ECF-Type ATP-Binding Cassette Transporters.

Energy-coupling factor (ECF)-type ATP-binding cassette (ABC) transporters catalyze membrane transport of micronutrients in prokaryotes. Crystal structures and biochemical characterization have revealed that ECF transporters are mechanistically distinct from other ABC transport systems. Notably, ECF transporters make use of small integral membrane subunits (S-components) that are predicted to topple over in the membrane when carrying the bound substrate from the extracellular side of the bilayer to the cytosol. Here, we review the phylogenetic diversity of ECF transporters as well as recent structural and biochemical advancements that have led to the postulation of conceptually different mechanistic models. These models can be described as power stroke and thermal ratchet. Structural data indicate that the lipid composition and bilayer structure are likely to have great impact on the transport function. We argue that study of ECF transporters could lead to generic insight into membrane protein structure, dynamics, and interaction.

Genetic variants of phospholipase C-γ2 alter the phenotype and function of microglia and confer differential risk for Alzheimer's disease.

Genetic association studies have demonstrated the critical involvement of the microglial immune response in Alzheimer's disease (AD) pathogenesis. Phospholipase C-gamma-2 (PLCG2) is selectively expressed by microglia and functions in many immune receptor signaling pathways. In AD, PLCG2 is induced uniquely in plaque-associated microglia. A genetic variant of PLCG2, PLCG2P522R, is a mild hypermorph that attenuates AD risk. Here, we identified a loss-of-function PLCG2 variant, PLCG2M28L, that confers an increased AD risk. PLCG2P522R attenuated disease in an amyloidogenic murine AD model, whereas PLCG2M28L exacerbated the plaque burden associated with altered phagocytosis and Aß clearance. The variants bidirectionally modulated disease pathology by inducing distinct transcriptional programs that identified microglial subpopulations associated with protective or detrimental phenotypes. These findings identify PLCG2M28L as a potential AD risk variant and demonstrate that PLCG2 variants can differentially orchestrate microglial responses in AD pathogenesis that can be therapeutically targeted.

Pathways for macrophage uptake of cell-free circular RNAs.

Circular RNAs (circRNAs) are stable RNAs present in cell-free RNA, which may comprise cellular debris and pathogen genomes. Here, we investigate the phenomenon and mechanism of cellular uptake and intracellular fate of exogenous circRNAs. Human myeloid cells and B cells selectively internalize extracellular circRNAs. Macrophage uptake of circRNA is rapid, energy dependent, and saturable. CircRNA uptake can lead to translation of encoded sequences and antigen presentation. The route of internalization influences immune activation after circRNA uptake, with distinct gene expression programs depending on the route of RNA delivery. Genome-scale CRISPR screens and chemical inhibitor studies nominate macrophage scavenger receptor MSR1, Toll-like receptors, and mTOR signaling as key regulators of receptor-mediated phagocytosis of circRNAs, a dominant pathway to internalize circRNAs in parallel to macropinocytosis. These results suggest that cell-free circRNA serves as an "eat me" signal and danger-associated molecular pattern, indicating orderly pathways of recognition and disposal.

ERMA (TMEM94) is a P-type ATPase transporter for Mg2+ uptake in the endoplasmic reticulum.

Intracellular Mg2+ (iMg2+) is bound with phosphometabolites, nucleic acids, and proteins in eukaryotes. Little is known about the intracellular compartmentalization and molecular details of Mg2+ transport into/from cellular organelles such as the endoplasmic reticulum (ER). We found that the ER is a major iMg2+ compartment refilled by a largely uncharacterized ER-localized protein, TMEM94. Conventional and AlphaFold2 predictions suggest that ERMA (TMEM94) is a multi-pass transmembrane protein with large cytosolic headpiece actuator, nucleotide, and phosphorylation domains, analogous to P-type ATPases. However, ERMA uniquely combines a P-type ATPase domain and a GMN motif for ERMg2+ uptake. Experiments reveal that a tyrosine residue is crucial for Mg2+ binding and activity in a mechanism conserved in both prokaryotic (mgtB and mgtA) and eukaryotic Mg2+ ATPases. Cardiac dysfunction by haploinsufficiency, abnormal Ca2+ cycling in mouse Erma+/- cardiomyocytes, and ERMA mRNA silencing in human iPSC-cardiomyocytes collectively define ERMA as an essential component of ERMg2+ uptake in eukaryotes.

