INAUGURAL ARTICLE by a Recently Elected Academy Member:Structural insights into the human PA28–20S proteasome enabled by efficient tagging and purification of endogenous proteins

The ability to produce folded and functional proteins is a necessity for structural biology and many other biological sciences. This task is particularly challenging for numerous biomedically important targets in human cells, including membrane proteins and large macromolecular assemblies, hampering mechanistic studies and drug development efforts. Here we describe a method combining CRISPR-Cas gene editing and fluorescence-activated cell sorting to rapidly tag and purify endogenous proteins in HEK cells for structural characterization. We applied this approach to study the human proteasome from HEK cells and rapidly determined cryogenic electron microscopy structures of major proteasomal complexes, including a high-resolution structure of intact human PA28αβ–20S. Our structures reveal that PA28 with a subunit stoichiometry of 3α/4β engages tightly with the 20S proteasome. Addition of a hydrophilic peptide shows that polypeptides entering through PA28 are held in the antechamber of 20S prior to degradation in the proteolytic chamber. This study provides critical insights into an important proteasome complex and demonstrates key methodologies for the tagging of proteins from endogenous sources.

Recent technological breakthroughs in single-particle cryogenic electron microscopy (cryo-EM) have greatly accelerated the pace of high-resolution structure determination of biological macromolecules. However, a major bottleneck in the structural study of important targets including membrane proteins, protein–DNA complexes, and large protein assemblies is sample production. The challenge of producing functional proteins extends beyond structural biology and impacts many areas of biological sciences that currently rely on techniques of protein overexpression. Conventional methods of protein production involve overexpression of a target gene from a plasmid within heterologous systems, including bacterial (Escherichia coli), insect (Sf9), and mammalian (HEK) cells. Overexpression approaches frequently encounter issues with proteins that are misfolded, nonfunctional, or degraded by the host cells. Overexpression of certain proteins may also alter protein homeostasis within cells. Substantial optimization is often necessary to identify the right conditions to produce suitable proteins for study, which is time-consuming and challenging in many cases. The problem is compounded further by multiprotein complexes where multiple subunits need to be assembled in the correct order and stoichiometries, making the coexpression of multiple subunits and purification of protein assemblies even more challenging.Studies of challenging proteins and large protein complexes have sometimes been possible by extracting samples from natural sources. While this approach has proven successful in special cases, examples of native proteins purified in this way have been largely limited to highly abundant and large protein assemblies that can be isolated by sucrose gradient ultracentrifugation (1) or where a known binding partner can be used as bait (2, 3). A general and efficient method to isolate proteins from endogenous sources with high specificity and at sufficient quantities for structural studies remains a goal of substantial interest. A rapid and efficient approach to add an affinity tag onto a target protein in human cells for affinity purification of endogenous complexes would greatly accelerate structural characterization of challenging and biomedically important targets. Furthermore, this strategy could be combined with complementary approaches such as mass spectrometry to better understand protein function under more native-like conditions.Recent technological advances in CRISPR-Cas–mediated genome engineering have provided new avenues to target endogenous proteins from native sources by genetically incorporating an affinity tag onto a protein of interest for purification and downstream analysis (4, 5). However, the time-consuming process of generating and selecting gene-edited cells with an endogenously tagged protein has created a substantial barrier for this approach to be widely adopted by the structural biology community and other fields of protein science. A simple, fast, and minimally perturbing way to target and extract endogenous proteins would make this approach more widely accessible to the general scientific community. Here we describe a simple and highly efficient method that leverages the unique properties of split fluorescent proteins (6) to select cells harboring an affinity tag on target endogenous proteins for purification.We demonstrate this approach on the study of the human proteasome. The proteasome plays an essential role in protein degradation and is critical to maintaining cellular protein homeostasis (7). Changes to proteostasis in cells and lowered protein degradation due to decreases in proteasomal activity have been linked to aging and neurodegeneration (8). While the proteasome has been a topic of extensive studies using a variety of strategies (9–12), the study of some human proteasomal complexes has remained challenging. In this study, by tagging, purifying, and determining structures of various human proteasomal complexes from HEK cells we demonstrate that the efficient protein tagging method presented here can facilitate rapid structural studies of endogenous human protein complexes in HEK cells. Analyzing cryo-EM structures of PA28–20S complexes reveals insights into PA28 stoichiometry and sheds light on how unfolded polypeptide substrates traverse through the antechamber of the 20S core particle before entering the proteolytic chamber for degradation.     

Sociobiologic factors influencing low birth weight at a rural project hospital

The incidence of low birth weight (less than or equal to 2500 g) was 38.9% among 2292 singleton live births at a rural project hospital in Haryana. Only 7.0% of the newborns weighed 2000 g or less. Female babies had a higher incidence of low birth weight. Other factors considered were maternal age, parity and literacy and father's literacy. Young mothers (less than 20 years) had a higher incidence of low birth weight. Similarly parents who were illiterate or educated to below the primary grade also had a higher incidence of low birth weight. The maximum percentage of low birth weight was seen in the primiparous mothers. An increase of low birth weight was also seen after the 4th parity. The best outcome was at para 4.     

