Engineering Papers⌕ Search

SEARCH · Engineering Papers

Results for “PEPTIDE HORMONES”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 91 records · Page 5

Harnessing Aromatic‐Histidine Interactions through Synergistic Backbone Extension and Side Chain Modification

Abstract Peptide engineering efforts have delivered drugs for diverse human diseases. Side chain alteration is among the most common approaches to designing new peptides for specific applications. The peptide backbone can be modified as well, but this strategy has received relatively little attention. Here we show that new and favorable contacts between a His side chain on a target protein and an aromatic side chain on a synthetic peptide ligand can be engineered by rational and coordinated side chain modification and backbone extension. Side chain modification alone was unsuccessful. Binding measurements, high‐resolution structural studies and pharmacological outcomes all support the synergy between backbone and side chain modification in engineered ligands of the parathyroid hormone receptor‐1, which is targeted by osteoporosis drugs. These results should motivate other structure‐based designs featuring coordinated side chain modification and backbone extension to enhance the engagement of peptide ligands with target proteins.

36 MATERIALS SCIENCE↗

Harnessing Aromatic‐Histidine Interactions through Synergistic Backbone Extension and Side Chain Modification

Abstract Peptide engineering efforts have delivered drugs for diverse human diseases. Side chain alteration is among the most common approaches to designing new peptides for specific applications. The peptide backbone can be modified as well, but this strategy has received relatively little attention. Here we show that new and favorable contacts between a His side chain on a target protein and an aromatic side chain on a synthetic peptide ligand can be engineered by rational and coordinated side chain modification and backbone extension. Side chain modification alone was unsuccessful. Binding measurements, high‐resolution structural studies and pharmacological outcomes all support the synergy between backbone and side chain modification in engineered ligands of the parathyroid hormone receptor‐1, which is targeted by osteoporosis drugs. These results should motivate other structure‐based designs featuring coordinated side chain modification and backbone extension to enhance the engagement of peptide ligands with target proteins.

36 MATERIALS SCIENCE↗

Endocrine relationships during human spaceflight

Human spaceflight is associated with a chronic loss of protein from muscle. The objective of this study was to determine whether changes in urinary hormone excretion could identify a hormonal role for this loss. Urine samples were collected from the crews of two Life Sciences Space Shuttle missions before and during spaceflight. Data are means +/- SE with the number of subjects in parentheses. The first value is the mean preflight measurement, and the second value is the mean inflight measurement. Adrenocorticotropic hormone (ACTH) [27.7 +/- 4.4 (9) vs. 25.1 +/- 3.4 (9) ng/day], growth hormone [724 +/- 251 (9) vs. 710 +/- 206 (9) ng/day], insulin-like growth factor I [6.81 +/- 0.62 vs. 6.04 +/- 0.51 (8) nM/day], and C-peptide [44.9 +/- 8.3 (9) vs. 50.7 +/- 10.3 (9) micrograms/day] were unchanged with spaceflight. In contrast, free 3,5,3'-triiodothyronine [791 +/- 159 (9) vs. 371 +/- 41 (9) pg/day, P < 0.05], prostaglandin E2 (PGE2) [1, 064 +/- 391 (8) vs. 465 +/- 146 (8) ng/day, P < 0.05], and its metabolite PGE-M [1,015 +/- 98 (9) vs. 678 +/- 105 (9) ng/day, P < 0. 05] were decreased inflight. The urinary excretion of most hormones returned to their preflight levels during the postflight period, with the exception of ACTH [47.5 +/- 10.3 (9) ng/day], PGE2 [1,433 +/- 327 (8) ng/day], PGF2alpha, [2,786 +/- 313 (8) ng/day], and its metabolite PGF-M [4,814 +/- 402 (9) ng/day], which were all increased compared with the preflight measurement (P < 0.05). There was a trend for urinary cortisol to be elevated inflight [55.3 +/- 5. 9 (9) vs. 72.5 +/- 11.1 micrograms/day, P = 0.27] and postflight [82.7 +/- 8.6 (8) micrograms/day, P = 0.13]. The inflight human data support ground-based in vitro work showing that prostaglandins have a major role in modulating the changes in muscle protein content in response to tension or the lack thereof.

