Mammalian Complex Research Articles

The gastrointestinal tract is a major source of the acute metformin-stimulated rise in GDF15

The hormone GDF15 is secreted in response to cellular stressors. Metformin elevates circulating levels of GDF15, an action important for the drug’s beneficial effects on body weight. Metformin can also inhibit mammalian respiratory complex I, leading to decreases in ATP:AMP ratio, activation of AMP Kinase (AMPK), and increased GDF15 production. We undertook studies using a range of mice with tissue-specific loss of Gdf15 (namely gut, liver and global deletion) to determine the relative contributions of two classical metformin target tissues, the gut and liver, to the elevation of GDF15 seen with metformin. In addition, we performed comparative studies with another pharmacological agent, the AMP kinase pan-activator, MK-8722. Deletion of Gdf15 from the intestinal epithelium significantly reduced the circulating GDF15 response to oral metformin, whereas deletion of Gdf15 from the liver had no effect. In contrast, deletion of Gdf15 from the liver, but not the gut, markedly reduced circulating GDF15 responses to MK-8722. Further, our data show that, while GDF15 restricts high-fat diet-induced weight gain, the intestinal production of GDF15 is not necessary for this effect. These findings add to the body of evidence implicating the intestinal epithelium in key aspects of the pharmacology of metformin action.

DGARM/C10orf76/ARMH3 for Ceramide Transfer Zone at the Endoplasmic Reticulum-Distal Golgi Contacts.

Phosphatidylinositol 4-monophosphate (PtdIns(4)P) is one of the key membrane components which mark the membrane contact sites. In the mammalian Golgi complex, PtdIns(4)P is produced at various subregions via specific mechanisms for each site. Particularly, PtdIns(4)P pools generated at the distal Golgi regions are pivotal for the determination of membrane contacts between the endoplasmic reticulum (ER) and Golgi, at which inter-organelle lipid transport takes place. In this short review, we will focus on C10orf76 (or ARMH3), which we propose to rename as DGARM after a distal Golgi armadillo repeat protein, for its function in generating a PtdIns(4)P pool crucial for ER-to-distal Golgi ceramide transport. We further discuss from the viewpoint of the evolutionary conservation of DGARM.

Comparative genomics of the Natural Killer Complex in carnivores.

The mammalian Natural Killer Complex (NKC) harbors genes and gene families encoding a variety of C-type lectin-like proteins expressed on various immune cells. The NKC is a complex genomic region well-characterized in mice, humans and domestic animals. The major limitations of automatic annotation of the NKC in non-model animals include short-read based sequencing, methods of assembling highly homologous and repetitive sequences, orthologues missing from reference databases and weak expression. In this situation, manual annotations of complex genomic regions are necessary. This study presents a manual annotation of the genomic structure of the NKC region in a high-quality reference genome of the domestic cat and compares it with other felid species and with representatives of other carnivore families. Reference genomes of Carnivora, irrespective of sequencing and assembly methods, were screened by BLAST to retrieve information on their killer cell lectin-like receptor (KLR) gene content. Phylogenetic analysis of in silico translated proteins of expanded subfamilies was carried out. The overall genomic structure of the NKC in Carnivora is rather conservative in terms of its C-type lectin receptor gene content. A novel KLRH-like gene subfamily (KLRL) was identified in all Carnivora and a novel KLRJ-like gene was annotated in the Mustelidae. In all six families studied, one subfamily (KLRC) expanded and experienced pseudogenization. The KLRH gene subfamily expanded in all carnivore families except the Canidae. The KLRL gene subfamily expanded in carnivore families except the Felidae and Canidae, and in the Canidae it eroded to fragments. Knowledge of the genomic structure and gene content of the NKC region is a prerequisite for accurate annotations of newly sequenced genomes, especially of endangered wildlife species. Identification of expressed genes, pseudogenes and gene fragments in the context of expanded gene families would allow the assessment of functionally important variability in particular species.