CD36 as a gatekeeper of myocardial lipid metabolism and therapeutic target for metabolic disease.

The multifunctional membrane glycoprotein CD36 is expressed in different types of cells and plays a key regulatory role in cellular lipid metabolism, especially in cardiac muscle. CD36 facilitates the cellular uptake of long-chain fatty acids, mediates lipid signaling, and regulates storage and oxidation of lipids in various tissues with active lipid metabolism. CD36 deficiency leads to marked impairments in peripheral lipid metabolism, which consequently impact on the cellular utilization of multiple different fuels because of the integrated nature of metabolism. The functional presence of CD36 at the plasma membrane is regulated by its reversible subcellular recycling from and to endosomes and is under the control of mechanical, hormonal, and nutritional factors. Aberrations in this dynamic role of CD36 are causally associated with various metabolic diseases, in particular insulin resistance, diabetic cardiomyopathy, and cardiac hypertrophy. Recent research in cardiac muscle has disclosed the endosomal proton pump vacuolar-type H+-ATPase (v-ATPase) as a key enzyme regulating subcellular CD36 recycling and being the site of interaction between various substrates to determine cellular substrate preference. In addition, evidence is accumulating that interventions targeting CD36 directly or modulating its subcellular recycling are effective for the treatment of metabolic diseases. In conclusion, subcellular CD36 localization is the major adaptive regulator of cellular uptake and metabolism of long-chain fatty acids and appears a suitable target for metabolic modulation therapy to mend failing hearts.

Astrocytic Insulin Signaling Couples Brain Glucose Uptake with Nutrient Availability.

We report that astrocytic insulin signaling co-regulates hypothalamic glucose sensing and systemic glucose metabolism. Postnatal ablation of insulin receptors (IRs) in glial fibrillary acidic protein (GFAP)-expressing cells affects hypothalamic astrocyte morphology, mitochondrial function, and circuit connectivity. Accordingly, astrocytic IR ablation reduces glucose-induced activation of hypothalamic pro-opio-melanocortin (POMC) neurons and impairs physiological responses to changes in glucose availability. Hypothalamus-specific knockout of astrocytic IRs, as well as postnatal ablation by targeting glutamate aspartate transporter (GLAST)-expressing cells, replicates such alterations. A normal response to altering directly CNS glucose levels in mice lacking astrocytic IRs indicates a role in glucose transport across the blood-brain barrier (BBB). This was confirmed in vivo in GFAP-IR KO mice by using positron emission tomography and glucose monitoring in cerebral spinal fluid. We conclude that insulin signaling in hypothalamic astrocytes co-controls CNS glucose sensing and systemic glucose metabolism via regulation of glucose uptake across the BBB.

Haploinsufficiency underlies the neurodevelopmental consequences of SLC6A1 variants.

Heterozygous variants in SLC6A1, encoding the GAT-1 GABA transporter, are associated with seizures, developmental delay, and autism. The majority of affected individuals carry missense variants, many of which are recurrent germline de novo mutations, raising the possibility of gain-of-function or dominant-negative effects. To understand the functional consequences, we performed an in vitro GABA uptake assay for 213 unique variants, including 24 control variants. De novo variants consistently resulted in a decrease in GABA uptake, in keeping with haploinsufficiency underlying all neurodevelopmental phenotypes. Where present, ClinVar pathogenicity reports correlated well with GABA uptake data; the functional data can inform future reports for the remaining 72% of unscored variants. Surface localization was assessed for 86 variants; two-thirds of loss-of-function missense variants prevented GAT-1 from being present on the membrane while GAT-1 was on the surface but with reduced activity for the remaining third. Surprisingly, recurrent de novo missense variants showed moderate loss-of-function effects that reduced GABA uptake with no evidence for dominant-negative or gain-of-function effects. Using linear regression across multiple missense severity scores to extrapolate the functional data to all potential SLC6A1 missense variants, we observe an abundance of GAT-1 residues that are sensitive to substitution. The extent of this missense vulnerability accounts for the clinically observed missense enrichment; overlap with hypermutable CpG sites accounts for the recurrent missense variants. Strategies to increase the expression of the wild-type SLC6A1 allele are likely to be beneficial across neurodevelopmental disorders, though the developmental stage and extent of required rescue remain unknown.

