A rapid approach for characterization of thiol-conjugated antibody–drug conjugates and calculation of drug–antibody ratio by liquid chromatography mass spectrometry

We present the demonstration of a rapid “middle-up” liquid chromatography mass spectrometry (LC–MS)-based workflow for use in the characterization of thiol-conjugated maleimidocaproyl-monomethyl auristatin F (mcMMAF) and valine-citrulline-monomethyl auristatin E (vcMMAE) antibody–drug conjugates. Deconvoluted spectra were generated following a combination of deglycosylation, IdeS (immunoglobulin-degrading enzyme from Streptococcus pyogenes) digestion, and reduction steps that provide a visual representation of the product for rapid lot-to-lot comparison—a means to quickly assess the integrity of the antibody structure and the applied conjugation chemistry by mass. The relative abundance of the detected ions also offer information regarding differences in drug conjugation levels between samples, and the average drug–antibody ratio can be calculated. The approach requires little material (<100 μg) and, thus, is amenable to small-scale process development testing or as an early component of a complete characterization project facilitating informed decision making regarding which aspects of a molecule might need to be examined in more detail by orthogonal methodologies.     

Protein signatures—A novel way to classify proteins

A method is developed to classify proteins by establishing their familial identity based on the presence and the absence of characteristic signature dipeptides. It is hoped that this method will lead to the establishment of a theoretical basis for the determination of interrelationships among proteins.     

QuantiSpec — Quantitative mass spectrometry data analysis of 15N-metabolically labeled proteins

For relative protein quantitation by mass spectrometry we metabolically labeled E. coli bacteria with ¹⁵N-enriched diets. Proteins extracted from ¹⁵N-labeled and unlabeled E. coli bacteria were mixed, separated by two-dimensional gel electrophoresis and enzymatically digested. The resulting tryptic peptides were analyzed by MALDI mass spectrometry. For the relative protein quantitation we developed fully automated software, QuantiSpec (Quantitative Mass Spectrometry Analysis Software), which uses data from MALDI TOF mass spectrometry and the Mascot database search engine. QuantiSpec detects natural as well as partially or fully labeled peptide isotope distributions. For each identified peptide the ¹⁵N incorporation rate is determined by comparing the experimental to a set of theoretical isotope patterns based on the peptide sequence. Relative quantitation is accomplished by calculating the signal intensity ratios for each ¹⁴N/¹⁵N peptide pair.     

A novel approach to microbial breeding—low-energy ion implantation

Low-energy ions exist widely in the natural world. People had neglected the interaction between low-energy ions and material; it was even more out of the question to study the relation of low-energy ions and the complicated organism until the biological effects of low-energy ion implantation were discovered in 1989. Nowadays, the value of low-energy ion beam implantation, as a new breeding way, has drawn extensive attention of biologists and breeding experts. In this review, the understanding and utilization of microbial breeding by low-energy ion beam irradiation is summarized, including the characteristics of an ion beam bioengineering facility, present status of the technology of low-energy ions for microbial breeding, and new insights into microbial biotechnology.     

Understanding the mechanism of copurification of “difficult to remove” host cell proteins in rituximab biosimilar products

Host cell proteins (HCPs) are considered a critical quality attribute and are linked to safety and efficacy of biotherapeutic products. Researchers have identified 10 HCPs in Chinese hamster ovary (CHO) that exhibit common characteristics of product association, coelution, and age-dependent expression and therefore are “difficult to remove” during downstream purification. These include cathepsin D, clusterin, galectin-3-binding protein, G-protein coupled receptor 56, lipoprotein lipase, metalloproteinase inhibitor, nidogen-1 secreted protein acidic and rich in cysteine (SPARC), sulfated glycoprotein, and insulin-like growth factor-2 RNA-binding protein. While the levels of HCPs in the investigated biosimilars were within the acceptable range of <100 ppm, certain “difficult to remove” HCPs were found in the biosimilar samples. This article aims to elucidate the underlying interactions between these “difficult to remove” HCPs and the mAb product. Surface study of rituximab exhibited unstable discontinuous patches of residues on the protein surface that have high propensity to get buried and lower the solvent exposed area. The higher order structure and the receptor binding were not affected, except for one of the biosimilars, owing to extremely low-HCP levels in its final drug product. Finally, based on the combined experimental and computational data from this study, a probable mechanism of retention for the 10 HCPs is proposed. The results presented here can guide downstream process design or avenues for protein engineering during product discovery to achieve more effective removal of the impurities.     