Epimorphic regeneration in the mammalian tympanic membrane

Adult mammals are generally believed to have limited ability to regenerate complex tissues and instead, repair wounds by forming scars. In humans and across mammalian species, the tympanic membrane (TM) rapidly repairs perforations without intervention. Using mouse models, we demonstrate that the TM repairs itself through a process that bears many hallmarks of epimorphic regeneration rather than typical wound healing. Following injury, the TM forms a wound epidermis characterized by EGFR ligand expression and signaling. After the expansion of the wound epidermis that emerges from known stem cell regions of the TM, a multi-lineage blastema-like cellular mass is recruited. After two weeks, the tissue architecture of the TM is largely restored, but with disorganized collagen. In the months that follow, the organized and patterned collagen framework of the TM is restored resulting in scar-free repair. Finally, we demonstrate that deletion of Egfr in the epidermis results in failure to expand the wound epidermis, recruit the blastema-like cells, and regenerate normal TM structure. This work establishes the TM as a model of mammalian complex tissue regeneration.

Vimentin intermediate filaments provide structural stability to the mammalian Golgi complex.

The Golgi complex comprises a connected ribbon of stacked cisternal membranes localized to the perinuclear region in most vertebrate cells. The position and morphology of this organelle depends upon interactions with microtubules and the actin cytoskeleton. In contrast, we know relatively little about the relationship of the Golgi complex with intermediate filaments (IFs). In this study, we show that the Golgi is in close physical proximity to vimentin IFs in cultured mouse and human cells. We also show that the trans-Golgi network coiled-coil protein GORAB can physically associate with vimentin IFs. Loss of vimentin and/or GORAB had a modest effect upon Golgi structure at the steady state. The Golgi underwent more rapid disassembly upon chemical disruption with brefeldin A or nocodazole, and slower reassembly upon drug washout, in vimentin knockout cells. Moreover, loss of vimentin caused reduced Golgi ribbon integrity when cells were cultured on high-stiffness hydrogels, which was exacerbated by loss of GORAB. These results indicate that vimentin IFs contribute to the structural stability of the Golgi complex and suggest a role for GORAB in this process.

First person – Teresa Vitali

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. Teresa Vitali is first author on ‘ Vimentin intermediate filaments provide structural stability to the mammalian Golgi complex’, published in JCS. Teresa undertook the work while a Research Associate in the lab of Martin Lowe at the School of Biological Sciences, University of Manchester, UK. Teresa has since spent time as a Senior Research Assistant in the lab of Dr Inhee Chung at George Washington University, Washington, USA and will soon be starting a new role at Washington University Saint Louis, USA. She is interested in cytoskeletal organization and function in diverse cellular processes (at the molecular and cellular level), and in pathological contexts like cancer and infection.

C9orf72-catalyzed GTP loading of Rab39A enables HOPS-mediated membrane tethering and fusion in mammalian autophagy

The multi-subunit homotypic fusion and vacuole protein sorting (HOPS) membrane-tethering complex is required for autophagosome-lysosome fusion in mammals, yet reconstituting the mammalian HOPS complex remains a challenge. Here we propose a “hook-up” model for mammalian HOPS complex assembly, which requires two HOPS sub-complexes docking on membranes via membrane-associated Rabs. We identify Rab39A as a key small GTPase that recruits HOPS onto autophagic vesicles. Proper pairing with Rab2 and Rab39A enables HOPS complex assembly between proteoliposomes for its tethering function, facilitating efficient membrane fusion. GTP loading of Rab39A is important for the recruitment of HOPS to autophagic membranes. Activation of Rab39A is catalyzed by C9orf72, a guanine exchange factor associated with amyotrophic lateral sclerosis and familial frontotemporal dementia. Constitutive activation of Rab39A can rescue autophagy defects caused by C9orf72 depletion. These results therefore reveal a crucial role for the C9orf72-Rab39A-HOPS axis in autophagosome-lysosome fusion.

Thioredoxin regulates the redox state and the activity of the human tRNA ligase complex.

