To understand the health impact of long-duration spaceflight, one identical twin astronaut was monitored before, during, and after a 1-year mission onboard the International Space Station; his twin served as a genetically matched ground control. Longitudinal assessments identified spaceflight-specific changes, including decreased body mass, telomere elongation, genome instability, carotid artery distension and increased intima-media thickness, altered ocular structure, transcriptional and metabolic changes, DNA methylation changes in immune and oxidative stress–related pathways, gastrointestinal microbiota alterations, and some cognitive decline postflight. Although average telomere length, global gene expression, and microbiome changes returned to near preflight levels within 6 months after return to Earth, increased numbers of short telomeres were observed and expression of some genes was still disrupted. These multiomic, molecular, physiological, and behavioral datasets provide a valuable roadmap of the putative health risks for future human spaceflight.

Summary Points List:1. Proteases are essential for proteolytic processing of proneuropeptide precursors into active peptide neurotransmitters and hormones.2. Secretory vesicles represent the primary subcellular site of neuropeptide biosynthesis, which are produced, stored, and secreted to mediate cell-cell communication. 3.Protease pathways for proneuropeptide processing have been elucidated consisting of (a) the newly identified cysteine protease cathepsin L with aminopeptidase B in secretory vesicles, and (b) the well-established, proprotein convertase family that include the neuroendocrine-specific prohormone convertases 1 and 2 (PC1/3 and PC2) with carboxypeptidase E. 4.Protease gene knockout experiments have validated the roles of PC1/3, PC2, as well as cathepsin L for the production of neuropeptides in nervous and endocrine tissues. 5.Endogenous regulators consisting of inhibitors and activators participate in the in vivo control of processing enzyme functions.6. Structural biology of protease and proneuropeptides will be important to understand interacting mechanisms for proneuropeptide processing. 7.Neuropeptidomics has recently been applied to investigations of neuropeptide systems for their primary sequence and structural identification, as well as quantitation by LC-MS/MS tandem mass spectrometry. 8.Proteomic studies have revealed functional protein families that participate in secretory vesicle functions for the production, storage, and secretion of neuropeptides.9. Pharmacological evaluation of unique specificities among neuropeptide processing systems will be valuable for design of future strategies to develop selective small molecule modulators of processing enzymes for therapeutic applications in health and disease.Future Issues: Areas of Neuropeptide Research for Exploration. 1.How are cathepsin L and prohormone convertase protease pathways coordinately regulated? 2.What is the proteolytic basis for tissue-specific processing of proneuropeptides, such as that for POMC?3. Selective and potent inhibitors of protease components for processing prohormones should be developed to facilitate basic and pharmacological research. 4.What are the structural features of prohormone and protease interactions for functional processing? Peptide neurotransmitters and peptide hormones, collectively known as neuropeptides, are required for cell-cell communication in neurotransmission and for regulation of endocrine functions. Neuropeptides are synthesized from protein precursors (termed proneuropeptides or prohormones) that require proteolytic processing primarily within secretory vesicles that store and secrete the mature neuropeptides to control target cellular and organ systems. This review describes interdisciplinary strategies that have elucidated two primary protease pathways for prohormone processing consisting of the cysteine protease pathway mediated by secretory vesicle cathepsin L and the well-known subtilisin-like proprotein convertase pathway that together support neuropeptide biosynthesis. Importantly...