GDP-Capture Compound — A novel tool for the profiling of GTPases in pro- and eukaryotes by capture compound mass spectrometry (CCMS)

The functional isolation of proteome subsets based on small molecule–protein interactions is an increasingly popular and promising field in functional proteomics. Entire protein families may be profiled on the basis of their common interaction with a metabolite or small molecule inhibitor. This is enabled by novel multifunctional small molecule probes. One platform approach in this field are Capture Compounds that contain a small molecule of interest to bind target proteins, a photo-activatable reactivity function to covalently trap bound proteins, and a sorting function to isolate Capture Compound–protein conjugates from complex biological samples for direct trypsinisation and protein identification by liquid chromatography/mass spectrometry (CCMS). We here present the synthesis and application of a novel GDP-Capture Compound for the functional enrichment of GTPases, a pivotal protein family that exerts key functions in signal transduction. We present data from CCMS experiments on two biological lysates from Escherichia coli and from human-derived Hek293 cells. The GDP-Capture Compound robustly captures a wide range of different GTPases from both systems and will be a valuable tool for the proteomic profiling of this important protein family.     

An integrative approach combining ion mobility mass spectrometry,X-ray crystallography,and nuclear magnetic resonance spectroscopy to study the conformational dynamics of α1-antitrypsin upon ligand binding

Native mass spectrometry (MS) methods permit the study of multiple protein species within solution equilibria, whereas ion mobility (IM)-MS can report on conformational behavior of specific states. We used IM-MS to study a conformationally labile protein (α₁-antitrypsin) that undergoes pathological polymerization in the context of point mutations. The folded, native state of the Z-variant remains highly polymerogenic in physiological conditions despite only minor thermodynamic destabilization relative to the wild-type variant. Various data implicate kinetic instability (conformational lability within a native state ensemble) as the basis of Z α₁-antitrypsin polymerogenicity. We show the ability of IM-MS to track such disease-relevant conformational behavior in detail by studying the effects of peptide binding on α₁-antitrypsin conformation and dynamics. IM-MS is, therefore, an ideal platform for the screening of compounds that result in therapeutically beneficial kinetic stabilization of native α₁-antitrypsin. Our findings are confirmed with high-resolution X-ray crystallographic and nuclear magnetic resonance spectroscopic studies of the same event, which together dissect structural changes from dynamic effects caused by peptide binding at a residue-specific level. IM-MS methods, therefore, have great potential for further study of biologically relevant thermodynamic and kinetic instability of proteins and provide rapid and multidimensional characterization of ligand interactions of therapeutic interest.PDB Code(s): 4PYW     

A novel and high-throughput approach to assess photosynthetic thermal tolerance of kelp using chlorophyll α fluorometry

Foundation seaweed species are experiencing widespread declines and localized extinctions due to increased instability of sea surface temperature. Characterizing temperature thresholds are useful for predicting patterns of change and identifying species most vulnerable to extremes. Existing methods for characterizing seaweed thermal tolerance produce diverse metrics and are often time-consuming, making comparisons between species and techniques difficult, hindering insight into global patterns of change. Using three kelp species, we adapted a high-throughput method – previously used in terrestrial plant thermal biology – for use on kelps. This method employs temperature-dependent fluorescence (T–F₀) curves under heating or cooling regimes to determine the critical temperature (T_crit) of photosystem II (PSII), i.e., the breakpoint between slow and fast rise fluorescence response to changing temperature, enabling rapid assays of photosynthetic thermal tolerance using a standardized metric. This method enables characterization of T_crit for up to 48 samples per two-hour assay, demonstrating the capacity of T–F₀ curves for high-throughput assays of thermal tolerance. Temperature-dependent fluorescence curves and their derived metric, T_crit, may offer a timely and powerful new method for the field of phycology, enabling characterization and comparison of photosynthetic thermal tolerance of seaweeds across many populations, species, and biomes.     

The UPEC pore-forming toxin α-hemolysin triggers proteolysis of host proteins to disrupt cell adhesion, inflammatory, and survival pathways

Uropathogenic Escherichia coli (UPEC), which are the leading cause of both acute and chronic urinary tract infections, often secrete a labile pore-forming toxin known as α-hemolysin (HlyA). We show that stable insertion of HlyA into epithelial cell and macrophage membranes triggers degradation of the cytoskeletal scaffolding protein paxillin and other host regulatory proteins, as well as components of the proinflammatory NFκB signaling cascade. Proteolysis of these factors requires host serine proteases, and paxillin degradation specifically involves the serine protease mesotrypsin. The induced activation of mesotrypsin by HlyA is preceded by redistribution of mesotrypsin precursors from the cytosol into foci along microtubules and within nuclei. HlyA intoxication also stimulated caspase activation, which occurred independently of effects on host serine proteases. HlyA-induced proteolysis of host proteins likely allows UPEC to not only modulate epithelial cell?functions, but also disable macrophages and suppress inflammatory responses.     