Flight Experiment↗

Elevated central venous pressure: A consequence of exercise training-induced hypervolemia

Resting plasma volumes, and arterial and central venous pressures (CVP) were measured in 16 men before and after exercise training to determine if training-induced hypervolemia could be explained by a change in total vascular capacitance. In addition, resting levels of plasma vasopressin (AVP), atrial natriuretic peptide (ANP), aldosterone (ALD), and norepinephrine (NE) were measured before and after training. The same measurements of vacular volume, pressures, and plasma hormones were measured in 8 subjects who did not undergo exercise and acted as controls. The exercise training program consisted of 10 weeks of controlled cycle exercise for 30 min/d, 4 d/wk at 75 to 80 percent of maximal oxygen uptake (VO2max). A training effect was verified by a 20 percent increase in VO2max, a resting bradycardia, and a 370 ml (9 percent) increase in blood volume. Mean arterial blood pressure was unaltered by exercise training, but resting CVP increased. The percent change in blood volume from before to after training was linearly related to the percent change in CVP. As a consequence of elevations in both blood volume and CVP, the volume-to-pressure ratio was essentially unchanged following exercise training. Plasma AVP, ANP, ALD, and NE were unaltered. Results indicate that elevated CVP is a consequence of training-induced hypervolemia without alteration in total effective venous capacitance. This may represent a resetting of the pressure-volume stimulus-response relation for regulation of blood volume.

Convertino, Victor A.↗

Regulation of body fluid volume and electrolyte concentrations in spaceflight

Despite a number of difficulties in performing experiments during weightlessness, a great deal of information has been obtained concerning the effects of spaceflight on the regulation of body fluid and electrolytes. Many paradoxes and questions remain, however. Although body mass, extracellular fluid volume, and plasma volume are reduced during spaceflight and remain so at landing, the changes in total body water are comparatively small. Serum or plasma sodium and osmolality have generally been unchanged or reduced during the spaceflight, and fluid intake is substantially reduced, especially during the first of flight. The diuresis that was predicted to be caused by weightlessness, has only rarely been observed as an increased urine volume. What has been well established by now, is the occurrence of a relative diuresis, where fluid intake decreases more than urine volume does. Urinary excretion of electrolytes has been variable during spaceflight, but retention of fluid and electrolytes at landing has been consistently observed. The glomerular filtration rate was significantly elevated during the SLS missions, and water and electrolyte loading tests have indicated that renal function is altered during readaptation to Earth's gravity. Endocrine control of fluid volumes and electrolyte concentrations may be altered during weightlessness, but levels of hormones in body fluids do not conform to predictions based on early hypotheses. Antidiuretic hormone is not suppressed, though its level is highly variable and its secretion may be affected by space motion sickness and environmental factors. Plasma renin activity and aldosterone are generally elevated at landing, consistent with sodium retention, but inflight levels have been variable. Salt intake may be an important factor influencing the levels of these hormones. The circadian rhythm of cortisol has undoubtedly contributed to its variability, and little is known yet about the influence of spaceflight on circadian rhythms. Atrial natriuretic peptide does not seem to play an important role in the control of natriuresis during spaceflight. Inflight activity of the sympathetic nervous system, assessed by measuring catecholamines and their metabolites and precursors in body fluids, generally seems to be no greater than on Earth, but this system is usually activated at landing. Collaborative experiments on the Mir and the International Space Station should provide more of the data needed from long-term flights, and perhaps help to resolve some of the discrepancies between U.S. and Russian data. The use of alternative methods that are easier to execute during spaceflight, such as collection of saliva instead of blood and urine, should permit more thorough study of circadian rhythms and rapid hormone changes in weightlessness. More investigations of dietary intake of fluid and electrolytes must be performed to understand regulatory processes. Additional hormones that may participate in these processes, such as other natriuretic hormones, should be determined during and after spaceflight. Alterations in body fluid volume and blood electrolyte concentrations during spaceflight have important consequences for readaptation to the 1-G environment. The current assessment of fluid and electrolyte status during weightlessness and at landing and our still incomplete understanding of the processes of adaptation to weightlessness and readaptation to Earth's gravity have resulted in the development of countermeasures that are only partly successful in reducing the postflight orthostatic intolerance experienced by astronauts and cosmonauts. More complete knowledge of these processes can be expected to produce countermeasures that are even more successful, as well as expand our comprehension of the range of adaptability of human physiologic processes.