De novo variants in DENND5B cause a neurodevelopmental disorder.

The Rab family of guanosine triphosphatases (GTPases) includes key regulators of intracellular transport and membrane trafficking targeting specific steps in exocytic, endocytic, and recycling pathways. DENND5B (Rab6-interacting Protein 1B-like protein, R6IP1B) is the longest isoform of DENND5, an evolutionarily conserved DENN domain-containing guanine nucleotide exchange factor (GEF) that is highly expressed in the brain. Through exome sequencing and international matchmaking platforms, we identified five de novo variants in DENND5B in a cohort of five unrelated individuals with neurodevelopmental phenotypes featuring cognitive impairment, dysmorphism, abnormal behavior, variable epilepsy, white matter abnormalities, and cortical gyration defects. We used biochemical assays and confocal microscopy to assess the impact of DENND5B variants on protein accumulation and distribution. Then, exploiting fluorescent lipid cargoes coupled to high-content imaging and analysis in living cells, we investigated whether DENND5B variants affected the dynamics of vesicle-mediated intracellular transport of specific cargoes. We further generated an in silico model to investigate the consequences of DENND5B variants on the DENND5B-RAB39A interaction. Biochemical analysis showed decreased protein levels of DENND5B mutants in various cell types. Functional investigation of DENND5B variants revealed defective intracellular vesicle trafficking, with significant impairment of lipid uptake and distribution. Although none of the variants affected the DENND5B-RAB39A interface, all were predicted to disrupt protein folding. Overall, our findings indicate that DENND5B variants perturb intracellular membrane trafficking pathways and cause a complex neurodevelopmental syndrome with variable epilepsy and white matter involvement.

Mechanisms of DNA Uptake by Naturally Competent Bacteria.

Transformation is a widespread mechanism of horizontal gene transfer in bacteria. DNA uptake to the periplasmic compartment requires a DNA-uptake pilus and the DNA-binding protein ComEA. In the gram-negative bacteria, DNA is first pulled toward the outer membrane by retraction of the pilus and then taken up by binding to periplasmic ComEA, acting as a Brownian ratchet to prevent backward diffusion. A similar mechanism probably operates in the gram-positive bacteria as well, but these systems have been less well characterized. Transport, defined as movement of a single strand of transforming DNA to the cytosol, requires the channel protein ComEC. Although less is understood about this process, it may be driven by proton symport. In this review we also describe various phenomena that are coordinated with the expression of competence for transformation, such as fratricide, the kin-discriminatory killing of neighboring cells, and competence-mediated growth arrest.

The evolving biology of cross-presentation.

Cross-priming was first recognized in the context of in vivo cytotoxic T lymphocyte (CTL) responses generated against minor histocompatibility antigens induced by immunization with lymphoid cells. Even though the basis for T cell antigen recognition was still largely unclear at that time, these early studies recognized the implication that such minor histocompatibility antigens were derived from the immunizing cells and were obtained exogenously by the host's antigen presenting cells (APCs) that directly prime the CTL response. As antigen recognition by the T cell receptor became understood to involve peptides derived from antigens processed by the APCs and presented by major histocompatibility molecules, the "cross-priming" phenomenon was subsequently recast as "cross-presentation" and the scope considered for examining this process gradually broadened to include many different forms of antigens, including soluble proteins, and different types of APCs that may not be involved in in vivo CTL priming. Many studies of cross-presentation have relied on in vitro cell models that were recently found to differ from in vivo APCs in particular mechanistic details. A recent trend has focused on the APCs and pathways of cross-presentation used in vivo, especially the type 1 dendritic cells. Current efforts are also being directed towards validating the in vivo role of various putative pathways and gene candidates in cross-presentation garnered from various in vitro studies and to determine the relative contributions they make to CTL responses across various forms of antigens and immunologic settings. Thus, cross-presentation appears to be carried by different pathways in various types of cells for different forms under different physiologic settings, which remain to be evaluated in an in vivo physiologic setting.

Mortality risk information and health-seeking behavior during an epidemic.