The mammalian tRNA ligase complex (tRNA-LC) catalyzes the splicing of intron-containing pre-tRNAs in the nucleus and the splicing of XBP1 mRNA during the unfolded protein response (UPR) in the cytoplasm. We recently reported that the tRNA-LC coevolved with PYROXD1, an essential oxidoreductase that protects the catalytic cysteine of RTCB, the catalytic subunit of the tRNA-LC, against aerobic oxidation. In this study, we show that the oxidoreductase Thioredoxin (TRX) preserves the enzymatic activity of RTCB under otherwise inhibiting concentrations of oxidants. TRX physically interacts with oxidized RTCB, and reduces and reactivates RTCB through the action of its redox-active cysteine pair. We further show that TRX interacts with RTCB at late stages of UPR. Since the interaction requires oxidative conditions, our findings suggest that prolonged UPR generates reactive oxygen species. Thus, our results support a functional role for TRX in securing and repairing the active site of the tRNA-LC, thereby allowing pre-tRNA splicing and UPR to occur when cells encounter mild, but still inhibitory levels of reactive oxygen species.

Preserved respiratory chain capacity and physiology in mice with profoundly reduced levels of mitochondrial respirasomes

The mammalian respiratory chain complexes I, III2, and IV (CI, CIII2, and CIV) are critical for cellular bioenergetics and form a stable assembly, the respirasome (CI-CIII2-CIV), that is biochemically and structurally well documented. The role of the respirasome in bioenergetics and the regulation of metabolism is subject to intense debate and is difficult to study because the individual respiratory chain complexes coexist together with high levels of respirasomes. To critically investigate the invivo role of the respirasome, we generated homozygous knockin mice that have normal levels of respiratory chain complexes but profoundly decreased levels of respirasomes. Surprisingly, the mutant mice are healthy, with preserved respiratory chain capacity and normal exercise performance. Our findings show that high levels of respirasomes are dispensable for maintaining bioenergetics and physiology in mice but raise questions about their alternate functions, such as those relating to the regulation of protein stability and prevention of age-associated protein aggregation.

Kar4, the yeast homolog of METTL14, is required for mRNA m6A methylation and meiosis.

KAR4, the yeast homolog of the mammalian mRNA N6A-methyltransferase complex component METTL14, is required for two disparate developmental programs in Saccharomyces cerevisiae: mating and meiosis. To understand KAR4's role in yeast mating and meiosis, we used a genetic screen to isolate 25 function-specific mutant alleles, which map to non-overlapping surfaces on a predicted structure of the Kar4 protein (Kar4p). Most of the mating-specific alleles (Mat-) abolish Kar4p's interaction with the transcription factor Ste12p, indicating that Kar4p's mating function is through Ste12p. In yeast, the mRNA methyltransferase complex was previously defined as comprising Ime4p (Kar4p's paralog and the homolog of mammalian METTL3), Mum2p (homolog of mammalian WTAP), and Slz1p (MIS), but not Kar4p. During meiosis, Kar4p interacts with Ime4p, Mum2p, and Slz1p. Moreover, cells lacking Kar4p have highly reduced levels of mRNA methylation during meiosis indicating that Kar4p is a key member of the methyltransferase complex, as it is in humans. Analysis of kar4Δ/Δ and 7 meiosis-specific alleles (Mei-) revealed that Kar4p is required early in meiosis, before initiation of S-phase and meiotic recombination. High copy expression of the meiotic transcriptional activator IME1 rescued the defect of these Mei- alleles. Surprisingly, Kar4p was also found to be required at a second step for the completion of meiosis and sporulation. Over-expression of IME1 in kar4Δ/Δ permits pre-meiotic S-phase, but most cells remained arrested with a monopolar spindle. Analysis of the function-specific mutants revealed that roughly half became blocked after premeiotic DNA synthesis and did not sporulate (Spo-). Loss of Kar4p's Spo function was suppressed by overexpression of RIM4, a meiotic translational regulator. Overexpression of IME1 and RIM4 together allowed sporulation of kar4Δ/Δ cells. Taken together, these data suggest that Kar4p regulates meiosis at multiple steps, presumably reflecting requirements for methylation in different stages of meiotic gene expression.

Mitochondrial integrated stress response controls lung epithelial cell fate.

