Near-physiological in vitro assembly of 50S ribosomes involves parallel pathways.

Understanding the assembly principles of biological macromolecular complexes remains a significant challenge, due to the complexity of the systems and the difficulties in developing experimental approaches. As a ribonucleoprotein complex, the ribosome serves as a model system for the profiling of macromolecular complex assembly. In this work, we report an ensemble of large ribosomal subunit intermediate structures that accumulate during synthesis in a near-physiological and co-transcriptional in vitro reconstitution system. Thirteen pre-50S intermediate maps covering the entire assembly process were resolved using cryo-EM single-particle analysis and heterogeneous subclassification. Segmentation of the set of density maps reveals that the 50S ribosome intermediates assemble based on fourteen cooperative assembly blocks, including the smallest assembly core reported to date, which is composed of a 600-nucleotide-long folded rRNA and three ribosomal proteins. The cooperative blocks assemble onto the assembly core following defined dependencies, revealing the parallel pathways at both early and late assembly stages of the 50S subunit.

Functional and clinical studies reveal pathophysiological complexity of CLCN4-related neurodevelopmental condition.

Missense and truncating variants in the X-chromosome-linked CLCN4 gene, resulting in reduced or complete loss-of-function (LOF) of the encoded chloride/proton exchanger ClC-4, were recently demonstrated to cause a neurocognitive phenotype in both males and females. Through international clinical matchmaking and interrogation of public variant databases we assembled a database of 90 rare CLCN4 missense variants in 90 families: 41 unique and 18 recurrent variants in 49 families. For 43 families, including 22 males and 33 females, we collated detailed clinical and segregation data. To confirm causality of variants and to obtain insight into disease mechanisms, we investigated the effect on electrophysiological properties of 59 of the variants in Xenopus oocytes using extended voltage and pH ranges. Detailed analyses revealed new pathophysiological mechanisms: 25% (15/59) of variants demonstrated LOF, characterized by a "shift" of the voltage-dependent activation to more positive voltages, and nine variants resulted in a toxic gain-of-function, associated with a disrupted gate allowing inward transport at negative voltages. Functional results were not always in line with in silico pathogenicity scores, highlighting the complexity of pathogenicity assessment for accurate genetic counselling. The complex neurocognitive and psychiatric manifestations of this condition, and hitherto under-recognized impacts on growth, gastrointestinal function, and motor control are discussed. Including published cases, we summarize features in 122 individuals from 67 families with CLCN4-related neurodevelopmental condition and suggest future research directions with the aim of improving the integrated care for individuals with this diagnosis.

Correction to: Iron-free and iron-saturated bovine lactoferrin inhibit survivin expression and differentially modulate apoptosis in breast cancer.

After publication of the original article [1], the authors found that Fig. 3 contained an incorrect version of Fig. 3c. This does not affect the Figure legend, results and conclusions of the article.

CIUSuite: A Quantitative Analysis Package for Collision Induced Unfolding Measurements of Gas-Phase Protein Ions.

Ion mobility-mass spectrometry (IM-MS) is a technology of growing importance for structural biology, providing complementary 3D structure information for biomolecules within samples that are difficult to analyze using conventional analytical tools through the near-simultaneous acquisition of ion collision cross sections (CCSs) and masses. Despite recent advances in IM-MS instrumentation, the resolution of closely related protein conformations remains challenging. Collision induced unfolding (CIU) has been demonstrated as a useful tool for resolving isocrossectional protein ions, as they often follow distinct unfolding pathways when subjected to collisional heating in the gas phase. CIU has been used for a variety of applications, from differentiating binding modes of activation state-selective kinase inhibitors to characterizing the domain structure of multidomain proteins. With the growing utilization of CIU as a tool for structural biology, significant challenges have emerged in data analysis and interpretation, specifically the normalization and comparison of CIU data sets. Here, we present CIUSuite, a suite of software modules designed for the rapid processing, analysis, comparison, and classification of CIU data. We demonstrate these tools as part of a series of workflows for applications in comparative structural biology, biotherapeutic analysis, and high throughput screening of kinase inhibitors. These examples illustrate both the potential for CIU in general protein analysis as well as a demonstration of best practices in the interpretation of CIU data.

Iron-free and iron-saturated bovine lactoferrin inhibit survivin expression and differentially modulate apoptosis in breast cancer.

