Jennifer L. Garrison

Cotranslocational Degradation Protects the Stressed Endoplasmic Reticulum from Protein Overload

Summary The ERs capacity to process proteins is limited, and stress caused by accumulation of unfolded and misfolded proteins (ER stress) contributes to human disease. ER stress elicits the unfolded protein response (UPR), whose components attenuate protein synthesis, increase folding capacity, and enhance misfolded protein degradation. Here, we report that P58 IPK /DNAJC3 , a UPR-responsive gene previously implicated in translational control, encodes a cytosolic cochaperone that associates with the ER protein translocation channel Sec61. P58 IPK recruits HSP70 chaperones to the cytosolic face of Sec61 and can be crosslinked to proteins entering the ER that are delayed at the translocon. Proteasome-mediated cytosolic degradation of translocating proteins delayed at Sec61 is cochaperone dependent. In P58 IPK−/− mice, cells with a high secretory burden are markedly compromised in their ability to cope with ER stress. Thus, P58 IPK is a key mediator of cotranslocational ER protein degradation, and this process likely contributes to ER homeostasis in stressed cells.

Substrate-specific translocational attenuation during ER stress defines a pre-emptive quality control pathway.

Eukaryotic proteins entering the secretory pathway are translocated into the ER by signal sequences that vary widely in primary structure. We now provide a functional rationale for this long-observed sequence diversity by demonstrating that differences among signals facilitate substrate-selective modulation of protein translocation. We find that during acute ER stress, translocation of secretory and membrane proteins is rapidly and transiently attenuated in a signal sequence-selective manner. Their cotranslational rerouting to the cytosol for degradation reduces the burden of misfolded substrates entering the ER and represents a pathway for pre-emptive quality control (pQC). Bypassing the pQC pathway for the prion protein increases its rate of aggregation in the ER lumen during prolonged stress and renders cells less capable of viable recovery. Conversely, pharmacologically augmenting pQC during ER stress proved protective. Thus, protein translocation is a physiologically regulated process that is utilized for pQC as part of the ER stress response.

Oxytocin/Vasopressin-Related Peptides Have an Ancient Role in Reproductive Behavior

Social Neuropeptides in Nematodes The neuropeptides oxytocin and vasopressin stimulate maternal, reproductive, aggressive, and affiliative behaviors in mammals. They are implicated in behaviors ranging from ewe-lamb bonding in sheep to pair bonding in voles (see the Perspective by Emmons). Now, Garrison et al. (p. 540) and Beets et al. (p. 543) extend the evolutionary reach of these social neuropeptides to the invertebrate nematode worm, Caenorhabditis elegans. A similar neuropeptide was found to function in mating and also to modulate salt-taste preference, based on prior experience, suggesting an ancient role in associative learning. Oxytocin/vasopressin-related peptides and their receptors coordinate male mating programs in Caenorhabditis elegans. Many biological functions are conserved, but the extent to which conservation applies to integrative behaviors is unknown. Vasopressin and oxytocin neuropeptides are strongly implicated in mammalian reproductive and social behaviors, yet rodent loss-of-function mutants have relatively subtle behavioral defects. Here we identify an oxytocin/vasopressin-like signaling system in Caenorhabditis elegans, consisting of a peptide and two receptors that are expressed in sexually dimorphic patterns. Males lacking the peptide or its receptors perform poorly in reproductive behaviors, including mate search, mate recognition, and mating, but other sensorimotor behaviors are intact. Quantitative analysis indicates that mating motor patterns are fragmented and inefficient in mutants, suggesting that oxytocin/vasopressin peptides increase the coherence of mating behaviors. These results indicate that conserved molecules coordinate diverse behavioral motifs in reproductive behavior.

Selection of cell binding and internalizing epidermal growth factor receptor antibodies from a phage display library.

