Amino Acid Sequences of Neuropeptides in the Sinus Gland of the Land Crab Cardisoma carnifex: A Novel Neuropeptide Proteolysis Site

The sinus gland is a major neurosecretory structure in Crustacea. Five peptides, labeled C, D, E, F, and I, isolated from the sinus gland of the land crab have been hypothesized to arise from the incomplete proteolysis at two internal sites on a single biosynthetic intermediate peptide "H", based on amino acid composition additivities and pulse-chase radiolabeling studies. The presence of only a single major precursor for the sinus gland peptides implies that peptide H may be synthesized on a common precursor with crustacean hyperglycemic hormone forms, "J" and "L," and a peptide, "K," similar to peptides with molt inhibiting activity. Here I report amino acid sequences of these peptides. The amino terminal sequence of the parent peptide, H, (and the homologous fragments) proved refractory to Edman degradation. Data from amino acid analysis and carboxypeptidase digestion of the naturally occurring fragments and of fragments produced by endopeptidase digestion were used together with Edman degradation to obtain the sequences. Amino acid analysis of fragments of the naturally occurring "overlap" peptides (those produced by internal cleavage at one site on H) was used to obtain the sequences across the cleavage sites. The amino acid sequence of the land crab peptide H is Arg-Ser-Ala-Asp-Gly-Phe-Gly-Arg-Met-Glu-Ser-Leu-Leu-Thr-Ser-Leu-Arg-Gly- Ser-Ala-Glu- Ser-Pro-Ala-Ala-Leu-Gly-Glu-Ala-Ser-Ala-Ala-His-Pro-Leu-Glu. In vivo cleavage at one site involves excision of arginine from the sequence Leu-Arg-Gly, whereas cleavage at the other site involves excision of serine from the sequence Glu-Ser-Leu. Proteolysis at the latter sequence has not been previously reported in intact secretory granules. The aspartate at position 4 is possibly covalently modified.     

Temporal Profiles of Proteins Responsive to Transient Ischemia

The responses of long and short half-lived proteins to ischemia were measured in rat brain during 6 days of recovery from 30 min of transient forebrain ischemia produced by four-vessel occlusion. At the end of the ischemic interval, the neocortical activities of four vulnerable enzymes [ornithine (ODC) and S-adenosylmethionine (SAMDC) decarboxylases, and RNA polymerases I and II] were unchanged, but within 30 min of reperfusion, their activities dropped by 25-50%. The loss of substance P in the striatum and substantia nigra was slower, reaching about 50% by 12 h. On the other hand, the activities of 5 long half-lived enzymes did not change in the neocortex at 5 and 15 h of reperfusion and regional protein concentrations were essentially unaffected over 6 days survival. The rate and extent of normalization of the amounts or activities of the vulnerable proteins varied. RNA polymerase II and ODC activities were restored within 4 h, and ODC showed a biphasic increase in activity, with peaks at 10 h and 2-3 days. RNA polymerase I and SAMDC activities were restored by 18 h and 5 days, respectively, whereas substance P concentrations did not completely recover, even at 6-15 days. The greater the regional reduction of blood flow during ischemia, the larger the net change (gain or loss) of SAMDC or ODC activity and the longer the time required to normalize the activities of these enzymes. The average rate of proteolysis, assessed by measuring the rate of clearance of 14C from protein prelabeled with [14C]bicarbonate, was abnormal during the first 2 days of reperfusion. Postischemic changes in both protein synthesis and degradation could affect the amounts of some of the proteins responsive to transient ischemia.     

Purification from Synaptosomal Plasma Membranes of Calpain I, a Thiol Protease Activated by Micromolar Calcium Concentrations

Synaptosomal plasma membranes (SPMs) were prepared from whole rat brain and assayed for calcium-stimulated proteolytic activity. Addition of calcium to SPMs caused a dose-dependent increase in trichloroacetic acid-soluble protein. Two peaks of protease activity directed against a casein substrate were detectable when SPMs were incubated with low-ionic-strength buffer and the extract was fractionated on DEAE-cellulose. The enzyme in peak 1 required less than 1/10 the calcium concentration for activation as the peak 2 protease (Kact1 = 35 microM; Kact2 = 500 microM). The specific thiol-protease inhibitors leupeptin and antipain and the alkylator iodoacetate blocked enzyme activity. The low-sensitivity protease was converted to a high-sensitivity enzyme (Kact = 20 microM) by substrate affinity chromatography in the presence of calcium. This protease was purified 550-fold from SPMs. The high- and low-sensitivity membrane-associated calcium-dependent proteases are part of a family of enzymes, the calpains, previously reported in cytosolic fractions of several tissues.     