A novel approach to identification of somatic retroelements’ insertions in human genome

The activity of retroelements is one of the factors leading to genetic variability of the modern humans. Insertions of retroelements may result in alteration of gene expression and functional diversity between cells. In recent years an increasing amount of data indicating an elevated level of retroelements’ mobilisation in some human and animal tissues has been reported. Therefore, the development of a system for the detection of somatic retroposition events is required. Here we describe a novel approach to the whole-genome identification of somatic retroelement insertions in human genome. The developed approach was applied for the comparisons of somatic mosaicism levels in two tissues of the investigated individual. A total of 3410 insertions of retroelements belonging to AluYa5 subfamily were identified.     

Hydrophilicity and antigenicity of proteins — A case study of myoglobin and hemoglobin

Hydrophilicity index is used to locate antigenic determinants on two related groups of proteins-myoglobin and hemoglobin. The data on 41 species (including 34 mammals) of myoglobin show that average hydrophilicity for the complete myoglobin molecules as well as the average hydrophilicity for all hydrophilic regions put together seem to remain constant; the variation in the size and location of the antigenic determinants in these species is very small indicating that the antigenic sites are not shifted during evolution. In the case of both the proteins there is a good agreement between the antigenic sites picked up by using hydrophilicity index and the experimentally determined antigenic sites. The data on 56 species of hemoglobin α-chains and 44 species of hemoglobinβ-chains showed that although there are few sites on hemoglobin which have remained invariant during evolution, there is a significant variation in other sites in terms of either a splitting of a site, or a drastic change in the hydrophilicity values and/or a length of the site. Comparison of the hydrophilicity data on these two groups of proteins suggests that hemoglobins which perform a variety of functions as compared to myoglobins are evolving faster than myoglobins supporting the contention of earlier workers.     

Liquid chromatography–fluorescence and liquid chromatography–mass spectrometry detection of tryptophan degradation products of a recombinant monoclonal antibody

Light exposure is one of several conditions used to study the degradation pathways of recombinant monoclonal antibodies. Tryptophan is of particular interest among the 20 amino acids because it is the most photosensitive. Tryptophan degradation forms several products, including an even stronger photosensitizer and several reactive oxygen species. The current study reports a specific peptide mapping procedure to monitor tryptophan degradation. Instead of monitoring peptides using UV 214 nm, fluorescence detection with an excitation wavelength of 295 nm and an emission wavelength of 350 nm was used to enable specific detection of tryptophan-containing peptides. Peaks that decreased in area over time are likely to contain susceptible tryptophan residues. This observation can allow further liquid chromatography–mass spectrometry (LC–MS) analysis to focus only on those peaks to confirm tryptophan degradation products. After confirmation of tryptophan degradation, susceptibility of tryptophan residues can be compared based on the peak area decrease.     

ATG8ylation of proteins: A way to cope with cell stress?

The ATG8 family of proteins regulates autophagy in a variety of ways. Recently, ATG8s were demonstrated to conjugate directly to cellular proteins in a process termed “ATG8ylation,” which is amplified by mitochondrial damage and antagonized by ATG4 proteases. ATG8s may have an emerging role as small protein modifiers.