The first step in developing a targeted cancer therapeutic is generating a ligand that binds to a receptor which is either tumor specific or sufficiently overexpressed in tumors to provide targeting specificity. For this work, we generated human monoclonal antibodies to the EGF receptor (EGFR), an antigen overexpressed on many solid tumors. Single chain Fv (scFv) antibody fragments were directly selected by panning a phage display library on tumor cells (A431) overexpressing EGFR or Chinese hamster ovary cells (CHO/EGFR cells) transfected with the EGFR gene and recovering endocytosed phage from within the cell. Three unique scFvs were isolated, two from selections on A431 cells and two from selections on CHO/EGFR cells. All three scFv bound native receptor as expressed on a panel of tumor cells and did not bind EGFR negative cells. Phage antibodies and multivalent immunoliposomes constructed from scFv were endocytosed by EGFR expressing cells as shown by confocal microscopy. Native scFv primarily stained the cell surface, with less staining intracellularly. The results demonstrate how phage antibodies binding native cell surface receptors can be directly selected on overexpressing cell lines or transfected cells. Use of a transfected cell line allows selection of antibodies to native receptors without the need for protein expression and purification, significantly speeding the generation of targeting antibodies to genomic sequences. Depending upon the format used, the antibodies can be used to deliver molecules to the cell surface or intracellularly.

Molecular Profiling of Activated Neurons by Phosphorylated Ribosome Capture

The mammalian brain is composed of thousands of interacting neural cell types. Systematic approaches to establish the molecular identity of functional populations of neurons would advance our understanding of neural mechanisms controlling behavior. Here, we show that ribosomal protein S6, a structural component of the ribosome, becomes phosphorylated in neurons activated by a wide range of stimuli. We show that these phosphorylated ribosomes can be captured from mouse brain homogenates, thereby enriching directly for the mRNAs expressed in discrete subpopulations of activated cells. We use this approach to identify neurons in the hypothalamus regulated by changes in salt balance or food availability. We show that galanin neurons are activated by fasting and that prodynorphin neurons restrain food intake during scheduled feeding. These studies identify elements of the neural circuit that controls food intake and illustrate how the activity-dependent capture of cell-type-specific transcripts can elucidate the functional organization of a complex tissue.

A substrate-specific inhibitor of protein translocation into the endoplasmic reticulum

The segregation of secretory and membrane proteins to the mammalian endoplasmic reticulum is mediated by remarkably diverse signal sequences that have little or no homology with each other. Despite such sequence diversity, these signals are all recognized and interpreted by a highly conserved protein-conducting channel composed of the Sec61 complex. Signal recognition by Sec61 is essential for productive insertion of the nascent polypeptide into the translocation site, channel gating and initiation of transport. Although subtle differences in these steps can be detected between different substrates, it is not known whether they can be exploited to modulate protein translocation selectively. Here we describe cotransin, a small molecule that inhibits protein translocation into the endoplasmic reticulum. Cotransin acts in a signal-sequence-discriminatory manner to prevent the stable insertion of select nascent chains into the Sec61 translocation channel. Thus, the range of substrates accommodated by the channel can be specifically and reversibly modulated by a cell-permeable small molecule that alters the interaction between signal sequences and the Sec61 complex.

Efficient secretion of small proteins in mammalian cells relies on Sec62-dependent posttranslational translocation.

The study reveals a role of mammalian Sec62 in ER translocation. It elucidates the choice between SRP- and Sec62-dependent translocation routes, showing that short proteins rely on Sec62 and proteins longer than 160 amino acids rely on SRP for maximal translocation efficiency, implying that the Sec62 path is a fail-safe route for small preproteins.

Linking smell to metabolism and aging

The olfactory system can have direct effects on energy homeostasis The sense of smell, or olfaction, allows animals to survey the chemical landscape of the outside world and use this information to guide behavior. Olfactory cues are particularly important for the regulation of feeding, but how odor perception influences other aspects of energy homeostasis remains poorly understood. Recent work has begun to uncover some of these connections, revealing an unexpected role for olfaction in the control of metabolism and longevity.