Elucidation of A␤-lowering agents that inhibit processing of the wild-type (WT) ␤-secretase amyloid precursor protein (APP) site, present in most Alzheimer disease (AD) patients, is a logical approach for improving memory deficit in AD. The cysteine protease inhibitors CA074Me and E64d were selected by inhibition of ␤-secretase activity in regulated secretory vesicles that produce ␤-amyloid (A␤). The regulated secretory vesicle activity, represented by cathepsin B, selectively cleaves the WT ␤-secretase site but not the rare Swedish mutant ␤-secretase site. In vivo treatment of London APP mice, expressing the WT ␤-secretase site, with these inhibitors resulted in substantial improvement in memory deficit assessed by the Morris water maze test. After inhibitor treatment, the improved memory function was accompanied by reduced amyloid plaque load, decreased A␤40 and A␤42, and reduced C-terminal ␤-secretase fragment derived from APP by ␤-secretase. However, the inhibitors had no effects on any of these parameters in mice expressing the Swedish mutant ␤-secretase site of APP. The notable efficacy of these inhibitors to improve memory and reduce A␤ in an AD animal model expressing the WT ␤-secretase APP site present in the majority of AD patients provides support for CA074Me and E64d inhibitors as potential AD therapeutic agents. Alzheimer disease (AD)2 is an age-related neurodegenerative disorder that results in loss of memory and accumulation of neurotoxic ␤-amyloid (A␤) peptides in brain (1-4). Expression of mutant forms of human amyloid precursor protein (APP) in mouse models of AD results in increased A␤ and amyloid plaques in brain, with memory deficits that resemble AD (2, 3, 5-7). Such studies demonstrate that overproduction of A␤ peptides participates as a major factor in the development of AD.A␤ peptides are produced by proteolytic processing of APP, resulting in production of A␤40 and A␤42 (1-40 and 1-42 residues, respectively). Proteases referred to as ␤-secretase and ␥-secretase cleave at the N-and C termini of A␤ within APP to generate A␤ peptides. A␤ peptides then undergo secretion to provide extracellular A␤ that produces neurotoxic effects with aggregation and accumulation in amyloid plaques of AD brains. Compounds that inhibit ␤-secretase activity are particularly attractive as potential therapeutic agents for AD.The majority of AD patients express the wild-type (WT) ␤-secretase site of APP. Therefore, compounds that inhibit cleavage of the WT ␤-secretase site are relevant to the AD population. Significantly, findings in this study showed that the endogenous ␤-secretase activity in A␤-containing regulated secretory vesicles (RSV) possesses high selectivity for cleaving the WT ␤-secretase site but does not cleave the Swedish (Swe) mutant ␤-secretase site. The RSV provide production and synthesis of a major portion of secreted A␤ (8, 9). Although many previous studies of ␤-secretase have utilized substrates containing the Swe mutant ␤-secretase site (10 -14) expressed in one extended family (15), it i...

Multistep proteolytic mechanisms are essential for converting proprotein precursors into active peptide neurotransmitters and hormones. Cysteine proteases have been implicated in the processing of proenkephalin and other neuropeptide precursors. Although the papain family of cysteine proteases has been considered the primary proteases of the lysosomal degradation pathway, more recent studies indicate that functions of these enzymes are linked to specific biological processes. However, few protein substrates have been described for members of this family. We show here that secretory vesicle cathepsin L is the responsible cysteine protease of chromaffin granules for converting proenkephalin to the active enkephalin peptide neurotransmitter. The cysteine protease activity was identified as cathepsin L by affinity labeling with an activity-based probe for cysteine proteases followed by mass spectrometry for peptide sequencing. Production of T he biosynthesis of enkephalin opioid peptides as well as numerous peptide neurotransmitters and hormones requires proteolytic processing of respective proprotein precursors within regulated secretory vesicles (1-4). The mature, processed enkephalin peptide is stored within these vesicles and undergoes stimulated secretion to mediate neurotransmission and cell-cell communication in the regulation of analgesia, behavior, and immune-cell functions. Secretory vesicles of neuroendocrine chromaffin cells (also known as chromaffin granules) contain enkephalin and its precursor proenkephalin (PE) (5, 6), with relevant prohormone convertases for converting PE into active enkephalin.The primary PE-cleaving activity in chromaffin granules has been characterized as a cysteine protease complex known as ''prohormone thiol protease'' (PTP) (7-10). The cysteine protease activity cleaves PE and enkephalin-containing peptide substrates at paired basic residues, as well as at certain monobasic residues, to generate appropriate enkephalin-related peptide products. Cellular inhibition of PTP by a cysteine protease inhibitor results in reduced production of enkephalin (11). Molecular identification of the protease component responsible for this cysteine protease activity will facilitate our understanding of multiple proteolytic enzymes that produce active peptides including the opioid [Met]enkephalin (ME) (12,13).In this study the protease responsible for PE-cleaving activity in chromaffin granules was identified by using an activity-based probe for cysteine proteases (14, 15) combined with mass spectrometry (MS) for peptide sequencing. Results identified secretory vesicle cathepsin L as the enzyme responsible for the previously described PTP cysteine protease activity involved in enkephalin and neuropeptide production (7-10). Cathepsin L generated the active peptide ME by cleaving enkephalin-containing peptide substrates at native dibasic and monobasic sites. Notably, cathepsin L colocalized with ME in the regulated secretory pathway of chromaffin cells. In cathepsin L gene knockout (KO) mice (16-1...

Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder resulting in selective neuronal loss and dysfunction in the striatum and cortex. The molecular pathways leading to the selectivity of neuronal cell death in HD are poorly understood. Proteolytic processing of full-length mutant huntingtin (Htt) and subsequent events may play an important role in the selective neuronal cell death found in this disease. Despite the identification of Htt as a substrate for caspases, it is not known which caspase(s) cleaves Htt in vivo or whether regional expression of caspases contribute to selective neuronal cells loss. Here, we evaluate whether specific caspases are involved in cell death induced by mutant Htt and if this correlates with our recent finding that Htt is cleaved in vivo at the caspase consensus site 552. We find that caspase-2 cleaves Htt selectively at amino acid 552. Further, Htt recruits caspase-2 into an apoptosome-like complex. Binding of caspase-2 to Htt is polyglutamine repeat-length dependent, and therefore may serve as a critical initiation step in HD cell death. This hypothesis is supported by the requirement of caspase-2 for the death of mouse primary striatal cells derived from HD transgenic mice expressing full-length Htt (YAC72). Expression of catalytically inactive (dominant-negative) forms of caspase-2, caspase-7, and to some extent caspase-6, reduced the cell death of YAC72 primary striatal cells, while the catalytically inactive forms of caspase-3, -8, and -9 did not. Histological analysis of post-mortem human brain tissue and YAC72 mice revealed activation of caspases and enhanced caspase-2 immunoreactivity in medium spiny neurons of the striatum and the cortical projection neurons when compared to controls. Further, upregulation of caspase-2 correlates directly with decreased levels of brain-derived neurotrophic factor in the cortex and striatum of 3-month YAC72 transgenic mice and therefore suggests that these changes are early events in HD pathogenesis. These data support the involvement of caspase-2 in the selective neuronal cell death associated with HD in the striatum and cortex.

Peptide hormones and neurotransmitters constitute a large class of neurohumoral agents that mediate cell-cell communication in neuroendocrine systems. Their biosynthesis requires proteolytic processing of inactive protein precursors into active neuropeptides. Elucidation of the proteolytic components required for prohormone processing is important for identifying key proteases that may control the production of neuropeptides. This article compares the subtilisin-like PC1/3 and PC2 processing enzymes identified through molecular biological approaches, and several candidate processing enzymes identified biochemically, including the 'proopiomelanocortin converting enzyme' (PCE) and the 'prohormone thiol protease' (PTP), as well as others of different classes (aspartyl, cysteine, metallo, and serine proteases). A role for PTP in cellular proenkephalin processing is suggested by blockade of forskolin-stimulated (Met)enkephalin production by Ep453 that is converted intracellularly to E-64c, a selective cysteine protease inhibitor that potently inhibits PTP. A possible role for endogenous protease inhibitors in prohormone processing represents a new aspect of cellular mechanisms that may regulate neuropeptide biosynthesis. Future studies of the enzymology and molecular biology of processing enzymes and endogenous protease inhibitors will be necessary to elucidate mechanisms of prohormone processing.

The regulated secretory pathway of neurons is the major source of extracellular A beta that accumulates in Alzheimer's disease (AD). Extracellular A beta secreted from that pathway is generated by beta-secretase processing of amyloid precursor protein (APP). Previously, cysteine protease activity was demonstrated as the major beta-secretase activity in regulated secretory vesicles of neuronal chromaffin cells. In this study, the representative cysteine protease activity in these secretory vesicles was purified and identified as cathepsin B by peptide sequencing. Immunoelectron microscopy demonstrated colocalization of cathepsin B with A beta in these vesicles. The selective cathepsin B inhibitor, CA074, blocked the conversion of endogenous APP to A beta in isolated regulated secretory vesicles. In chromaffin cells, CA074Me (a cell permeable form of CA074) reduced by about 50% the extracellular A beta released by the regulated secretory pathway, but CA074Me had no effect on A beta released by the constitutive pathway. Furthermore, CA074Me inhibited processing of APP into the COOH-terminal beta-secretase-like cleavage product. These results provide evidence for cathepsin B as a candidate beta-secretase in regulated secretory vesicles of neuronal chromaffin cells. These findings implicate cathepsin B as beta-secretase in the regulated secretory pathway of brain neurons, suggesting that inhibitors of cathepsin B may be considered as therapeutic agents to reduce A beta in AD.

Selective degradation of cellular proteins offers an important mechanism to coordinate cellular processes including cell differentiation, defense, metabolic control, signal transduction and proliferation. While much is known about eukaryotic ubiquitination, we know little about the recently discovered ubiquitin-like protein in prokaryotes (PUP). Through expression of His7 tagged PUP and exploitation of the characteristic +243 Da mass shift attributed to trypsinized PUPylated peptides, a global pull-down of protein targets for PUPylation in Mycobacterium smegmatis revealed 103 candidate PUPylation targets and 52 confirmed targets. Similar to eukaryotic ubiquitination, further analysis of these targets revealed neither primary sequence nor secondary structure homology at the point of attachment. Pathways containing PUPylated proteins include many central to rapid cell growth, such as glycolysis, gluconeogenesis, amino acid and mycolic acid metabolism and biosynthesis, as well as translation. Seventeen of the 29 nitrosylated protein targets previously identified in Mycobacterium tuberculosis were also identified as PUPylation candidates indicating a connection between PUP-mediated remodeling of critical metabolic pathways and the mycobacterial response to exogenous stress.