Alveolar epithelial type 1 (AT1) cells are necessary to transfer oxygen and carbon dioxide between the blood and air. Alveolar epithelial type 2 (AT2) cells serve as a partially committed stem cell population, producing AT1 cells during postnatal alveolar development and repair after influenza A and SARS-CoV-2 pneumonia1-6. Little is known about the metabolic regulation of the fate of lung epithelial cells. Here we report that deleting the mitochondrial electron transport chain complex I subunit Ndufs2 in lung epithelial cells during mouse gestation led to death during postnatal alveolar development. Affected mice displayed hypertrophic cells with AT2 and AT1 cell features, known as transitional cells. Mammalian mitochondrial complex I, comprising 45 subunits, regenerates NAD+ and pumps protons. Conditional expression of yeast NADH dehydrogenase (NDI1) protein that regenerates NAD+ without proton pumping7,8 was sufficient to correct abnormal alveolar development and avert lethality. Single-cell RNA sequencing revealed enrichment of integrated stress response (ISR) genes in transitional cells. Administering an ISR inhibitor9,10 or NAD+ precursor reduced ISR gene signatures in epithelial cells and partially rescued lethality in the absence of mitochondrial complex I function. Notably, lung epithelial-specific loss of mitochondrial electron transport chain complex II subunit Sdhd, which maintains NAD+ regeneration, did not trigger high ISR activation or lethality. These findings highlight an unanticipated requirement for mitochondrial complex I-dependent NAD+ regeneration in directing cell fate during postnatal alveolar development by preventing pathological ISR induction.

Investigation of hydrated channels and proton pathways in a high-resolution cryo-EM structure of mammalian complex I.

Respiratory complex I, a key enzyme in mammalian metabolism, captures the energy released by reduction of ubiquinone by NADH to drive protons across the inner mitochondrial membrane, generating the proton-motive force for ATP synthesis. Despite remarkable advances in structural knowledge of this complicated membrane-bound enzyme, its mechanism of catalysis remains controversial. In particular, how ubiquinone reduction is coupled to proton pumping and the pathways and mechanisms of proton translocation are contested. We present a 2.4-Å resolution cryo-EM structure of complex I from mouse heart mitochondria in the closed, active (ready-to-go) resting state, with 2945 water molecules modeled. By analyzing the networks of charged and polar residues and water molecules present, we evaluate candidate pathways for proton transfer through the enzyme, for the chemical protons for ubiquinone reduction, and for the protons transported across the membrane. Last, we compare our data to the predictions of extant mechanistic models, and identify key questions to answer in future work to test them.

Decision tree classifier based on topological characteristics of subgraph for the mining of protein complexes from large scale PPI networks

The growing accessibility of large-scale protein interaction data demands extensive research to understand cell organization and its functioning at the network level. Bioinformatics and data mining researchers have extensively studied network clustering to examine the structural and operational features of protein protein interaction (PPI) networks. Clustering PPI networks has proven useful in numerous research over the past two decades for identifying functional modules, understanding the roles of previously unknown proteins, and other purposes. Protein complexes represent one of the essential cellular components for creating biological activities. Inferring protein complexes has been made more accessible by experimental approaches. We offer a novel method that integrates the classification model with local topological data, making it more reliable and efficient. This article describes a decision tree classifier based on topological characteristics of the subgraph for mining protein complexes. The proposed graph-based algorithm is an effective and efficient way to identify protein complexes from large-scale PPI networks. The performance of the proposed algorithm is observed in protein–protein interaction networks of yeast and human in the Database of Interacting Proteins (DIP) and the Biological General Repository for Interaction Datasets (BioGRID) using widely accepted benchmark protein complexes from the comprehensive resource of mammalian protein complexes (CORUM) and the comprehensive catalogue of yeast protein complexes (CYC2008). The outcomes demonstrate that our method can outperform the best-performing supervised, semi-supervised, and unsupervised approaches to detecting protein complexes.