ATG8 proteins directly conjugate to cellular proteinsAutophagy describes the capture of intracellular material by autophagosomes and their delivery to lysosomes for destruction (Kaur and Debnath, 2015). This process homeostatically remodels the intracellular environment and is necessary for an organism to overcome starvation (Kaur and Debnath, 2015). The autophagy pathway is coordinated by autophagy-related (ATG) proteins that are controlled by diverse post-translational modifications (e.g., phosphorylation, acetylation, ubiquitination, and lipidation; Ichimura et al., 2000; McEwan and Dikic, 2011). Recently, a previously uncharacterized post-translational modification termed “ATG8ylation” was uncovered (Agrotis et al., 2019; Nguyen et al., 2021). ATG8ylation is the direct covalent attachment of the small ubiquitin-like family of ATG8 proteins to cellular proteins (Agrotis et al., 2019; Nguyen et al., 2021). Until now, the only known instances of ATG8 conjugation to proteins were of a transient nature, as E1- and E2-like intermediates with ATG7 and ATG3, respectively, as a way of ligating ATG8 to the lipid phosphatidylethanolamine during autophagy (Ichimura et al., 2000). Therefore, ATG8ylation may represent an underappreciated regulatory mechanism for many cellular proteins that coordinate pathways such as mitophagy.ATG8s play many roles in the autophagy pathwayDuring canonical autophagy, the ATG8 family (comprising LC3A, -B, and -C and GABARAP, -L1, and -L2) undergoes molecular processing that concludes with their attachment to phosphatidylethanolamine, enabling proper construction of autophagosomes and subsequent autophagosome–lysosome fusion (Nguyen et al., 2016). The ATG4 family of cysteine proteases (ATG4A, -B, -C, and -D) cleaves ATG8 proteins immediately after a conserved glycine residue in their C terminus in a process dubbed “priming,” which leads to the formation of ATG8-I (Skytte Rasmussen et al., 2017; Tanida et al., 2004). ATG7 then attaches to the exposed glycine residue of ATG8-I via a thioester linkage to form an E1 ubiquitin-like complex that transfers ATG8-I to ATG3 in a similar way to generate an E2-like complex (Ichimura et al., 2000). The ATG5–ATG12–ATG16L1 complex then catalyzes the E3-like transfer of ATG8-I from ATG3 to phosphatidylethanolamine to form ATG8-II, which is the lipidated species that is incorporated into double membrane–bound compartments such as autophagosomes (Hanada et al., 2007). The lipidation of ATG8s and their recruitment to the phagophore are not essential for the formation of autophagosomes but are important for phagophore expansion, the selective capture of autophagic substrates, and autophagosome–lysosome fusion (Kirkin and Rogov, 2019; Nguyen et al., 2016). Intriguingly, ATG8 lipidation is multifaceted, as ATG8s can be alternatively lipidated with phosphatidylserine (instead of phosphatidylethanolamine) to enable their recruitment to single membrane–bound compartments during LC3-associated phagocytosis, influenza infection, and lysosomal dysfunction (Durgan et al., 2021).The discovery of ATG8ylationKey insights into ATG8ylation came from the observation that various ATG8s form high-molecular-weight species in cells following the expression of their primed forms that have their C-terminal glycine exposed (for example, LC3B-G), bypassing the need for cleavage by ATG4 (Agrotis et al., 2019; Nguyen et al., 2021). Indeed, on an immunoblot, ATG8⁺ “smears” resemble that of ubiquitinated proteins (Agrotis et al., 2019; Nguyen et al., 2021). Traditionally, in the autophagy field, ATG8⁺ smears were thought to arise from poor antibody specificity. However, in light of recent findings, this widely accepted interpretation has been challenged, given that ATG8⁺ smears are enriched following ATG8 overexpression and disappear in the absence of ATG8s (Agrotis et al., 2019; Nguyen et al., 2021). Smearing has also been detected after immunoprecipitation of epitope-tagged ATG8s from cell extracts under denaturing conditions, ruling out noncovalent interactions accounting for this upshift (Agrotis et al., 2019; Nguyen et al., 2021). Further, smearing is not abolished by deubiquitinase treatment, arguing strongly against ATG8 ubiquitination as the cause (Nguyen et al., 2021). Everything considered, the most plausible explanation is that ATG8 itself undergoes covalent linkage to cellular proteins, akin to ubiquitin and NEDD8 modifiers, which are structurally similar to ATG8s. Remarkably, the protease ATG4 antagonizes the ATG8ylation state of many proteins (Agrotis et al., 2019; Nguyen et al., 2021).ATG4 displays isoform-specific proteolytic cleavage of ATG8ATG4 is required for the formation of autophagosomes, but its protease activity is not (Nguyen et al., 2021). The protease activity of ATG4 is, however, required for ATG8 processing, such as priming ahead of lipidation and de-lipidation, which removes excess ATG8 from autophagosomes and other membranes (Nguyen et al., 2021; Tanida et al., 2004; Fig. 1 A). Apart from these functions, ATG4 regulates the deubiquitinase-like removal of ATG8 from