In a context where pessimistic survival perceptions have been widespread as a result of the HIV/AIDS epidemic (Fig. 1 A), we study vaccine uptake and other health behaviors during the recent COVID-19 pandemic. Leveraging a longitudinal cohort study in rural Malawi that has been followed for up to 25 y, we document that a 2017 mortality risk information intervention designed to reduce pessimistic mortality perceptions (Fig. 1 B) resulted in improved health behavior, including COVID-19 vaccine uptake (Fig. 1 C). We also report indirect effects for siblings and household members. This was likely the result of a reinforcing process where the intervention triggered engagement with the healthcare system and stronger beliefs in the efficacy of modern biomedical treatments, which led to the adoption of health risk reduction behavior, including vaccine uptake. Our findings suggest that health information interventions focused on survival perceptions can be useful in promoting health behavior and participation in the formal healthcare system, even during health crises-such as the COVID-19 pandemic-that are unanticipated at the time of the intervention. We also note the importance of the intervention design, where establishing rapport, tailoring the content to the local context, and spending time with respondents to convey the information contributed to the salience of the message.

Conformational changes in the Niemann-Pick type C1 protein NCR1 drive sterol translocation.

The membrane protein Niemann-Pick type C1 (NPC1, named NCR1 in yeast) is central to sterol homeostasis in eukaryotes. Saccharomyces cerevisiae NCR1 is localized to the vacuolar membrane, where it is suggested to carry sterols across the protective glycocalyx and deposit them into the vacuolar membrane. However, documentation of a vacuolar glycocalyx in fungi is lacking, and the mechanism for sterol translocation has remained unclear. Here, we provide evidence supporting the presence of a glycocalyx in isolated S. cerevisiae vacuoles and report four cryo-EM structures of NCR1 in two distinct conformations, named tense and relaxed. These two conformations illustrate the movement of sterols through a tunnel formed by the luminal domains, thus bypassing the barrier presented by the glycocalyx. Based on these structures and on comparison with other members of the Resistance-Nodulation-Division (RND) superfamily, we propose a transport model that links changes in the luminal domains with a cycle of protonation and deprotonation within the transmembrane region of the protein. Our model suggests that NPC proteins work by a generalized RND mechanism where the proton motive force drives conformational changes in the transmembrane domains that are allosterically coupled to luminal/extracellular domains to promote sterol transport.

MICU1 Interacts with the D-Ring of the MCU Pore to Control Its Ca2+ Flux and Sensitivity to Ru360.

Proper control of the mitochondrial Ca2+ uniporter's pore (MCU) is required to allow Ca2+-dependent activation of oxidative metabolism and to avoid mitochondrial Ca2+ overload and cell death. The MCU's gatekeeping and cooperative activation is mediated by the Ca2+-sensing MICU1 protein, which has been proposed to form dimeric complexes anchored to the EMRE scaffold of MCU. We unexpectedly find that MICU1 suppresses inhibition of MCU by ruthenium red/Ru360, which bind to MCU's DIME motif, the selectivity filter. This led us to recognize in MICU1's sequence a putative DIME interacting domain (DID), which is required for both gatekeeping and cooperative activation of MCU and for cell survival. Thus, we propose that MICU1 has to interact with the D-ring formed by the DIME domains in MCU to control the uniporter.

The magnitude and pace of photosynthetic recovery after wildfire in California ecosystems.

Wildfire modifies the short- and long-term exchange of carbon between terrestrial ecosystems and the atmosphere, with impacts on ecosystem services such as carbon uptake. Dry western US forests historically experienced low-intensity, frequent fires, with patches across the landscape occupying different points in the fire-recovery trajectory. Contemporary perturbations, such as recent severe fires in California, could shift the historic stand-age distribution and impact the legacy of carbon uptake on the landscape. Here, we combine flux measurements of gross primary production (GPP) and chronosequence analysis using satellite remote sensing to investigate how the last century of fires in California impacted the dynamics of ecosystem carbon uptake on the fire-affected landscape. A GPP recovery trajectory curve of more than five thousand fires in forest ecosystems since 1919 indicated that fire reduced GPP by [Formula: see text] g C m[Formula: see text] y[Formula: see text]([Formula: see text]) in the first year after fire, with average recovery to prefire conditions after [Formula: see text] y. The largest fires in forested ecosystems reduced GPP by [Formula: see text] g C m[Formula: see text] y[Formula: see text] (n = 401) and took more than two decades to recover. Recent increases in fire severity and recovery time have led to nearly [Formula: see text] MMT CO[Formula: see text] (3-y rolling mean) in cumulative forgone carbon uptake due to the legacy of fires on the landscape, complicating the challenge of maintaining California's natural and working lands as a net carbon sink. Understanding these changes is paramount to weighing the costs and benefits associated with fuels management and ecosystem management for climate change mitigation.