NASA Discipline Number 18-10↗

A temperature-sensitive FERONIA mutant allele that alters root hair growth

Abstract In plants, root hairs undergo a highly polarized form of cell expansion called tip-growth, in which cell wall deposition is restricted to the root hair apex. In order to identify essential cellular components that might have been missed in earlier genetic screens, we identified conditional temperature-sensitive (ts) root hair mutants by ethyl methanesulfonate mutagenesis in Arabidopsis thaliana. Here, we describe one of these mutants, feronia-temperature sensitive (fer-ts). Mutant fer-ts seedlings were unaffected at normal temperatures (20°C), but failed to form root hairs at elevated temperatures (30°C). Map based-cloning and whole-genome sequencing revealed that fer-ts resulted from a G41S substitution in the extracellular domain of FERONIA (FER). A functional fluorescent fusion of FER containing the fer-ts mutation localized to plasma membranes, but was subject to enhanced protein turnover at elevated temperatures. While tip-growth was rapidly inhibited by addition of rapid alkalinization factor 1 (RALF1) peptides in both wild-type and fer-ts mutants at normal temperatures, root elongation of fer-ts seedlings was resistant to added RALF1 peptide at elevated temperatures. Additionally, at elevated temperatures fer-ts seedlings displayed altered reactive oxygen species (ROS) accumulation upon auxin treatment and phenocopied constitutive fer mutant responses to a variety of plant hormone treatments. Molecular modeling and sequence comparison with other Catharanthus roseus receptor-like kinase 1L (CrRLK1L) receptor family members revealed that the mutated glycine in fer-ts is highly conserved, but is not located within the recently characterized RALF23 and LORELI-LIKE-GLYCOPROTEIN 2 binding domains, perhaps suggesting that fer-ts phenotypes may not be directly due to loss of binding to RALF1 peptides.

Plant Sciences↗

Characterization of atrial natriuretic peptide receptors in brain microvessel endothelial cells

Atrial natriuretic peptide (ANP) binding and ANP-induced increases in cyclic guanosine monophosphate (cGMP) levels have been observed in brain microvessels (Chabrier et al., 1987; Steardo and Nathanson, 1987), suggesting that this fluid-regulating hormone may play a role in the fluid homeostasis of the brain. This study was initiated to characterize the ANP receptors in primary cultures of brain microvessel endothelial cells (BMECs). The apparent equilibrium dissociation constant, Kd, for ANP increased from 0.25 nM to 2.5 nM, and the number of ANP binding sites as determined by Scatchard analysis increased from 7,100 to 170,000 sites/cell between 2 and 10 days of culture following monolayer formation. Time- and concentration-dependent studies on the stimulation of cGMP levels by ANP indicated that guanylate cyclase-linked ANP receptors were present in BMECs. The relative abilities of ANP, brain natriuretic peptide (BNP), and a truncated analog of ANP containing amino acids 5-27 (ANP 5-27) to modulate the accumulation of cGMP was found to be ANP greater than BNP much greater than ANP 5-27. Affinity cross-linking with disuccinimidyl suberate and radiolabeled ANP followed by gel electrophoresis under reducing conditions demonstrated a single band corresponding to the 60-70 kD receptor, indicating the presence of the nonguanylate cyclase-linked ANP receptor. Radiolabeled ANP binding was examined in the presence of various concentrations of either ANP, BNP, or ANP 5-27 and suggested that a large proportion of the ANP receptors present in blood-brain barrier endothelial cells bind all of these ligands similarly. These data indicate both guanylate cyclase linked and nonguanylate cyclase linked receptors are present on BMECs and that a higher proportion of the nonguanylate cyclase linked receptors is expressed. This in vitro culture system may provide a valuable tool for the examination of ANP receptor expression and function in blood-brain barrier endothelial cells.