BACKGROUND: Iron binding, naturally occurring protein bovine lactoferrin (bLf) has attracted attention as a safe anti-cancer agent capable of inducing apoptosis. Naturally, bLf exists partially saturated (15-20%) with Fe(3+) however, it has been demonstrated that manipulating the saturation state can enhance bLf's anti-cancer activities. METHODS: Apo-bLf (Fe(3+) free) and Fe-bLf (>90% Fe(3+) Saturated) were therefore, tested in MDA-MB-231 and MCF-7 human breast cancer cells in terms of cytotoxicity, proliferation, migration and invasion. Annexin-V Fluos staining was also employed in addition to apoptotic protein arrays and Western blotting to determine the specific mechanism of bLf-induced apoptosis with a key focus on p53 and inhibitor of apoptosis proteins (IAP), specifically survivin. RESULTS: Apo-bLf induced significantly greater cytotoxicity and reduction in cell proliferation in both cancer cells showing a time and dose dependent effect. Importantly, no cytotoxicity was detected in normal MCF-10-2A cells. Both forms of bLf significantly reduced cell invasion in cancer cells. Key apoptotic molecules including p53, Bcl-2 family proteins, IAP members and their inhibitors were significantly modulated by both forms of bLf, though differentially in each cell line. Most interestingly, both Apo-bLf and Fe-bLf completely inhibited the expression of survivin protein (key IAP), after 48 h at 30 and 40 nM in cancer cells. CONCLUSIONS: The capacity of these forms of bLf to target survivin expression and modulation of apoptosis demonstrates an exciting potential for bLf as an anti-cancer therapeutic in the existing void of survivin inhibitors, with a lack of successful inhibitors in the clinical management of cancer.

Making creativity explicit: A workshop to foster creativity in biomedical science education.

Previously we identified that biomedical science students commonly misunderstand "creativity," mistaking it for "freedom." In the present study, we describe and evaluate a workshop designed to increase students' awareness of creativity as a highly sought-after employability skill and cognitive process applicable to scientific endeavors. To achieve this, we developed and introduced students to a process called the "Diamond Model," utilizing a case study to contextualize and signpost the creative processes of divergent and convergent thinking. This model was introduced to students in the first workshop of a 12-week undergraduate biochemistry unit (subject) within the Bachelor of Biomedical Science at Monash University, Australia. Students completed pre- and post-workshop surveys to gauge the impact of the workshop on their conceptions of creativity and Bloom's taxonomy of learning. In addition, reflective journals were completed by a small subset of students (n = 9) following the workshop. Following the workshop, over 65% of students indicated that their conception of creativity had changed. Thematic analysis of students' survey responses and reflections indicated that this change in the conception of creativity included broadening their definition of creativity, increased awareness of creativity as a skill and science as a creative process, and that creativity can be applied to different areas of life. Students attributed the signposting of creative elements as a contributing factor to their increased awareness. These results indicate the positive impact the workshop and our novel Diamond model had on student conception of creativity, highlighting the importance of explicit communication and signposting in skill development.

Assembly landscape for the bacterial large ribosomal subunit.

Assembly of ribosomes in bacteria is highly efficient, taking ~2-3 min, but this makes the abundance of assembly intermediates very low, which is a challenge for mechanistic understanding. Genetic perturbations of the assembly process create bottlenecks where intermediates accumulate, facilitating structural characterization. We use cryo-electron microscopy, with iterative subclassification to identify intermediates in the assembly of the 50S ribosomal subunit from E. coli. The analysis of the ensemble of intermediates that spans the entire biogenesis pathway for the 50 S subunit was facilitated by a dimensionality reduction and cluster picking approach using PCA-UMAP-HDBSCAN. The identity of the cooperative folding units in the RNA with associated proteins is revealed, and the hierarchy of these units reveals a complete assembly map for all RNA and protein components. The assembly generally proceeds co-transcriptionally, with some flexibility in the landscape to ensure efficiency for this central cellular process under a variety of growth conditions.

Cell-penetrating properties of the transactivator of transcription and polyarginine (R9) peptides, their conjugative effect on nanoparticles and the prospect of conjugation with arsenic trioxide.

Cell-penetrating peptides (CPPs) are short chains of amino acids with the distinct ability to cross cell plasma membranes. They are usually between seven and 30 residues in length. The mechanism of action is still a highly debated subject among researchers; it seems that a commonality between all CPPs is the presence of positively charged residues within the amino acid chain. Polyarginine and the transactivator of transcription peptide are two widely used CPPs. One distinct application of these CPPs is the ability to further enhance the therapeutic properties of a range of different agents. One group of agents of particular importance are nanoparticles (NPs). Most NPs have no mechanism for cellular uptake. Hence, by conjugating CPPs to NPs, the amount of NPs taken up by cells can be increased, and therefore, the therapeutic benefits can be maximized. Some examples of this will be explored further in this review. In addition to CPPs, the concept of conjugation with the anticancer drug arsenic trioxide is reviewed and the prospect of transactivator of transcription-conjugated arsenic trioxide albumin microspheres is also discussed. Recent locked nucleic acid technology to stabilize nucleotides (RNA or DNA) aptamer complexes able to target cancer cells more specifically and selectively to kill tumour cells and spare normal body cells. NPs tagged with modified locked nucleic acid-aptamers have the potential to kill cancer cells more specifically and effectively while sparing normal cells.

