a thiol redox protein. It is not essential for aerobic or anaerobic growth and appears to have a marginal role in protection against oxidative stress. In vitro, S. aureus NrdH was found to be an efficient reductant of disulfide bonds in low-molecular-weight substrates and proteins using dithiothreitol as the source of reducing power and an effective reductant for the homologous class Ib RNR employing thioredoxin reductase and NADPH as the source of the reducing power. Its ability to reduce NrdEF is comparable to that of thioredoxin-thioredoxin reductase. Hence, S. aureus contains two alternative thiol redox proteins, NrdH and thioredoxin, with both proteins being able to function in vitro with thioredoxin reductase as the immediate hydrogen donors for the class Ib RNR. It remains to be clarified under which in vivo physiological conditions the two systems are used.Ribonucleotide reductases (RNRs) are essential enzymes in all living cells, providing the only known de novo pathway for the biosynthesis of deoxyribonucleotides, the immediate precursors of DNA synthesis and repair. RNRs catalyze the controlled reduction of all four ribonucleotides to maintain a balanced pool of deoxyribonucleotides during the cell cycle (29). Three main classes of RNRs are known. Class I RNRs are oxygen-dependent enzymes, class II RNRs are oxygen-independent enzymes, and class III RNRs are oxygen-sensitive enzymes. Class I RNRs are divided into two subclasses, subclasses Ia and Ib.Staphylococcus aureus is a Gram-positive facultative aerobe and a major human pathogen (24). S. aureus contains class Ib and class III RNRs, which are essential for aerobic and anaerobic growth, respectively (26). The class Ib NrdEF RNR is encoded by the nrdE and nrdF genes: NrdE contains the substrate binding and allosteric binding sites, and NrdF contains the catalytic site for ribonucleotide reduction. The S. aureus nrdEF genes form an operon containing a third gene, nrdI, the product of which, NrdI, is a flavodoxin (5, 33). Many other bacteria such as Escherichia coli (16), Lactobacillus lactis (17), and Mycobacterium and Corynebacterium spp. possess class Ib RNR operons that contain a fourth gene, nrdH (30, 44, 50), whose product, NrdH, is a thiol-disulfide redoxin (16, 17, 40, 43, 49). More-complex situations are found for some bacteria, where the class Ib RNR operon may be duplicated and one or more of the nrdI and nrdH genes may be missing or located in another part of the chromosome (29).NrdH proteins are glutaredoxin-like protein disulfide oxidoreductases that alter the redox state of target proteins via the reversible oxidation of their active-site dithiol proteins. NrdH proteins function with high specificity as electron donors for class I RNRs (9, 16-18). They are widespread in bacteria, particularly in those bacteria that lack glutathione (GSH), where they function as a hydrogen donor for the class Ib RNR (12, 16, 17). In E. coli, which possesses class Ia and class Ib RNRs, NrdH functions in vivo as the primary electron donor for the class Ib RNR, whereas thioredoxin or glutaredoxin is used by the class Ia NrdAB RNR (12, 17). NrdH redoxins contain a conserved CXXC motif, have a low redox potential, and can reduce insulin disulfides. NrdH proteins possess an amino acid sequence similar to that of glutaredoxins but behave functionally more like thioredoxins. NrdH proteins are reduced by thioredoxin reductase but not by GSH and lack those residues in glutaredoxin that bind GSH and the GSH binding cleft (39, 40). The structures of the E. coli and Corynebacterium ammoniagenes NrdH redoxins reveal the presence of a wide hydrophobic pocket at the surface, like that in thioredoxin, that is presumed to be involved in binding to thioredoxin reductase (39, 40). NrdI proteins are flavodoxin proteins that function as electron donors for class Ib RNRs and are involved in the maintenance of the NrdF diferric tyrosyl radical (5, 33). In Streptococcus pyogenes, NrdI is essential for the activity of the NrdHEF system in a heterologous E. coli in vivo complementation assay (33). Class Ib RNRs are proposed to depend on two specific electron donors, NrdH, which provides reducing power to the NrdE subunit, and NrdI, which supplies electrons to the NrdF subunit (33).In this report we identify an open reading frame (ORF) in S. aureus encoding an NrdH-like protein with partial sequence relatedness to the E. coli, Salmonella enterica serovar Typhimurium, L. lactis, and C. ammoniagenes NrdH proteins. In contrast to these bacteria, the S. aureus nrdH gene does not form part of the class Ib RNR operon. The S. aureus NrdH protein differs in its structure from the canonical NrdH in its redox-active site, C-P-P-C instead of C-M/V-Q-C, and in the absence of the C-terminal [WF]SGFRP[DE] structural motif. We show that in vitro, S. aureus NrdH reduces protein disulfides and is an electron donor for the homologous class Ib NrdEF ribonucleotide reductase.     

PKCeta associates with cyclin E/Cdk2 complex in serum-starved MCF-7 and NIH-3T3 cells

Protein kinase C (PKC) encodes a family of enzymes implicated in cellular differentiation, growth control, and tumor promotion. However, very little is known with respect to the molecular mechanisms that link protein kinase C to cell cycle control. Here we report that PKCeta associates with the cyclin E/Cdk2 complex. This is shown for the ectopically overexpressed PKCeta in NIH-3T3 cells, the inducibly expressed PKCeta in MCF-7 cells (under control of the tetracycline-responsive promoter), and the endogenously expressed PKCeta in mouse mammary epithelial HC11 cells. Subcellular cell fractionation experiments revealed that the complex with cyclin E is formed mostly in the nuclear fractions, although in these cells PKCeta is predominantly expressed in the cytosolic fractions. The complex of PKCeta and cyclin E was studied at various phases of the cell cycle, in serum-starved quiescent cells and in cells stimulated with serum to reenter the cell cycle. Interestingly, the interaction between PKCeta and cyclin E was most prominent in serum-starved cells and was disintegrated when cells entered the cells cycle. Immunofluorescence staining demonstrated that in serum-starved cells PKCeta is concentrated at the perinuclear zone, which is also the site of its colocalization with cyclin E. Colocalization of PKCeta and cyclin E in the perinuclear region was observed in serum-starved cells, and less in proliferating cells. These experiments suggest that the interaction between PKCeta and cyclin E is carefully regulated, and is correlated with the inactivated form of the cyclin E/Cdk2 complex. Thus, our studies support an important link between PKC and cell cycle control.