Cryo-EM structure of the Saccharomyces cerevisiae Rpd3L histone deacetylase complex

The Rpd3L histone deacetylase (HDAC) complex is an ancient 12-subunit complex conserved in a broad range of eukaryotes that performs localized deacetylation at or near sites of recruitment by DNA-bound factors. Here we describe the cryo-EM structure of this prototypical HDAC complex that is characterized by as many as seven subunits performing scaffolding roles for the tight integration of the only catalytic subunit, Rpd3. The principal scaffolding protein, Sin3, along with Rpd3 and the histone chaperone, Ume1, are present in two copies, with each copy organized into separate lobes of an asymmetric dimeric molecular assembly. The active site of one Rpd3 is completely occluded by a leucine side chain of Rxt2, while the tips of the two lobes and the more peripherally associated subunits exhibit varying levels of flexibility and positional disorder. The structure reveals unexpected structural homology/analogy between unrelated subunits in the fungal and mammalian complexes and provides a foundation for deeper interrogations of structure, biology, and mechanism of these complexes, as well as for the discovery of HDAC complex-specific inhibitors.

Inactivation of Minar2 in mice hyperactivates mTOR signaling and results in obesity

ObjectiveObesity is a complex disorder and is linked to chronic diseases such as type 2 diabetes. Major intrinsically disordered NOTCH2-associated receptor2 (MINAR2) is an understudied protein with an unknown role in obesity and metabolism. The purpose of this study was to determine the impact of Minar2 on adipose tissues and obesity. MethodWe generated Minar2 knockout (KO) mice and used various molecular, proteomic, biochemical, histopathology, and cell culture studies to determine the pathophysiological role of Minar2 in adipocytes. ResultsWe demonstrated that the inactivation of Minar2 results in increased body fat with hypertrophic adipocytes. Minar2 KO mice on a high-fat diet develop obesity and impaired glucose tolerance and metabolism. Mechanistically, Minar2 interacts with Raptor, a specific and essential component of mammalian TOR complex 1 (mTORC1) and inhibits mTOR activation. mTOR is hyperactivated in the adipocytes deficient for Minar2 and over-expression of Minar2 in HEK-293 cells inhibited mTOR activation and phosphorylation of mTORC1 substrates, including S6 kinase, and 4E-BP1. ConclusionOur findings identified Minar2 as a novel physiological negative regulator of mTORC1 with a key role in obesity and metabolic disorders. Impaired expression or activation of MINAR2 could lead to obesity and obesity-associated diseases.

Structure of complex III with bound antimalarial agent CK-2-68 provides insights into selective inhibition of Plasmodium cytochrome bc1 complexes

Among the various components of the protozoan Plasmodium mitochondrial respiratory chain, only Complex III is a validated cellular target for antimalarial drugs. The compound CK-2-68 was developed to specifically target the alternate NADH dehydrogenase of the malaria parasite respiratory chain, but the true target for its antimalarial activity has been controversial. Here, we report the cryo-EM structure of mammalian mitochondrial Complex III bound with CK-2-68 and examine the structure-function relationships of the inhibitor's selective action on Plasmodium. We show that CK-2-68 binds specifically to the quinol oxidation site of Complex III, arresting the motion of the iron-sulfur protein subunit, which suggests an inhibition mechanism similar to that of Pf-type Complex III inhibitors such as atovaquone, stigmatellin, and UHDBT. Our results shed light on the mechanisms of observed resistance conferred by mutations, elucidate the molecular basis of the wide therapeutic window of CK-2-68 for selective action of Plasmodium vs. host cytochrome bc1, and provide guidance for future development of antimalarials targeting Complex III.

Deficiency in mammalian STN1 promotes colon cancer development via inhibiting DNA repair.

Despite the high lethality of colorectal cancers (CRCs), only a limited number of genetic risk factors are identified. The mammalian ssDNA-binding protein complex CTC1-STN1-TEN1 protects genome stability, yet its role in tumorigenesis is unknown. Here, we show that attenuated CTC1/STN1 expression is common in CRCs. We generated an inducible STN1 knockout mouse model and found that STN1 deficiency in young adult mice increased CRC incidence, tumor size, and tumor load. CRC tumors exhibited enhanced proliferation, reduced apoptosis, and elevated DNA damage and replication stress. We found that STN1 deficiency down-regulated multiple DNA glycosylases, resulting in defective base excision repair (BER) and accumulation of oxidative damage. Collectively, this study identifies STN1 deficiency as a risk factor for CRC and implicates the previously unknown STN1-BER axis in protecting colon tissues from oxidative damage, therefore providing insights into the CRC tumor-suppressing mechanism.

