High-Resolution Structural Proteomics of Mitochondria Using the 'Build and Retrieve' Methodology.

The application of integrated systems biology to the field of structural biology is a promising new direction, although it is still in the infant stages of development. Here we report the use of single particle cryo-EM to identify multiple proteins from three enriched heterogeneous fractions prepared from human liver mitochondrial lysate. We simultaneously identify and solve high-resolution structures of nine essential mitochondrial enzymes with key metabolic functions, including fatty acid catabolism, reactive oxidative species clearance, and amino acid metabolism. Our methodology also identified multiple distinct members of the acyl-CoA dehydrogenase family. This work highlights the potential of cryo-EM to explore tissue proteomics at the atomic level.

A cryo-electron microscopic approach to elucidate protein structures from human brain microsomes.

We recently developed a "Build and Retrieve" cryo-electron microscopy (cryo-EM) methodology, which is capable of simultaneously producing near-atomic resolution cryo-EM maps for several individual proteins from a heterogeneous, multiprotein sample. Here we report the use of "Build and Retrieve" to define the composition of a raw human brain microsomal lysate. From this sample, we simultaneously identify and solve cryo-EM structures of five different brain enzymes whose functions affect neurotransmitter recycling, iron metabolism, glycolysis, axonal development, energy homeostasis, and retinoic acid biosynthesis. Interestingly, malfunction of these important proteins has been directly linked to several neurodegenerative disorders, such as Alzheimer's, Huntington's, and Parkinson's diseases. Our work underscores the importance of cryo-EM in facilitating tissue and organ proteomics at the atomic level.

Principles of operation of a cerebellar learning circuit.

We provide behavioral evidence using monkey smooth pursuit eye movements for four principles of cerebellar learning. Using a circuit-level model of the cerebellum, we link behavioral data to learning's neural implementation. The four principles are: (1) early, fast, acquisition driven by climbing fiber inputs to the cerebellar cortex, with poor retention; (2) learned responses of Purkinje cells guide transfer of learning from the cerebellar cortex to the deep cerebellar nucleus, with excellent retention; (3) functionally different neural signals are subject to learning in the cerebellar cortex versus the deep cerebellar nuclei; and (4) negative feedback from the cerebellum to the inferior olive reduces the magnitude of the teaching signal in climbing fibers and limits learning. Our circuit-level model, based on these four principles, explains behavioral data obtained by strategically manipulating the signals responsible for acquisition and recall of direction learning in smooth pursuit eye movements across multiple timescales.

The human brain can do many things, from reading and remembering the words written on a page to adapting and improving movements. When a movement misses its goal, the strength of the connections between cells in a part of the brain known as the cerebellum changes. The cerebellum is important for coordinating movements, including eye movements. When the connections between the cells in the cerebellum ­ known as neurons ­ strengthen or weaken, the cerebellum changes how it will respond in the future, leading to more accurate movements. However, the speed of the changes in the connections and how the connections between different neurons evolve and coordinate were unknown. Herzfeld et al. have now combined eye-tracking studies in monkeys with computer modeling based on what is known about the neural circuits in the cerebellum to learn more about the changes in these connections. Monkeys watched a moving target that would abruptly change direction. In the next movement, the eye-tracking equipment monitored how well the monkey's eyes anticipated the unexpected change in the target's direction ­ a form of motor learning. Using the experimental data, Herzfeld et al. produced a model that outlines general principles of how the cerebellum might manage this process. The model suggested that neurons in one region in the cerebellum, known as Purkinje cells, learn from mistakes quickly, but have poor long-term retention. If the movement is repeated, Purkinje cells teach another area of the cerebellum, the cerebellar nucleus, which takes longer to learn but has much better retention. Although these findings are based on a simple motor learning task, they are the first step to understanding how the brain forms memories and how we might learn more complex behaviors.

The newly-arisen Devil facial tumour disease 2 (DFT2) reveals a mechanism for the emergence of a contagious cancer.

Devil Facial Tumour 2 (DFT2) is a recently discovered contagious cancer circulating in the Tasmanian devil (Sarcophilus harrisii), a species which already harbours a more widespread contagious cancer, Devil Facial Tumour 1 (DFT1). Here we show that in contrast to DFT1, DFT2 cells express major histocompatibility complex (MHC) class I molecules, demonstrating that loss of MHC is not necessary for the emergence of a contagious cancer. However, the most highly expressed MHC class I alleles in DFT2 cells are common among host devils or non-polymorphic, reducing immunogenicity in a population sharing these alleles. In parallel, MHC class I loss is emerging in vivo, thus DFT2 may be mimicking the evolutionary trajectory of DFT1. Based on these results we propose that contagious cancers may exploit partial histocompatibility between the tumour and host, but that loss of allogeneic antigens could facilitate widespread transmission of DFT2.

The effects of 5-hydroxytryptophan on attention and central serotonin neurochemistry in the rhesus macaque.