Endothelium, Vascular/chemistry↗

Acute effects of head-down tilt and hypoxia on modulators of fluid homeostasis

In an effort to understand the interaction between acute postural fluid shifts and hypoxia on hormonal regulation of fluid homeostasis, the authors measured the responses to head-down tilt with and without acute exposure to normobaric hypoxia. Plasma atrial natriuretic peptide (ANP), cyclic guanosine monophosphate (cGMP), cyclic adenosine monophosphate (cAMP), plasma aldosterone (ALD), and plasma renin activity (PRA) were measured in six healthy male volunteers who were exposed to a head-down tilt protocol during normoxia and hypoxia. The tilt protocol consisted of a 17 degrees head-up phase (30 minutes), a 28 degrees head-down phase (1 hour), and a 17 degrees head-up recovery period (2 hours, with the last hour normoxic in both experiments). Altitude equivalent to 14,828 ft was simulated by having the subjects breathe an inspired gas mixture with 13.9% oxygen. The results indicate that the postural fluid redistribution associated with a 60-minute head-down tilt induces the release of ANP and cGMP during both hypoxia and normoxia. Hypoxia increased cGMP, cAMP, ALD, and PRA throughout the protocol and significantly potentiated the increase in cGMP during head-down tilt. Hypoxia had no overall effect on the release of ANP, but appeared to attenuate the increase with head-down tilt. This study describes the acute effects of hypoxia on the endocrine response during fluid redistribution and suggests that the magnitude, but not the direction, of these changes with posture is affected by hypoxia.

Posture↗

Changes in renal function and fluid and electrolyte regulation in space flight

The cephalad fluid redistribution resulting from weightlessness has a number of physiologic consequences. Plasma volume is reduced soon after weightlessness is reached, and red blood cell mass reduction follows. Plasma atrial natriuretic peptide, which inhibits aldosterone secretion, was elevated during space flight while plasma aldosterone was below preflight levels. Serum sodium was also reduced and potassium was elevated. Antidiuretic hormone (ADH) was markedly elevated at almost all measurement times in the first eight days of flight, but plasma volume did not return to preflight levels.

Leach, C. S.↗

Herpesviruses in Saliva and Their Clinical Significance

Saliva has been used as a source of biological markers for a wide spectrum of normal and disease states for a long time. It is a non-invasive, easily accessible, and self-collected body fluid that contains a variety of measurable biological substances. While mostly water, saliva also contains ions, carbohydrates, proteins and peptides, exfoliated cells, nucleic acids, and microorganisms. Saliva can reflect tissue levels of some natural substances and a large variety of molecules introduced for therapeutic use, emotional status; hormonal status, immunological status, neurological effects, and nutritional and metabolic status. It can also be used to monitor a variety of drugs including marijuana, cocaine, and alcohol. It is the most cost-effective approach for screening large population in community mass screening programs and for longitudinal sampling of hospitalized individuals aimed at monitoring viral load dynamics and treatment response. During the COVID-19 pandemic, scientific evidence emerged indicating that molecular tests performed on saliva have diagnostic sensitivity and specificity comparable to those observed with nasopharyngeal swabs for SARS-CoV-2 RNA detection. The presence of IgA and IgG antibodies at the mucosal level has been demonstrated to influence the progression of viral infection and the severity of clinical manifestation. As saliva contains both respiratory secretions and immunological components, it has wide applications, ranging from clinical diagnostics to post-vaccine disease burden and immunity surveillance.

