A novel and cost-effective methodology for enhanced production of industrially valuable alkaline xylano-pectinolytic enzymes cocktail in short solid-state fermentation cycle

The aim of this study was to enhance the production of xylano-pectinolytic enzymes concurrently and also to reduce the fermentation period. In this study, the effect of agro-residues extract-based inoculum on yield and fermentation time of xylano-pectinolytic enzymes was studied. Microbial inoculum and fermentation media were supplemented with xylan and pectin polysaccharides derived from agro-based residues. Enzymes production parameters were optimized through two-stage statistical design approach. Under optimized conditions (temperature 37°C, pH 7.2, K₂HPO₄ 0.22%, MgSO₄ 0.1%, gram flour 5.6%, substrate: moisture ratio 1:2, inoculum size 20%, agro-based crude xylan in production media 0.45%, and agro-based crude xylan–pectin in inoculum 0.13%), nearly 28,255 ± 565 and 9,202 ± 193 IU of xylanase and pectinase, respectively, were obtained per gram of substrate in a time interval of 6 days only. The yield of both xylano-pectinolytic enzymes was enhanced along with a reduction of nearly 24 h in fermentation time in comparison with control, using polysaccharides extracted from agro-residues. The activity of different types of pectinase enzymes such as exo-polymethylgalacturonase (exo-PMG), endo-PMG, exo-polygalacturonase (exo-PG), endo-PG, pectin lyase, pectate lyase, and pectin esterase was obtained as 1,601, 12.13, 5637, 24.86, 118.62, 124.32, and 12.56 IU/g, respectively, and was nearly twofold higher than obtained for all seven types in control samples. This is the first report mentioning the methodology for enhanced production of xylano-pectinolytic enzymes in short solid-state fermentation cycle using agro-residues extract-based inoculum and production media.     

3D structure generation,virtual screening and docking of human Ras-associated binding (Rab3A) protein involved in tumourigenesis

Rab3A is expressed predominantly in brain and synaptic vesicles. Rab3A is involved specifically in tethering and docking of synaptic vesicles prior to fusion which is a critical step in regulated release of neurotransmitters. The precise function of Rab3A is still not known. However, up-regulation of Rab3A has been reported in malignant neuroendocrine and breast cancer cells. In the present study, the structure of Rab3A protein was generated using MODELLER 9v8 software. The modeled protein structure was validated and subjected to molecular docking analyses. Docking with GTP was carried out on the binding site of Rab3A using GOLD software. The Rab3A-GTP complex has best GOLD fitness value of 77.73. Ligplot shows hydrogen bondings (S16, S17, V18, G19, K20, T21, S22, S31, T33, A35, S38, T39 and G65) and hydrophobic interacting residues (F25, F32, P34, F36, V37, D62 and A64) with the GTP ligands in the binding site of Rab3A protein. Here, the ligand molecules of NCI diversity set II from the ZINC database against the active site of the Rab3A protein were screened. For this purpose, the incremental construction algorithm of GLIDE and the genetic algorithm of GOLD were used. Docking results were analyzed for top ranking compounds using a consensus scoring function of X-Score to calculate the binding affinity and Ligplot was used to measure protein–ligand interactions. Five compounds which possess good inhibitory activity and may act as potential high affinity inhibitors against Rab3A active site were identified. The top ranking molecule (ZINC13152284) has a Glide score of ?6.65 kcal/mol, X-Score of ?3.02 kcal/mol and GOLD score of 64.54 with 03 hydrogen bonds and 09 hydrophobic contacts. This compound is thus a good starting point for further development of strong inhibitors.     

Inhibition of 5′-UTR RNA Conformational Switching in HIV-1 Using Antisense PNAs

Background

The genome of retroviruses, including HIV-1, is packaged as two homologous (+) strand RNA molecules, noncovalently associated close to their 5′-end in a region called dimer linkage structure (DLS). Retroviral HIV-1 genomic RNAs dimerize through complex interactions between dimerization initiation sites (DIS) within the (5′-UTR). Dimer formation is prevented by so calledLong Distance Interaction (LDI) conformation, whereas Branched Multiple Hairpin (BMH) conformation leads to spontaneous dimerization.