cellular proteins (de-ATG8ylation; Agrotis et al., 2019; Nguyen et al., 2021; Fig. 1 A). Consistent with this role, deletion of all four ATG4 isoforms (A, B, C, and D) increases the abundance of ATG8ylated proteins (Nguyen et al., 2021). In contrast, overexpression of ATG4B has the opposite effect, but only if its protease activity is intact (Agrotis et al., 2019). As such, ATG4 inhibits the ATG8ylation state of many proteins, which is likely to modulate their downstream functions.Open in a separate windowFigure 1.The many roles of ATG4 in ATG8 processing. (A) Molecular processing of ATG8 proteins by ATG4 illustrating its roles in priming, de-lipidation, and de-ATG8ylation. The structure of LC3B (Protein Data Bank accession no. 1V49) was used to denote ATG8 (G, glycine; PE, phosphatidylethanolamine). (B) Heatmap summarizing relationships between ATG4 isoforms and ATG8 family members. Data were summarized for qualitative interpretation (Agrotis et al., 2019; Li et al., 2011; Nguyen et al., 2021). Int., intermediate; N.d., not determined. (C) Graphical summary of questions moving forward with ATG8ylation (P, phosphorylation).ATG4 is an important “gatekeeper” for ATG8 conjugation events. ATG4 primes ATG8s to expose their C-terminal glycine, which is required for conjugation to proteins or lipids; however, ATG4 also catalyzes de-ATG8ylation and de-lipidation events, respectively (Agrotis et al., 2019; Nguyen et al., 2021; Tanida et al., 2004). Because the C-terminal glycine of a single ATG8 is occupied when conjugated to a protein or lipid, it is unlikely that ATG8ylated proteins directly engage with phagophore membranes in the same way as ATG8-II. Indeed, protease protection assays with recombinant ATG4B reveal that de-ATG8ylation of cell lysates remains unchanged with or without organellar membrane disruption, suggesting that ATG8ylated proteins are largely cytoplasmic facing rather than intraluminal (Agrotis et al., 2019). Paradoxically, however, ATG8ylation is enhanced by lysosomal V-type ATPase inhibition, which blocks the degradation of lysosomal contents, indicating that ATG8ylated substrates may undergo lysosome-dependent turnover (Agrotis et al., 2019; Nguyen et al., 2021). One explanation for these differences may be that the process of ATG8ylation is itself sensitive to lysosomal dysfunction.Functional relationships between ATG4s and ATG8sIsoforms of ATG4 show clear preferences for proteolytically processing ATG8 subfamilies (i.e., LC3s and GABARAPs) for de-ATG8ylation and priming upstream of phosphatidylethanolamine ligation (Agrotis et al., 2019; Li et al., 2011; Nguyen et al., 2021; Fig. 1 B). ATG4A strongly reduces the abundance of proteins that have been ATG8ylated with the GABARAP family while promoting ligation of GABARAPs to phosphatidylethanolamine (Agrotis et al., 2019; Nguyen et al., 2021; Fig. 1 B). In contrast, ATG4B strongly reduces the abundance of proteins that have been ATG8ylated with LC3 proteins while promoting ligation of LC3s to phosphatidylethanolamine (Agrotis et al., 2019; Nguyen et al., 2021; Fig. 1 B). In comparison, ATG4C and -D lack obvious de-ATG8ylation activity, although the latter weakly promotes phosphatidylethanolamine ligation to GABARAPL1 only (Nguyen et al., 2021). These functional similarities between ATG4 isoforms are consistent with both their sequence and structural homology (i.e., ATG4A and -B are most similar; Maruyama and Noda, 2018; Satoo et al., 2009). Structurally, ATG4B adopts an auto-inhibited conformation with its regulatory loop and N-terminal tail blocking substrate entry to its proteolytic core (Maruyama and Noda, 2018). LC3B induces conformational rearrangements in ATG4B that involve displacement of its regulatory loop and its N-terminal tail, with the latter achieved by an interaction between the ATG8-interacting region in its N-terminal tail with a second copy of LC3B that functions allosterically (Maruyama and Noda, 2018; Satoo et al., 2009). These rearrangements permit entry of LC3B into the proteolytic core of ATG4B, where cleavage of LC3B following its C-terminal glycine occurs (Li et al., 2011; Maruyama and Noda, 2018). ATG4B^L232 is directly involved in LC3B binding and its selectivity for LC3s (Satoo et al., 2009). This residue corresponds to ATG4A^I233 and, when substituted for leucine, gives ATG4A^I233L the ability to efficiently process LC3 proteins, whereas without this mutation it preferentially processes GABARAPs (Satoo et al., 2009). Moreover, the ATG8–ATG4 interaction is necessary for the de-ATG8ylation of cellular proteins, as an LC3B-G^Q116P mutant that cannot bind to ATG4 leads to widespread ATG8ylation (Agrotis et al., 2019). Altogether, these observations hint toward a common mechanism of ATG8 cleavage that regulates priming, de-lipidation, and de-ATG8ylation.Mitochondrial damage promotes ATG8ylationATG8ylation of cellular proteins appears to be enhanced by mitochondrial depolarization and inhibition of the lysosomal V-type ATPase (Agrotis et al., 2019; Nguyen et al., 2021). This may be the consequence of acute ATG4A and -B