Psychiatric disorders, particularly depression and anxiety, are often associated with impaired serotonergic function. However, serotonergic interventions yield inconsistent effects on behavioral impairments. To better understand serotonin's role in these pathologies, we investigated the role of serotonin in a behavior frequently impaired in depression and anxiety, attention. In this study, we used a quantitative, repeated, within-subject, design to test how L-5-hydroxytryptophan (5-HTP), the immediate serotonin precursor, modulates central serotoninergic function and attention in macaques. We observed that intramuscular 5-HTP administration increased cisternal cerebrospinal fluid (CSF) 5-HTP and serotonin. In addition, individuals' baseline looking duration, during saline sessions, predicted the direction and magnitude in which 5-HTP modulated attention. We found that 5-HTP decreased looking duration in animals with high baseline attention, but increased looking duration in low baseline attention animals. Furthermore, individual differences in 5-HTP's effects were also reflected in how engaged individuals were in the task and how they allocated attention to salient facial features-the eyes and mouth-of stimulus animals. However, 5-HTP constricted pupil size in all animals, suggesting that the bi-directional effects of 5-HTP cannot be explained by serotonin-mediated changes in autonomic arousal. Critically, high and low baseline attention animals exhibited different baseline CSF concentrations of 5-HTP and serotonin, an index of extracellular functionally active serotonin. Thus, our results suggest that baseline central serotonergic functioning may underlie and predict variation in serotonin's effects on cognitive operation. Our findings may help inform serotonin's role in psychopathology and help clinicians predict how serotonergic interventions will influence pathologies.

Oxytocin under opioid antagonism leads to supralinear enhancement of social attention.

To provide new preclinical evidence toward improving the efficacy of oxytocin (OT) in treating social dysfunction, we tested the benefit of administering OT under simultaneously induced opioid antagonism during dyadic gaze interactions in monkeys. OT coadministered with a µ-opioid receptor antagonist, naloxone, invoked a supralinear enhancement of prolonged and selective social attention, producing a stronger effect than the summed effects of each administered separately. These effects were consistently observed when averaging over entire sessions, as well as specifically following events of particular social importance, including mutual eye contact and mutual reward receipt. Furthermore, attention to various facial regions was differentially modulated depending on social context. Using the Allen Institute's transcriptional atlas, we further established the colocalization of µ-opioid and κ-opioid receptor genes and OT genes at the OT-releasing sites in the human brain. These data across monkeys and humans support a regulatory relationship between the OT and opioid systems and suggest that administering OT under opioid antagonism may boost the therapeutic efficacy of OT for enhancing social cognition.

Crystal structure of the transcriptional regulator Rv0678 of Mycobacterium tuberculosis.

Recent work demonstrates that the MmpL (mycobacterial membrane protein large) transporters are dedicated to the export of mycobacterial lipids for cell wall biosynthesis. An MmpL transporter frequently works with an accessory protein, belonging to the MmpS (mycobacterial membrane protein small) family, to transport these key virulence factors. One such efflux system in Mycobacterium tuberculosis is the MmpS5-MmpL5 transporter. The expression of MmpS5-MmpL5 is controlled by the MarR-like transcriptional regulator Rv0678, whose open reading frame is located downstream of the mmpS5-mmpL5 operon. To elucidate the structural basis of Rv0678 regulation, we have determined the crystal structure of this regulator, to 1.64 Å resolution, revealing a dimeric two-domain molecule with an architecture similar to members of the MarR family of transcriptional regulators. Rv0678 is distinct from other MarR regulators in that its DNA-binding and dimerization domains are clustered together. These two domains seemingly cooperate to bind an inducing ligand that we identified as 2-stearoylglycerol, which is a fatty acid glycerol ester. The structure also suggests that the conformational change leading to substrate-mediated derepression is primarily caused by a rigid body rotational motion of the entire DNA-binding domain of the regulator toward the dimerization domain. This movement results in a conformational state that is incompatible with DNA binding. We demonstrate using electrophoretic mobility shift assays that Rv0678 binds to the mmpS5-mmpL5, mmpS4-mmpL4, and the mmpS2-mmpL2 promoters. Binding by Rv0678 was reversed upon the addition of the ligand. These findings provide new insight into the mechanisms of gene regulation in the MarR family of regulators.

Crystal structure of the transcriptional regulator Rv1219c of Mycobacterium tuberculosis.

The Rv1217c-Rv1218c multidrug efflux system, which belongs to the ATP-binding cassette superfamily, recognizes and actively extrudes a variety of structurally unrelated toxic chemicals and mediates the intrinsic resistance to these antimicrobials in Mycobacterium tuberculosis. The expression of Rv1217c-Rv1218c is controlled by the TetR-like transcriptional regulator Rv1219c, which is encoded by a gene immediately upstream of rv1218c. To elucidate the structural basis of Rv1219c regulation, we have determined the crystal structure of Rv1219c, which reveals a dimeric two-domain molecule with an entirely helical architecture similar to members of the TetR family of transcriptional regulators. The N-terminal domains of the Rv1219c dimer are separated by a large center-to-center distance of 64 Å. The C-terminal domain of each protomer possesses a large cavity. Docking of small compounds to Rv1219c suggests that this large cavity forms a multidrug binding pocket, which can accommodate a variety of structurally unrelated antimicrobial agents. The internal wall of the multidrug binding site is surrounded by seven aromatic residues, indicating that drug binding may be governed by aromatic stacking interactions. In addition, fluorescence polarization reveals that Rv1219c binds drugs in the micromolar range.