Methods and Results

We evaluated the role of SL1 (DIS), PolyA Hairpin signal and a long distance U5-AUG interaction by in-vitro dimerization, conformer assay and coupled dimerization and template-switching assays using antisense PNAs. Our data suggests evidence that PNAs targeted against SL1 produced severe inhibitory effect on dimerization and template-switching processes while PNAs targeted against U5 region do not show significant effect on dimerization and template switching, while PNAs targeted against AUG region showed strong inhibition of dimerization and template switching processes.

Conclusions

Our results demonstrate that PNA can be used successfully as an antisense to inhibit dimerization and template switching process in HIV -1 and both of the processes are closely linked to each other. Different PNA oligomers have ability of switching between two thermodynamically stable forms. PNA targeted against DIS and SL1 switch, LDI conformer to more dimerization friendly BMH form. PNAs targeted against PolyA haipin configuration did not show a significant change in dimerization and template switching process. The PNA oligomer directed against the AUG strand of U5-AUG duplex structure also showed a significant reduction in RNA dimerization as well as template- switching efficiency.The antisense PNA oligomers can be used to regulate the shift in the LDI/BMH equilibrium.     

Acute exposure of apigenin induces hepatotoxicity in Swiss mice

Apigenin, a dietary flavonoid, is reported to have several therapeutic effects in different diseases including cancer. Toxicity of Apigenin is however, least explored, and reports are scanty in literature. This warrants dose-specific evaluation of toxicity in vivo. In the present study, Apigenin was administered intraperitoneally to Swiss mice at doses of 25, 50, 100 and 200 mg/kg. Serum levels of alanine amino transferase (ALT), aspartate amino transferase (AST) and alkaline phosphatase (ALP) were measured along with the examination of liver histology, reactive oxygen species (ROS) in blood, lipid peroxidation (LPO), glutathione level, superoxide dismutase activity, catalase activity, glutathione S-transferase activity and gene expression in liver tissue. Increase in ALT, AST, ALP, ROS, ratio of oxidized to reduced glutathione (GSSG/GSH) and LPO, altered enzyme activities along with damaged histoarchitecture in the liver of 100 or 200 mg/kg Apigenin treated animals were found. Microarray analysis revealed the differential expression of genes that correspond to different biologically relevant pathways including oxidative stress and apoptosis. In conclusion, these results suggested the oxidative stress induced liver damage which may be due to the regulation of multiple genes by Apigenin at higher doses in Swiss mice.     

Piper relictum sp. nov. (Piperaceae) from northern Western Ghats,India

A new species of Piper, P. relictum Lekhak, S. S. Kambale & S. R. Yadav sp. nov. is described and illustrated. It grows as a climber on the edges of high altitude lateritic plateaus in evergreen forests of the northern Western Ghats in India. The new species is similar to P. galeatum, but differs in the characters of the male inflorescence.     

Protein tyrosine nitration of the flavin subunit is associated with oxidative modification of mitochondrial complex II in the post-ischemic myocardium

Increased O(2)* and NO production is a key mechanism of mitochondrial dysfunction in myocardial ischemia/reperfusion injury. A crucial segment of the mitochondrial electron transport chain is succinate ubiquinone reductase (SQR or Complex II). In SQR, oxidative impairment and deglutathionylation of the 70-kDa flavin protein occurs in the post-ischemic heart ( Chen, Y. R., Chen, C. L., Pfeiffer, D. R., and Zweier, J. L. (2007) J. Biol. Chem. 282, 32640-32654 ). To gain insights into the oxidative modification of the 70-kDa protein in the post-ischemic myocardium, we used the identified S-glutathionylated peptide ((77)AAFGLSEAGFNTACVTK(93)) of the 70-kDa protein as a chimeric epitope incorporating a "promiscuous" T cell epitope to generate a high titer polyclonal antibody, AbGSC90. Purified AbGSC90 showed a high binding affinity to isolated SQR. Antibodies of AbGSC90 moderately inhibited the electron transfer and superoxide generation activities of SQR. To test for protein nitration, rats were subjected to 30 min of coronary ligation followed by 24 h of reperfusion. Tissue homogenates were immunoprecipitated with AbGSC90 and probed with antibodies against 3-nitrotyrosine. Enhancement of protein tyrosine nitration was detected in the post-ischemic myocardium. Isolated SQR was subjected to in vitro protein nitration with peroxynitrite, leading to site-specific nitration at the 70-kDa polypeptide and impairment of SQR electron transfer activity. Protein nitration of SQR further impaired its protein-protein interaction with Complex III. Liquid chromatography/tandem mass spectrometry analysis indicated that Tyr-56 and Tyr-142 were involved in protein tyrosine nitration. When the isolated SQR was subjected to in vitro S-glutathionylation, oxidative modification and impairment mediated by peroxynitrite were significantly decreased, thus confirming the protective effect of S-glutathionylation from the oxidative damage of nitration.     