inhibition, given that cells lacking all ATG4 isoforms display an increased abundance of ATG8ylated proteins and are insensitive to further increase by mitochondrial depolarization or lysosomal V-type ATPase inhibition (Agrotis et al., 2019; Nguyen et al., 2021). Indeed, mitochondrial depolarization leads to activation of ULK1, which phosphorylates ATG4B^S316 to inhibit its protease activity (Pengo et al., 2017). Similarly, mitochondrial depolarization stimulates TBK1 activation, which prevents de-lipidation of ATG8s by blocking the ATG8–ATG4 interaction through phosphorylation of LC3C^S93/S96 and GABARAP-L2^S87/S88 (Herhaus et al., 2020; Richter et al., 2016). As such, ATG8 phosphorylation may render ATG8ylated substrates more resistant to de-ATG8ylation by ATG4s. This may be analogous to how chains of phosphorylated ubiquitin^S65 are more resistant to hydrolysis by deubiquitinating enzymes than unphosphorylated ones (Wauer et al., 2015). Moreover, ATG8ylation is insensitive to nutrient deprivation and pharmacological inhibition of mTOR, which rules out a functional contribution of this process to starvation-induced autophagy (Agrotis et al., 2019). Therefore, ATG8ylation may be a unique aspect of mitophagy (and perhaps also other forms of selective autophagy) given that depolarization potently activates Parkin-dependent mitophagy (Agrotis et al., 2019; Nguyen et al., 2021).Substrates of ATG8ylationBased on ATG8⁺ smearing, ATG4 regulates the de-ATG8ylation of numerous proteins (Agrotis et al., 2019; Nguyen et al., 2021). For the majority, their identity, induced structural and functional changes, and the cellular contexts during which these modifications occur await exploration. Considering that the ATG8 interactome is well characterized, it is likely that at least some ATG8ylated proteins have been mistaken for ATG8-binding partners (Behrends et al., 2010). Given their E2- and E3-like roles in ATG8 lipidation, it is remarkable that ATG3 and ATG16L1 are themselves modified by ATG8ylation (Agrotis et al., 2019; Hanada et al., 2007; Ichimura et al., 2000; Nguyen et al., 2021). Lysine mutagenesis indicates that ATG3^K243 is the “acceptor” site for ATG8ylation (Agrotis et al., 2019). ATG3^K243 is essential for its conjugation to either LC3B or ATG12 and is required for autophagosomes to form around damaged mitochondria (Agrotis et al., 2019; Radoshevich et al., 2010). This also raises the possibility that key functions originally attributed to ATG3–ATG12 conjugation may be, at least in part, due to ATG3–ATG8 conjugation. Because multiple high-molecular-weight species of ATG3 are enriched following immunoprecipitation of primed LC3B-G from cells lacking ATG4B, it is likely that ATG3 is either mono-ATG8ylated at several sites or poly-ATG8ylated (Agrotis et al., 2019). ATG8ylation of ATG3 may also reflect the stabilization of its E2-like intermediate (Ichimura et al., 2000). ATG8ylation of ATG16L1 may regulate whether canonical or noncanonical autophagy pathways are activated (Durgan et al., 2021; Nguyen et al., 2021). In line with this possibility, the WD40 domain mutant of ATG16L1^K490A prevents lipidation of ATG8s with phosphatidylserine (i.e., during noncanonical autophagy pathways) but not phosphatidylethanolamine (i.e., during canonical autophagy; Durgan et al., 2021). Moreover, given that ATG8ylation of protein targets correlates with the activation of mitophagy, it is tempting to speculate that it may stimulate the E2-/E3-like activity of the ATG8 conjugation machinery to amplify mitochondrial capture and destruction.Concluding remarksThe finding that numerous cellular proteins are modified by ATG8ylation poses several questions about how signaling networks are coordinated during selective autophagy (i.e., mitophagy). Whether ATG8ylation is augmented by mitochondrial injury per se or is the consequence of mitophagy activation is yet to be determined, as is whether this phenomenon occurs during other types of selective autophagy (e.g., ER-phagy, ribophagy, and lysophagy; Kirkin and Rogov, 2019; Fig. 1 C). While the in vivo relevance of ATG8ylation is not yet understood, it is plausible that this process could be altered in diseases with defective mitophagy (e.g., Parkinson’s disease and atherosclerosis). Exploring the mechanistic aspects of ATG8ylation (e.g., ATG8 ligases and regulatory proteins, linkage types, acceptor sites, etc.) and de-ATG8ylation by ATG4 will improve our understanding about how this modifier alters the structure and biological function of cellular proteins (Fig. 1 C). By identifying ATG8ylated substrates, or the ATG8ylome, insights into whether ATG8ylation is a ubiquitous epiphenomenon or a post-translational modification that is selective to proteins of distinct biological function(s) will become clearer (Fig. 1 C). Considering the similarity of ATG8s with bona fide modifier proteins (e.g., ubiquitin and ubiquitin-like proteins) and the diversity of their substrates (e.g., lipid species and proteins), only now are we beginning to understand the functional complexities of the ATG8 protein family.     