Quantitative mining of compositional heterogeneity in cryo-EM datasets of ribosome assembly intermediates.

Single-particle cryoelectron microscopy (cryo-EM) offers a unique opportunity to characterize macromolecular structural heterogeneity by virtue of its ability to place distinct particle populations into different groups through computational classification. However, there is a dearth of tools for surveying the heterogeneity landscape, quantitatively analyzing heterogeneous particle populations after classification, deciding how many unique classes are represented by the data, and accurately cross-comparing reconstructions. Here, we develop a workflow that contains discovery and analysis modules to quantitatively mine cryo-EM data for sets of structures with maximal diversity. This workflow was applied to a dataset of E. coli 50S ribosome assembly intermediates, which are characterized by significant structural heterogeneity. We identified more detailed branchpoints in the assembly process and characterized the interactions of an assembly factor with immature intermediates. While the tools described here were developed for ribosome assembly, they should be broadly applicable to the analysis of other heterogeneous cryo-EM datasets.

Discrimination between Functional and Non-functional Cellular Gag Complexes involved in HIV-1 Assembly.

HIV-1 Gag and Gag-Pol are responsible for viral assembly and maturation and represent a major paradigm for enveloped virus assembly. Numerous intracellular Gag-containing complexes (GCCs) have been identified in cellular lysates using sucrose gradient ultracentrifugation. While these complexes are universally present in Gag-expressing cells, their roles in virus assembly are not well understood. Here we demonstrate that most GCC species are predominantly comprised of monomeric or dimeric Gag molecules bound to ribosomal complexes, and as such, are not on-pathway intermediates in HIV assembly. Rather, these GCCs represent a population of Gag that is not yet functionally committed for incorporation into a viable virion precursor. We hypothesize that these complexes act as a reservoir of monomeric Gag that can incorporate into assembling viruses, and serve to mitigate non-specific intracellular Gag oligomerization. We have identified a subset of large GCC complexes, comprising more than 20 Gag molecules, that may be equivalent to membrane-associated puncta previously shown to be bona fide assembling-virus intermediates. This work provides a clear rationale for the existence of diverse GCCs, and serves as the foundation for characterizing on-pathway intermediates early in virus assembly.

Laboratory classes in biochemistry and molecular biology: A parallel session at the IUBMB/PSBMB 2019 "Harnessing Interdisciplinary Education in Biochemistry and Molecular Biology" conference.

Laboratory classes are a central element of all biochemistry and molecular biology programs. These play a role in developing students' hands-on and technical skills and also offer much more. The design of laboratory classes depends on many factors including the programs the students are enrolled in, the level they are at, employment destinations, and learning outcomes. This conference session considered the design and outcomes of laboratory experiences for undergraduate students.

SrmB Rescues Trapped Ribosome Assembly Intermediates.

RNA helicases play various roles in ribosome biogenesis depending on the ribosome assembly pathway and stress state of the cell. However, it is unclear how most RNA helicases interact with ribosome assembly intermediates or participate in other cell processes to regulate ribosome assembly. SrmB is a DEAD-box helicase that acts early in the ribosome assembly process, although very little is known about its mechanism of action. Here, we use a combined quantitative mass spectrometry/cryo-electron microscopy approach to detail the protein inventory, rRNA modification state, and structures of 40S ribosomal intermediates that form upon SrmB deletion. We show that the binding site of SrmB is unperturbed by SrmB deletion, but the peptidyl transferase center, the uL7/12 stalk, and 30S contact sites all show severe assembly defects. Taking into account existing data on SrmB function and the experiments presented here, we propose several mechanisms by which SrmB could guide assembling particles from kinetic traps to competent subunits during the 50S ribosome assembly process.

Processing Embryo, Eggshell, and Fungal Culture for Scanning Electron Microscopy.