Natural polyphenolic inhibitors against the antiapoptotic BCL-2

The apoptotic mechanism is regulated by the BCL-2 family of proteins, such as BCL-2 or Bcl-xL, which block apoptosis while Bad, Bak, Bax, Bid, Bim or Hrk induce apoptosis. The overexpression of BCL-2 was found to be related to the progression of cancer and also providing resistance towards chemotherapeutic treatments. In the present study, we found that all polyphenols (apigenin, fisetin, galangin and luteolin) bind to the hydrophobic groove of BCL-2 and the interaction is stable throughout MD simulation run. Luteolin was found to bind with highest negative binding energy and thus, claimed highest potency towards BCL-2 inhibition followed by fisetin. The hydrophobic interactions were found to be critical for stable complex formation as revealed by the vdW energy and ligplot analysis. Finally, on the basis of data obtained during the study, it can be concluded that these polyphenols have the potential to be used as lead molecules for BCL-2 inhibition.     

Comprehensive physiological analyses and reactive oxygen species profiling in drought tolerant rice genotypes under salinity stress

Rice being a staple cereal is extremely susceptible towards abiotic stresses. Drought and salinity are two vital factors limiting rice cultivation in Eastern Indo-Gangetic Plains (EIGP). Present study has intended to evaluate the consequences of salinity stress on selected drought tolerant rice genotypes at the most susceptible seedling stage with an aim to identify the potential multi-stress (drought and salt) tolerant rice genotype of this region. Genotypic variation was obvious in all traits related to drought and salt susceptibility. IR84895-B-127-CRA-5-1-1, one of the rice genotypes studied, exhibited exceptional drought and salinity tolerance. IR83373-B-B-25-3-B-B-25-3 also displayed enhanced drought and salt tolerance following IR84895-B-127-CRA-5-1-1. Variations were perceptible in different factors involving photosynthetic performance, proline content, lipid peroxidation, K⁺/Na⁺ ratio. Accumulation of reactive oxygen species (ROS) disintegrated cellular and sub-cellular membrane leading to decreased photosynthetic activities. Therefore, accumulation and detoxification of reactive oxygen species was also considered as a major determinant of salt tolerance. IR84895-B-127-CRA-5-1-1 showed improved ROS detoxification mediated by antioxidant enzymes. IR84895-B-127-CRA-5-1-1 seedlings also displayed significant recovery after removal of salt stress. The results established a direct association of ROS scavenging with improved physiological activities and salt tolerance. The study also recommended IR84895-B-127-CRA-5-1-1 for improved crop performance in both drought and saline environments of EIGP. These contrasting rice genotypes may assist in understanding the multiple stress associated factors in concurrent drought and salt tolerant rice genotypes.     

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.     

Comparison of membrane proteins of Mycobacterium tuberculosis H37Rv and H37Ra strains

Background  

The potential causes for variation in virulence between distinct M. tuberculosis strains are still not fully known. However, differences in protein expression are probably an important factor. In this study we used a label-free quantitative proteomic approach to estimate differences in protein abundance between two closely related M. tuberculosis strains; the virulent H37Rv strain and its attenuated counterpart H37Ra.