Cooperation and cheating orchestrate Vibrio assemblages and polymicrobial synergy in oysters infected with OsHV-1 virus.

Polymicrobial infections threaten the health of humans and animals but remain understudied in natural systems. We recently described the Pacific Oyster Mortality Syndrome (POMS), a polymicrobial disease affecting oyster production worldwide. In the French Atlantic coast, the disease involves coinfection with ostreid herpesvirus 1 (OsHV-1) and virulent Vibrio. However, it is unknown whether consistent Vibrio populations are associated with POMS in different regions, how Vibrio contribute to POMS, and how they interact with OsHV-1 during pathogenesis. By connecting field-based approaches in a Mediterranean ecosystem, laboratory infection assays and functional genomics, we uncovered a web of interdependencies that shape the structure and function of the POMS pathobiota. We show that Vibrio harveyi and Vibrio rotiferianus are predominant in OsHV-1-diseased oysters and that OsHV-1 drives the partition of the Vibrio community observed in the field. However only V. harveyi synergizes with OsHV-1 by promoting mutual growth and accelerating oyster death. V. harveyi shows high-virulence potential and dampens oyster cellular defenses through a type 3 secretion system, making oysters a more favorable niche for microbe colonization. In addition, V. harveyi produces a key siderophore called vibrioferrin. This important resource promotes the growth of V. rotiferianus, which cooccurs with V. harveyi in diseased oysters, and behaves as a cheater by benefiting from V. harveyi metabolite sharing. Our data show that cooperative behaviors contribute to synergy between bacterial and viral coinfecting partners. Additional cheating behaviors further shape the polymicrobial consortium. Controlling cooperative behaviors or countering their effects opens avenues for mitigating polymicrobial diseases.

Interactions of TonB-dependent transporter FoxA with siderophores and antibiotics that affect binding, uptake, and signal transduction.

The outer membrane of gram-negative bacteria prevents many antibiotics from reaching intracellular targets. However, some antimicrobials can take advantage of iron import transporters to cross this barrier. We showed previously that the thiopeptide antibiotic thiocillin exploits the nocardamine xenosiderophore transporter, FoxA, of the opportunistic pathogen Pseudomonas aeruginosa for uptake. Here, we show that FoxA also transports the xenosiderophore bisucaberin and describe at 2.5 Å resolution the crystal structure of bisucaberin bound to FoxA. Bisucaberin is distinct from other siderophores because it forms a 3:2 rather than 1:1 siderophore-iron complex. Mutations in a single extracellular loop of FoxA differentially affected nocardamine, thiocillin, and bisucaberin binding, uptake, and signal transduction. These results show that in addition to modulating ligand binding, the extracellular loops of siderophore transporters are of fundamental importance for controlling ligand uptake and its regulatory consequences, which have implications for the development of siderophore-antibiotic conjugates to treat difficult infections.

The Rho guanine dissociation inhibitor α inhibits skeletal muscle Rac1 activity and insulin action.

The molecular events governing skeletal muscle glucose uptake have pharmacological potential for managing insulin resistance in conditions such as obesity, diabetes, and cancer. With no current pharmacological treatments to target skeletal muscle insulin sensitivity, there is an unmet need to identify the molecular mechanisms that control insulin sensitivity in skeletal muscle. Here, the Rho guanine dissociation inhibitor α (RhoGDIα) is identified as a point of control in the regulation of insulin sensitivity. In skeletal muscle cells, RhoGDIα interacted with, and thereby inhibited, the Rho GTPase Rac1. In response to insulin, RhoGDIα was phosphorylated at S101 and Rac1 dissociated from RhoGDIα to facilitate skeletal muscle GLUT4 translocation. Accordingly, siRNA-mediated RhoGDIα depletion increased Rac1 activity and elevated GLUT4 translocation. Consistent with RhoGDIα's inhibitory effect, rAAV-mediated RhoGDIα overexpression in mouse muscle decreased insulin-stimulated glucose uptake and was detrimental to whole-body glucose tolerance. Aligning with RhoGDIα's negative role in insulin sensitivity, RhoGDIα protein content was elevated in skeletal muscle from insulin-resistant patients with type 2 diabetes. These data identify RhoGDIα as a clinically relevant controller of skeletal muscle insulin sensitivity and whole-body glucose homeostasis, mechanistically by modulating Rac1 activity.