Molecular basis of the TRAP complex function in ER protein biogenesis

The translocon-associated protein (TRAP) complex resides in the endoplasmic reticulum (ER) membrane and interacts with the Sec translocon and the ribosome to facilitate biogenesis of secretory and membrane proteins. TRAP plays a key role in the secretion of many hormones, including insulin. Here we reveal the molecular architecture of the mammalian TRAP complex and how it engages the translating ribosome associated with Sec61 translocon on the ER membrane. The TRAP complex is anchored to the ribosome via a long tether and its position is further stabilized by a finger-like loop. This positions a cradle-like lumenal domain of TRAP below the translocon for interactions with translocated nascent chains. Our structure-guided TRAP mutations in Caenorhabditis elegans lead to growth deficits associated with increased ER stress and defects in protein hormone secretion. These findings elucidate the molecular basis of the TRAP complex in the biogenesis and translocation of proteins at the ER.

Comparative genomics of the Leukocyte Receptor Complex in carnivores.

The mammalian Leukocyte Receptor Complex (LRC) chromosomal region may contain gene families for the killer cell immunoglobulin-like receptor (KIR) and/or leukocyte immunoglobulin-like receptor (LILR) collections as well as various framing genes. This complex region is well described in humans, mice, and some domestic animals. Although single KIR genes are known in some Carnivora, their complements of LILR genes remain largely unknown due to obstacles in the assembly of regions of high homology in short-read based genomes. As part of the analysis of felid immunogenomes, this study focuses on the search for LRC genes in reference genomes and the annotation of LILR genes in Felidae. Chromosome-level genomes based on single-molecule long-read sequencing were preferentially sought and compared to representatives of the Carnivora. Seven putatively functional LILR genes were found across the Felidae and in the Californian sea lion, four to five genes in Canidae, and four to nine genes in Mustelidae. They form two lineages, as seen in the Bovidae. The ratio of functional genes for activating LILRs to inhibitory LILRs is slightly in favor of inhibitory genes in the Felidae and the Canidae; the reverse is seen in the Californian sea lion. This ratio is even in all of the Mustelidae except the Eurasian otter, which has a predominance of activating LILRs. Various numbers of LILR pseudogenes were identified. The structure of the LRC is rather conservative in felids and the other Carnivora studied. The LILR sub-region is conserved within the Felidae and has slight differences in the Canidae, but it has taken various evolutionary paths in the Mustelidae. Overall, the process of pseudogenization of LILR genes seems to be more frequent for activating receptors. Phylogenetic analysis found no direct orthologues across the Carnivora which corroborate the rapid evolution of LILRs seen in mammals.

Visualizing the disordered nuclear transport machinery in situ

The approximately 120 MDa mammalian nuclear pore complex (NPC) acts as a gatekeeper for the transport between the nucleus and cytosol1. The central channel of the NPC is filled with hundreds of intrinsically disordered proteins (IDPs) called FG-nucleoporins (FG-NUPs)2,3. Although the structure of the NPC scaffold has been resolved in remarkable detail, the actual transport machinery built up by FG-NUPs—about 50 MDa—is depicted as an approximately 60-nm hole in even highly resolved tomograms and/or structures computed with artificial intelligence4–11. Here we directly probed conformations of the vital FG-NUP98 inside NPCs in live cells and in permeabilized cells with an intact transport machinery by using a synthetic biology-enabled site-specific small-molecule labelling approach paired with highly time-resolved fluorescence microscopy. Single permeabilized cell measurements of the distance distribution of FG-NUP98 segments combined with coarse-grained molecular simulations of the NPC allowed us to map the uncharted molecular environment inside the nanosized transport channel. We determined that the channel provides—in the terminology of the Flory polymer theory12—a ‘good solvent’ environment. This enables the FG domain to adopt expanded conformations and thus control transport between the nucleus and cytoplasm. With more than 30% of the proteome being formed from IDPs, our study opens a window into resolving disorder–function relationships of IDPs in situ, which are important in various processes, such as cellular signalling, phase separation, ageing and viral entry.