Douglass Diak↗

Characterization of Treefoil Peptide Genes in Iron-Ion or X-Irradiated Human Cells

The gastrointestinal (GI) tract is especially sensitive to ionizing radiation, probably because of its high rate of cell turn over. Most of the data in the literature concerns the histological/anatomical description of damage rather than functional studies. In fact, previous reports in humans have shown that, at doses of 2 Gy or more, functional abnormalities appear indicating that in radiation sensitive tissues the effects of radiation are not limited to cell death. GI functions are controlled in particular by GI peptides. One hypothesis is that ionizing radiation may modulate the synthesis and release of these peptides and consequently may contribute largely to abnormalities in GI function. However, no previous studies have been concerned with GI-specific gene expression in irradiated GI tissues. The family of human trefoil peptides comprises three members thus far, all of which are expressed in specific regions of the GI tract. In addition, two trefoil peptides, pS2 (TFFI) and HITF (TFF2) are expressed in breast tissue. Their exact function in GI and breast tissues is unclear but mucosal integrity, repair, mucin secretion and responsiveness to hormones have been shown. We recently isolated and characterized pS2 as a novel p53- and estrogen receptor-independent gene whose MRNA expression in several cells lines was found to be delayed 4 to 7 days after irradiation with X-rays, fission neutrons or 1 GeV/n Fe-ions. The aim of the present study was to determine whether pS2 and HITF have a similar induction kinetics in irradiated gastric and breast cell lines, and whether they have the phorbol ester (TPA) responsive element (TRE).

Balcer-Kubiczek, E. K.↗

Protective role of AgRP neuron's PDK1 against salt-induced hypertension

Highlights: • AgRP neuron specific PDK1 KO mice responded to high salt diet with hypertension. • The KO mice showed elevated urinary noradrenalin excretion under high salt diet. • The KO mice showed elevated NUCB2 mRNA expression in paraventricular nucleus (PVN). • PVN NUCB2 silencing counteracted elevated noradrenalin excretion and hypertension. • AgRP neuron's PDK1 serves to regulate PVN nesfatin-1 neurons and blood pressure. In the hypothalamic arcuate nucleus (ARC), orexigenic agouti-related peptide (AgRP) neurons regulate feeding behavior and energy homeostasis. The 3-phosphoinositide-dependent protein kinase-1 (PDK1) in AgRP neurons serves as a major signaling molecule for leptin and insulin, the hormones regulating feeding behavior, energy homeostasis and circulation. However, it is unclear whether PDK1 in AGRP neurons is also involved in regulation of blood pressure. This study explored it by generating and analyzing AgRP neuron-specific PDK1 knockout (Agrp-Pdk1{sup flox/flox}) mice and effect of high salt diet on blood pressure in KO and WT mice was analyzed. Under high salt diet feeding, systolic blood pressure (SBP) of Agrp-Pdk1{sup flox/flox} mice was significantly elevated compared to Agrp-Cre mice. When the high salt diet was switched to control low salt diet, SBP of Agrp-Pdk1{sup flox/flox} mice returned to the basal level observed in Agrp-Cre mice within 1 week. In Agrp-Pdk1{sup flox/flox} mice, urinary noradrenalin excretion and NUCB2 mRNA expression in hypothalamic paraventricular nucleus (PVN) were markedly upregulated. Moreover, silencing of NUCB2 in the PVN counteracted the rises in urinary noradrenalin excretions and SBP. These results demonstrate a novel role of PDK1 in AgRP neurons to counteract the high salt diet-induced hypertension by preventing hyperactivation of PVN nesfatin-1 neurons.

60 APPLIED LIFE SCIENCES↗

Ghrelin promotes human non-small cell lung cancer A549 cell proliferation through PI3K/Akt/mTOR/P70S6K and ERK signaling pathways