Comparison of Nanogel Drug Carriers and their Formulations with Nucleoside 5′-Triphosphates

Purpose The aim of the study is to synthesize and characterize nanogel carriers composed of amphiphilic polymers and cationic polyethylenimine for encapsulation and delivery of cytotoxic nucleoside analogs 5′-triphosphates (NTPs) into cancer cells. Methods Nanogels were synthesized by a novel micellar approach and compared with carriers prepared by the emulsification/evaporation method. Complexes of nanogels with NTP were prepared; particle size and in vitro drug release were characterized. Resistance of the nanogel-encapsulated NTP to enzymatic hydrolysis was analyzed by ion-pair high-performance liquid chromatography. Binding to isolated cellular membranes, cellular accumulation and cytotoxicity were compared using breast carcinoma cell lines CL-66, MCF-7, and MDA-MB-231. In vivo biodistribution of the ³H-labeled NTP encapsulated in different types of nanogels was evaluated in comparison to the injected NTP alone. Results Nanogels with a particle size of 100–300 nm in the unloaded form and less than 140 nm in the NTP-loaded form were prepared. An in vitro release of NTP was >50% during the first 24 h. Nanogel formulations ensured increased NTP drug stability against enzymatic hydrolysis as compared to the drug alone. Pluronic?-based nanogels NG(F68), NG(F127), NG(P85), and NGM(P123) demonstrated 2–2.5 times enhanced interaction with cellular membranes and association with various cancer cells compared to NG(PEG). Among them, NG(F68) and NG(F127) exhibited the lowest cytotoxicity. Injection of nanogel-formulated NTP significantly modulated the drug accumulation in different mouse organs. Conclusions Nanogels composed of Pluronic? F68 and P123 were shown to display certain advanced properties compared to NG(PEG) as a drug delivery system for NTP analogs. Formulations of nucleoside analogs in active NTP form with these nanogels will improve the delivery of these cytotoxic drugs to cancer cells and the therapeutic potential of this anticancer chemotherapy. Electronic supplementary material Supplementary material is available in the online version of this article at .     

Recognition of and response to postpartum hemorrhage in rural northern India

This study describes the results of a Morbidity and Performance Assessment (MAP) conducted to provide insight into the medical factors contributing to maternal and newborn morbidity and mortality in a rural district of northern India, and to use these insights to develop a locally appropriate, community-based safe motherhood program The MAP study was based on verbal autopsy method. Five hundred ninety-nine women (or in the case of 9 maternal deaths, a family member) participated in the study. This article describes a subsample of women who reported signs or symptoms suggesting excessive bleeding (n = 159). Findings include a poor knowledge of danger signs; poor problem recognition during labor, birth, and the immediate postpartum period; and a low level of health seeking that was consistent with poor recognition. Maternal sociodemographic characteristics, antenatal care use, and knowledge of danger signs were generally not associated with problem recognition and health seeking. The case fatality rate was 4%. These findings suggest an urgent need to understand the phenomenon of problem recognition and to integrate this into the design of interventions to reduce delays in health seeking.     

Transverse facial cleft: A series of 17 cases

Introduction:

Transverse facial cleft (Tessier type 7) or congenital macrostomia is a rare congenital anomaly seldom occurring alone and is frequently associated with deformities of the structures developing from the first and second branchial arches. The reported incidence of No. 7 cleft varies from 1 in 60,000 to 1 in 300,000 live births.

Material and Methods:

Seventeen patients of transeverse facial cleft who presented to us in last 5 years were included in the study. Their history regarding familial and environmental predispositions was recorded. The cases were analysed on basis of sex, laterality, severity, associated anomalies and were graded according to severity. They were operated by z plasty technique and were followed up for 2 years to look for effectiveness of the technique and its complications.

Result:

Out of the seventeen patients of transverse cleft, none had familial predilection or any environmental etiology like antenatal radiological exposure or intake of drugs of teratogenic potential. Most of the patients (9/17) were associated with hemifacial microsomia and 1 patient was associated with Treacher Colin''s Syndrome. Out of the 6 cases of Grade I clefts, 4 were isolated transverse clefts and of the 10 patients of Grade II clefts, 7 were associated with hemifacial microsomia. We encountered only one case of Grade III Transverse Cleft which was not only associated with hemifacial microsomia but also had cardiac anomaly. Out of the 17 cases, 15 were operated and in most of them the outcome was satisfactory.KEY WORDS: Macrostomia, No. 7 cleft, transverse facial cleft     

Molecular dynamics calculations suggest a conduction mechanism for the M2 proton channel from influenza A virus

The M2 protein of the influenza A virus is activated by low endosomal pH and performs the essential function of proton transfer into the viral interior. The resulting decrease in pH within the virion is essential for the uncoating and further replication of the viral genetic material. The x-ray crystal [Stouffer AL, et al. (2008) Nature 451:596–599] and solution NMR [Schnell JR, Chou JJ (2008) Nature 451:591–595] structures of the transmembrane region of the M2 homo-tetrameric bundle both revealed pores with narrow constrictions at one end, leaving a question as to how protons enter the channel. His-37, which is essential for proton-gating and selective conduction of protons, lies in the pore of the crystallographic and NMR structures. Here, we explore the different protonation states of the His-37 residues of the M2 bundle in a bilayer using molecular dynamics (MD) simulations. When the His-37 residues are neutral, the protein prefers an Open_out-Closed_in conformation in which the channel is open to the environment on the outside of the virus but closed to the interior environment of the virus. Diffusion of protons into the channel from the outside of the virus and protonation of His-37 residues in the tetramer stabilizes an oppositely gated Closed_out-Open_in conformation. Thus, protons might be conducted through a transporter-like mechanism, in which the protein alternates between Open_out-Closed_in and Closed_out-Open_in conformations, and His-37 is protonated/deprotonated during each turnover. The transporter-like mechanism is consistent with the known properties of the M2 bundle, including its relatively low rate of proton flux and its strong rectifying behavior.