Intracellular antibody capture: A molecular biology approach to inhibitors of protein–protein interactions

Many proteins of interest in basic biology, translational research studies and for clinical targeting in diseases reside inside the cell and function by interacting with other macromolecules. Protein complexes control basic processes such as development and cell division but also abnormal cell growth when mutations occur such as found in cancer. Interfering with protein–protein interactions is an important aspiration in both basic and disease biology but small molecule inhibitors have been difficult and expensive to isolate. Recently, we have adapted molecular biology techniques to develop a simple set of protocols for isolation of high affinity antibody fragments (in the form of single VH domains) that function within the reducing environment of higher organism cells and can bind to their target molecules. The method called Intracellular Antibody Capture (IAC) has been used to develop inhibitory anti-RAS and anti-LMO2 single domains that have been used for target validation of these antigens in pre-clinical cancer models and illustrate the efficacy of the IAC approach to generation of drug surrogates. Future use of inhibitory VH antibody fragments as drugs in their own right (we term these macrodrugs to distinguish them from small molecule drugs) requires their delivery to target cells in vivo but they can also be templates for small molecule drug development that emulate the binding sites of the antibody fragments. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.     

α-Helical transmembrane peptides: A “Divide and Conquer” approach to membrane proteins

α-Helical membrane proteins fulfill many vital roles in all living cells and constitute the majority of drug targets. However, their relevance is in no way paralleled by our current understanding of their structures and functions. This is because membrane proteins present a number of experimental obstacles that are difficult to surmount by classical methods developed for water-soluble proteins. Moreover, membrane proteins are not only challenging on their very own but, when embedded in a biological membrane, also reside in an outstandingly complex milieu. These difficulties have fostered a “divide and conquer” approach, in which a membrane protein is dissected into shorter and easier-to-handle transmembrane (TM) peptides. Under suitable conditions, such peptides fold independently and retain many of the properties displayed in the context of the full-length parent protein.This contribution reviews some of the most notable insights into α-helical membrane proteins gleaned from experiments on protein-derived TM peptides. We recapitulate some peculiar properties of lipid bilayers that render them such a complex and unique environment and discuss generic features pertaining to hydrophobic peptides derived from α-helical membrane proteins. The main part of the review is devoted to a critical discussion of particularly interesting examples of TM peptides studied in membrane-mimetic systems of increasing complexity: isotropic solvents, detergent micelles, lipid bilayers, and biological membranes. The unifying theme is to explore to what extent TM peptides in combination with different membrane-mimetic systems can aid in advancing our knowledge and comprehension of α-helical membrane proteins as well as in developing new pharmacological tools.     

In vitro Induction of primary antibody responses to particulate and soluble protein antigens in T cell—replaced murine spleen cell cultures

Specific antibody responses could be induced in serumfree condition.Specific anti-SRBC or anti-SRBC ghost antibody were induced from anti-Thy treated (T-depleted) murine spleen cells in serum-free culture in the presence of Con A conditioned medium.This induction system may facilitate the study of lymphokine functions on antigen triggered B cells. In T cell-replaced cultures,the antibody responses of B cells could be successfully induced when soluble SRBC membrane proteins were used as antigens.It thus indicates that antigen together with lymphokines are sufficient to drive B cells to become antibody secreting cells in the absence of T cells.The T cell-replaced system provides a more stable way for in vitro immunization and may be applied to monoclonal antibody production when in vivo immunization is difficult to be carried out.     