Highlights: • Ghrelin promotes A549 cells proliferation through binding to GHSR. • Ghrelin activates PI3K/Akt/mTOR/P70S6K and ERK signaling pathways. • PI3K/Akt/mTOR/P70S6K and ERK signaling mediates ghrelin-induced A549 cells proliferation. Ghrelin is a gastric acyl-peptide that plays an important role in cell proliferation. In the present study, we explored the role of ghrelin in A549 cell proliferation and the possible molecular mechanisms. We found that ghrelin promotes A549 cell proliferation, knockdown of the growth hormone secretagogue receptor (GHSR) attenuated A549 cell proliferation caused by ghrelin. Ghrelin induced the rapid phosphorylation of phosphatidylinositol 3-kinase (PI3K), Akt, ERK, mammalian target of rapamycin (mTOR) and P70S6K. PI3K inhibitor (LY 294002), ERK inhibitor (PD98059) and mTOR inhibitor (Rapamycin) inhibited ghrelin-induced A549 cell proliferation. Moreover, GHSR siRNA inhibited phosphorylation of PI3K, Akt, ERK, mTOR and P70S6K induced by ghrelin. Akt and mTOR/P70S6K phosphorylation was inhibited by LY 294002 but not by PD98059. These results indicate that ghrelin promotes A549 cell proliferation via GHSR-dependent PI3K/Akt/mTOR/P70S6K and ERK signaling pathways.

60 APPLIED LIFE SCIENCES↗

Body fluid alterations during head-down bed rest in men at moderate altitude

To determine the effects of hypoxia on fluid balance responses to simulated zero-gravity, measurements were made in six subjects before and during -5 deg continuous head-down bed rest (HDBR) over 8 d at 10,678 ft. The same subjects were studied again at this altitude without HDBR as a control (CON) using a cross-over design. During this time, they maintained normal upright day-time activities, sleeping in the horizontal position at night. Fluid balance changes during HDBR in hypoxia were more pronounced than similar measurements previously reported from HDBR studies at sea level. Plasma volume loss was slightly greater and the diuresis and natriuresis were doubled in magnitude as compared to previous studies in normoxia and sustained for 4 d during hypoxia. These changes were associated with an immediate but transient rise in plasma atrial natriuretic peptide (ANP) to day 4 of 140 percent in HDBR and 41 percent in CON (p less than 0.005), followed by a decline towards baseline. Differences were less striking between HDBR and CON for plasma antidiuretic hormone and aldosterone, which were transiently reduced by HDBR. Plasma catecholamines showed a similar pattern to ANP in both HDBR and CON, suggesting that elevated ANP and catecholamines together accounted for the enhanced fluid shifts with HDBR during hypoxia.

Loeppky, J. A.↗

Molecular ageing: Free radical initiated epimerization of thymopentin – A case study

The epimerization of amino acid residues increases with age in living organisms. In the present study, the structural consequences and thermodynamic functions of the epimerization of thymopentin (TP-5), the active site of the thymic hormone thymopoietin, were studied using molecular dynamics and density functional theory methods. The results show that free radical-initiated D-amino acid formation is energetically favoured (−130 kJmol{sup −1}) for each residue and induces significant changes to the peptide structure. In comparison to the wild-type (each residue in the L-configuration), the radius of gyration of the D-Asp{sup 3} epimer of the peptide decreased by 0.5 Å, and disrupted the intramolecular hydrogen bonding of the native peptide. Beyond establishing important structural, energetic and thermodynamic benchmarks and reference data for the structure of TP-5, these results disseminate the understanding of molecular ageing, the epimerization of amino acid residues.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

A novel exendin-4 human serum albumin fusion protein, E2HSA, with an extended half-life and good glucoregulatory effect in healthy rhesus monkeys