Although scanning electron microscopy (SEM) is being widely used for the ultra-structural analysis of various biological and non-biological samples, methods involved in processing different biological samples involve unique practices. All conventional practices described in the literature for processing samples still find useful applications, but subtle changes in the sample preparation can alter image quality, as well as, introduce artifacts. Hence, using a unique sample preparation technique specific to the type of tissue analyzed is required to obtain a good quality image with ultrastructural resolution. The focus of this study is to provide the optimal sample preparation protocols for imaging embryos, rigid eggshells, and fungal cultures using SEM. The following optimizations were recommended to yield good results for the three different delicate biological samples studied. Use of milder fixatives like 4% paraformaldehyde or 3% glutaraldehyde followed by dehydration with ethanol series is mandatory. Fungal mycelium on agar blocks obtained by slide cultures yields a better ultrastructural integrity compared to cultures taken directly from agar plates. Chemical drying of embryos with HMDS provides drying without introducing surface tension artifacts compared to critical point drying. HMDS prevents cracking caused by shrinkage as samples are less brittle during drying. However, for fungal culture, critical point drying provides acceptable image quality compared to chemical drying. Eggshells can be imaged with no special preparation steps except for thorough washing and air drying prior to mounting. Preparation methodologies were standardized based on acceptable image quality obtained with each trial.

Collision-Induced Unfolding Reveals Unique Fingerprints for Remote Protein Interaction Sites in the KIX Regulation Domain.

The kinase-inducible domain (KIX) of the transcriptional coactivator CBP binds multiple transcriptional regulators through two allosterically connected sites. Establishing a method for observing activator-specific KIX conformations would facilitate the discovery of drug-like molecules that capture specific conformations and further elucidate how distinct activator-KIX complexes produce differential transcriptional effects. However, the transient and low to moderate affinity interactions between activators and KIX are difficult to capture using traditional biophysical assays. Here, we describe a collision-induced unfolding-based approach that produces unique fingerprints for peptides bound to each of the two available sites within KIX, as well as a third fingerprint for ternary KIX complexes. Furthermore, we evaluate the analytical utility of unfolding fingerprints for KIX complexes using CIUSuite, and conclude by speculating as to the structural origins of the conformational families created from KIX:peptide complexes following collisional activation. Graphical Abstract ᅟ.

Sizing Up Protein-Ligand Complexes: The Rise of Structural Mass Spectrometry Approaches in the Pharmaceutical Sciences.

Capturing the dynamic interplay between proteins and their myriad interaction partners is critically important for advancing our understanding of almost every biochemical process and human disease. The importance of this general area has spawned many measurement methods capable of assaying such protein complexes, and the mass spectrometry-based structural biology methods described in this review form an important part of that analytical arsenal. Here, we survey the basic principles of such measurements, cover recent applications of the technology that have focused on protein-small-molecule complexes, and discuss the bright future awaiting this group of technologies.

Nanotechnology based platforms for survivin targeted drug discovery.

INTRODUCTION: Development of an effective, safe and targeted drug delivery system to fight cancer and other diseases is a prime focus in the area of drug discovery. The emerging field of nanotechnology has revolutionised the way cancer therapy and diagnosis is achieved primarily due to the recent advances in material engineering and drug availability. Further, the recognition of the crucial role played by anti-apoptotic proteins such as survivin, has initiated the development of therapeutics that can target this protein as an attempt to develop alternative cancer therapies. However, a key challenge faced in drug development is the efficient delivery of survivin-targeted molecules to specific areas in the body. AREAS COVERED: This review primarily focuses on the different strategies employing nanotechnology for targeting survivin expressed in human cancers. Different nanomaterials incorporating nucleic molecules or drugs targeted at survivin are discussed and the results obtained from studies are highlighted. EXPERT OPINION: There are extensive studies reporting different treatment regimens for cancer, however, they still result in systemic toxicity, reduced bioavailability and ineffective delivery. Novel approaches involve the use of biocompatible nanomaterials together with gene or drug molecules to target proteins such as survivin, which is overexpressed in cancerous cells. These nanoformulations allow the benefits of protecting easily degradable molecules, allow controlled release, and enhance targeted delivery and effectiveness. Hence, nanotherapy utilizing survivin targeting can be considered to play a key role in the development of personalized nanomedicine for cancer.

Lactoferrin and cancer in different cancer models.

Lactoferrin (Lf) is a multifunctional protein and an essential element of innate immunity. Cancer is a major killer in today's world accounting for around 13% of all deaths according to the World Health Organisation (W.H.O.). The five most common forms of cancer include lung, colorectal, stomach, liver and breast cancer. Lactoferrin is a natural forming iron-binding glycoprotein with antibacterial, antioxidant and anti-carcinogenic effects. It is produced in exocrine glands and is secreted in many external fluids as a first line of defence. Lactoferrin also has the capacity to induce apoptosis and inhibit proliferation in cancer cells as well as restore white and red blood cell levels after chemotherapy. This review focuses on the therapeutic effect bovine sourced lactoferrin has on various forms of cancer in various models. It also focuses on the benefits of 3D in vitro cell culture. 3D cell culture has vast advantages over 2D models including demonstration of realistic therapeutic results and heightened resistance that 2D models fail to display.