Growth ring analysis of multiple dicotyledonous herb species—A novel community-wide approach

Longevity is a key demographic characteristic of herbaceous plants, but often unknown. While root or rhizome growth ring analysis may allow assessment plant longevity directly and conveniently, so far it has only been used in a few case studies of herbaceous dicotyledonous species. To evaluate whether growth ring analysis is applicable to a large spectrum of herbaceous dicotyledonous plant species, we used plant communities of varying species richness in a 12-year-old grassland biodiversity experiment (Jena Experiment). Cross-sections of the oldest available part of the plants were analysed for all available dicotyledonous perennial herb species (S = 37), which represented three functional groups: legumes, small herbs and tall herbs. We studied 1664 individuals representing the genet in clearly distinguishable plant individuals, and the ramet in clonally growing plant species.Roots of eleven species with permanent primary root were harvested. They showed clearly visible growth rings. Longevity was extended with a mean age of 4.0 years (SE = 0.3). Seven species, which also had a permanent primary root, showed less distinct growth rings. They were shorter-lived (mean age 3.0 years (SE = 0.3)). In six species with obligate clonal growth mostly rhizomes were sampled, but individuals were still identifiable due to their growth habit. For these species growth rings were clearly visible. Longevity of rhizomes was extended (mean age 3.3 years (SE = 0.5)). In 13 species with obligate clonal growth also rhizomes were sampled, but plant individuals were not identifiable. For these species longevity was low (mean age 2.1 years (SE = 0.2)). Community mean age was significantly lower when small herbs were present and higher when tall herbs were present, while legumes had no effect on community mean age. In summary, anatomical analysis of roots and rhizomes is a suitable tool to study the population age structure of a large spectrum of perennial dicotyledonous herbaceous species and therefore opens new perspectives for demographic studies at the community level.     

When and how does cell division order influence cell allocation to the inner cell mass of the mouse blastocyst?

Aggregate 8-cell embryos were constructed from four 2/8 pairs of blastomeres, one of which was marked with a short-term cell lineage marker and was also either 4 h older (derived from an early-dividing 4-cell) or 4 h younger (derived from a late-dividing 4-cell) than the other three pairs. The aggregate embryos were cultured to the 16-cell stage, at which time a second marker was used to label the outside cell population. The embryos were then disaggregated and each cell was examined to determine its labelling pattern. From this analysis, we calculated the relative contributions to the inside cell population of the 16-cell embryo of older and younger cells. Older cells were found to contribute preferentially. However, if the construction of the aggregate 8-cell embryo was delayed until each of the contributing 2/8 cell pairs had undergone intercellular flattening and then had been exposed to medium low in calcium to reverse this flattening immediately prior to aggregation, the advantage possessed by the older cells was lost. These results support the suggestion that older cells derived from early-dividing 4-cell blastomeres contribute preferentially to the inner cell mass as a result of being early-flattening cells.     

Binding of β4γ5 by adenosine A1 and A2A receptors determined by stable isotope labeling with amino acids in cell culture and mass spectrometry

Characterization of G protein βγ dimer isoform expression in different cellular contexts has been impeded by low levels of protein expression, broad isoform heterogeneity, and antibodies of limited specificity, sensitivity, or availability. As a new approach, we used quantitative mass spectrometry to characterize native βγ dimers associated with adenosine A(1):α(i1) and adenosine A(2A):α(S) receptor fusion proteins expressed in HEK-293 cells. Cells expressing A(1):α(i1) were cultured in media containing [(13)C(6)]Arg and [(13)C(6)]Lys and βγ labeled with heavy isotopes purified. Heavy βγ was combined with either recombinant βγ purified from Sf9 cells, βγ purified from the A(2A):α(S) expressed in HEK-293 cells cultured in standard media, or an enriched βγ fraction from HEK-293 cells. Samples were separated by SDS-PAGE, protein bands containing β and γ were excised, digested with trypsin, and separated by HPLC, and isotope ratios were analyzed by mass spectrometry. Three β isoforms, β(1), β(2), and β(4), and seven γ isoforms, γ(2), γ(4), γ(5), γ(7), γ(10), γ(11), and γ(12), were identified in the analysis. β(1) and γ(5) were most abundant in the enriched βγ fraction, and this βγ profile was generally mirrored in the fusion proteins. However, both A(2A):α(S) and A(1):α(i1) bound more β(4) and γ(5) compared to the enriched βγ fraction; also, more β(4) was associated with A(2A):α(S) than A(1):α(i1). Both fusion proteins also contained less γ(2), γ(10), and γ(12) than the enriched βγ fraction. These results suggest that preferences for particular βγ isoforms may be driven in part by structural motifs common to adenosine receptor family members.