Highlights: • E2HSA has an extended half-life and good plasma stability. • E2HSA could improve glucose-dependent insulin secretion. • E2HSA has excellent glucoregulatory effects in vivo. • E2HSA could potentially be used as a new long-acting GLP-1 receptor agonist for type 2 diabetes management. - Abstract: Glucagon-like peptide-1 (GLP-1) has attracted considerable research interest in terms of the treatment of type 2 diabetes due to their multiple glucoregulatory functions. However, the short half-life, rapid inactivation by dipeptidyl peptidase-IV (DPP-IV) and excretion, limits the therapeutic potential of the native incretin hormone. Therefore, efforts are being made to develop the long-acting incretin mimetics via modifying its structure. Here we report a novel recombinant exendin-4 human serum albumin fusion protein E2HSA with HSA molecule extends their circulatory half-life in vivo while still retaining exendin-4 biological activity and therapeutic properties. In vitro comparisons of E2HSA and exendin-4 showed similar insulinotropic activity on rat pancreatic islets and GLP-1R-dependent biological activity on RIN-m5F cells, although E2HSA was less potent than exendin-4. E2HSA had a terminal elimation half-life of approximate 54 h in healthy rhesus monkeys. Furthermore, E2HSA could reduce postprandial glucose excursion and control fasting glucose level, dose-dependent suppress food intake. Improvement in glucose-dependent insulin secretion and control serum glucose excursions were observed during hyperglycemic clamp test (18 h) and oral glucose tolerance test (42 h) respectively. Thus the improved physiological characterization of E2HSA make it a new potent anti-diabetic drug for type 2 diabetes therapy.

60 APPLIED LIFE SCIENCES↗

Quantitative proteomics reveals extensive lysine ubiquitination and transcription factor stability states in Arabidopsis

Abstract Protein activity, abundance, and stability can be regulated by post-translational modification including ubiquitination. Ubiquitination is conserved among eukaryotes and plays a central role in modulating cellular function; yet, we lack comprehensive catalogs of proteins that are modified by ubiquitin in plants. In this study, we describe an antibody-based approach to enrich ubiquitinated peptides coupled with isobaric labeling to enable quantification of up to 18-multiplexed samples. This approach identified 17,940 ubiquitinated lysine sites arising from 6,453 proteins from Arabidopsis (Arabidopsis thaliana) primary roots, seedlings, and rosette leaves. Gene ontology analysis indicated that ubiquitinated proteins are associated with numerous biological processes including hormone signaling, plant defense, protein homeostasis, and metabolism. We determined ubiquitinated lysine residues that directly regulate the stability of three transcription factors, CRYPTOCHROME-INTERACTING BASIC-HELIX-LOOP-HELIX 1 (CIB1), CIB1 LIKE PROTEIN 2 (CIL2), and SENSITIVE TO PROTON RHIZOTOXICITY1 (STOP1) using in vivo degradation assays. Furthermore, codon mutation of CIB1 to create a K166R conversion to prevent ubiquitination, via CRISPR/Cas9-derived adenosine base editing, led to an early flowering phenotype and increased expression of FLOWERING LOCUS T (FT). These comprehensive site-level ubiquitinome profiles provide a wealth of data for future functional studies related to modulation of biological processes mediated by this post-translational modification in plants.

Biochemistry & Molecular Biology↗

Purification and sequence analysis of two rat tissue inhibitors of metalloproteinases

Two protein inhibitors of metalloproteinases (TIMP) were isolated from medium conditioned by the clonal rat osteosarcoma line UMR 106-01. Initial purification of both a 30-kDa inhibitor and a 20-kDa inhibitor was accomplished using heparin-Sepharose chromatography with dextran sulfate elution followed by DEAE-Sepharose and CM-Sepharose chromatography. Purification of the 20-kDa inhibitor to homogeneity was completed with reverse-phase high-performance liquid chromatography. The 20-kDa inhibitor was identified as rat TIMP-2. The 30-kDa inhibitor, although not purified to homogeneity, was identified as rat TIMP-1. Amino terminal amino acid sequence analysis of the 30-kDa inhibitor demonstrated 86% identity to human TIMP-1 for the first 22 amino acids while the sequence of the 20-kDa inhibitor was identical to that of human TIMP-2 for the first 22 residues. Treatment with peptide:N-glycosidase F indicated that the 30-kDa rat inhibitor is glycosylated while the 20-kDa inhibitor is apparently unglycosylated. Inhibition of both rat and human interstitial collagenase by rat TIMP-2 was stoichiometric, with a 1:1 molar ratio required for complete inhibition. Exposure of UMR 106-01 cells to 10(-7) M parathyroid hormone resulted in approximately a 40% increase in total inhibitor production over basal levels.

NASA Discipline Musculoskeletal↗