Magnetite nanoparticle coating chemistry regulates root uptake pathways and iron chlorosis in plants.

Understanding the fundamental interaction of nanoparticles at plant interfaces is critical for reaching field-scale applications of nanotechnology-enabled plant agriculture, as the processes between nanoparticles and root interfaces such as root compartments and root exudates remain largely unclear. Here, using iron deficiency-induced plant chlorosis as an indicator phenotype, we evaluated the iron transport capacity of Fe3O4 nanoparticles coated with citrate (CA) or polyacrylic acid (PAA) in the plant rhizosphere. Both nanoparticles can be used as a regulator of plant hormones to promote root elongation, but they regulate iron deficiency in plant in distinctive ways. In acidic root exudates secreted by iron-deficient Arabidopsis thaliana, CA-coated particles released fivefold more soluble iron by binding to acidic exudates mainly through hydrogen bonds and van der Waals forces and thus, prevented iron chlorosis more effectively than PAA-coated particles. We demonstrate through roots of mutants and visualization of pH changes that acidification of root exudates primarily originates from root tips and the synergistic mode of nanoparticle uptake and transformation in different root compartments. The nanoparticles entered the roots mainly through the epidermis but were not affected by lateral roots or root hairs. Our results show that magnetic nanoparticles can be a sustainable source of iron for preventing leaf chlorosis and that nanoparticle surface coating regulates this process in distinctive ways. This information also serves as an urgently needed theoretical basis for guiding the application of nanomaterials in agriculture.

AMPK and the Adaptation to Exercise.

Noncommunicable diseases are chronic diseases that contribute to death worldwide, but these diseases can be prevented and mitigated with regular exercise. Exercise activates signaling molecules and the transcriptional network to promote physiological adaptations, such as fiber type transformation, angiogenesis, and mitochondrial biogenesis. AMP-activated protein kinase (AMPK) is a master regulator that senses the energy state, promotes metabolism for glucose and fatty acid utilization, and mediates beneficial cellular adaptations in many vital tissues and organs. This review focuses on the current, integrative understanding of the role of exercise-induced activation of AMPK in the regulation of system metabolism and promotion of health benefits.

Iron-sulfur cluster assembly scaffold protein IscU is required for activation of ferric uptake regulator (Fur) in Escherichiacoli.

It was generally postulated that when intracellular free iron content is elevated in bacteria, the ferric uptake regulator (Fur) binds its corepressor a mononuclear ferrous iron to regulate intracellular iron homeostasis. However, the proposed iron-bound Fur had not been identified in any bacteria. In previous studies, we have demonstrated that Escherichia coli Fur binds a [2Fe-2S] cluster in response to elevation of intracellular free iron content and that binding of the [2Fe-2S] cluster turns on Fur as an active repressor to bind a specific DNA sequence known as the Fur-box. Here we find that the iron-sulfur cluster assembly scaffold protein IscU is required for the [2Fe-2S] cluster assembly in Fur, as deletion of IscU inhibits the [2Fe-2S] cluster assembly in Fur and prevents activation of Fur as a repressor in E. coli cells in response to elevation of intracellular free iron content. Additional studies reveal that IscU promotes the [2Fe-2S] cluster assembly in apo-form Fur and restores its Fur-box binding activity in vitro. While IscU is also required for the [2Fe-2S] cluster assembly in the Haemophilus influenzae Fur in E. coli cells, deletion of IscU does not significantly affect the [2Fe-2S] cluster assembly in the E. coli ferredoxin and siderophore-reductase FhuF. Our results suggest that IscU may have a unique role for the [2Fe-2S] cluster assembly in Fur and that regulation of intracellular iron homeostasis is closely coupled with iron-sulfur cluster biogenesis in E. coli.