Properties of Chicken Lens MIP Channels Reconstituted into Planar Lipid Bilayers

Membrane fractions highly enriched in chicken lens MIP (MIP28) were found to form ion channels when incorporated into planar lipid bilayers. The channels displayed prominent unitary conductances of about 60 and 290 pS in symmetric 150 mm KCl solution and were slightly anion selective. For both depolarizing and hyperpolarizing voltages, voltage sensitivity of the MIP28-induced conductance could be fit by a Boltzmann relation, symmetric around zero mV, with V ₀ = 18.5 mV, n= 4.5 and g _min/g _max= 0.17. Channel properties were not appreciably altered by pH in the range of 5.8 to 7, although channel incorporation was observed to occur more frequently at lower pH values. Calcium, at millimolar concentrations, decreased the channel mean open time. Partial proteolysis of MIP28 to yield MIP21 did not appreciably affect single-channel conductance or voltage sensitivity of the reconstituted channels. MIP28 was not phosphorylated by cAMP dependent protein kinase (PKA). Although unitary conductance and selectivity of the chicken MIP channel are similar to those reported for the bovine MIP (MIP26), the voltage sensitivity of MIP28 was higher than that of the bovine homologue, and voltage sensitivity of MIP28 was not modulated by treatments previously shown to affect MIP26 voltage gating (partial proteolysis and protein phosphorylation by PKA: (Ehring et al., 1990). The existence of such strikingly different functional properties in highly homologous channel isoforms may provide a useful system for exploration of the structure-function relations of MIP channels. Received: 27 March 1996/Revised: 5 August 1996     

Overexpressions of cDNAs for β-Amyloid Precursor Proteins 695, 751, and 770 Enhance the Secretion of β-Amyloid Precursor Protein Derivatives and the Survival of P19-Derived Neurons

Abstract: P19 is a C3H mouse-derived line of multipotent embryonic carcinoma cells that differentiate into neural cells. P19 cell clones overexpressing the three major forms of β-amyloid precursor protein from their cDNA constructs were established. Unlike a previous study in which P19-derived neurons had a limited α-secretase activity, all of these clones produced significant amounts of secreted β-amyloid precursor protein. When treated with retinoic acid, these transformed lines differentiated into neurons and survived better than did nontransformed parental P19 cells. Furthermore, P19-derived neurons survived better in medium conditioned by the transformed P19 line, and survival was reduced by immunoabsorption with an antibody to β-amyloid precursor protein. These results suggest neurotrophic effects of secreted β-amyloid precursor protein and contrast with a previous report in which overexpression of a full-length cDNA for β-amyloid precursor protein led to degeneration of P19-derived neurons. Western blot analysis suggested that this difference might result from different levels of expression of putative neurotoxic C-terminal fragments of β-amyloid precursor protein; moreover, P19-derived neurons differ from P19 stem cells in the processing of these C-terminal fragments.     

Light-dependent conformational change at rhodopsin's cytoplasmic surface detected by increased susceptibility to proteolysis

Rhodopsin in bovine photoreceptor disk membranes was subjected to limited proteolysis by thermolysin, removing twelve amino acids from rhodopsin's carboxyl terminus. (1) The rate of proteolysis is significantly faster with rhodopsin following exposure to light than with unbleached rhodopsin, provided that the incubation conditions (pH, temperature) favor the formation of metarhodopsin II. (2) If the disk membranes are illuminated under conditions in which metarhodopsin I is the predominant photoproduct (pH 8.5, 0°C), no increase in the rate of proteolysis is observed compared to unilluminated membranes. (3) The light-induced increase in the rate of proteolysis is transient: it slowly decays in the dark to the original rate found for unbleached rhodopsin. The enhanced susceptibility to proteolysis appears to measure a conformational change at rhodopsin's cytoplasmic surface which is first exhibited at the metarhodopsin II stage. This and possibly other light-dependent changes may allow rhodopsin to mediate its signal as a light-receptor protein by binding to and activating certain rod cell enzymes.     

Expression, purification, and characterization of a structurally disordered and functional C-terminal autoinhibitory domain (AID) of the 70 kDa 40S ribosomal protein S6 kinase-1 (S6K1)

S6K1 is a member of the AGC subfamily of serine-threonine protein kinases, whereby catalytic activation requires dual phosphorylation of critical residues in the conserved T-loop (T229) and hydrophobic motif (HM; T389) peptide regions of its catalytic kinase domain (residues 1-398). In addition to its kinase domain, S6K1 contains a C-terminal autoinhibitory domain (AID; residues 399-502), which prevents T-loop and HM phosphorylation; and autoinhibition is relieved on multi-site Ser-Thr phosphorylation of the AID (S411, S418, T421, and S424). Interestingly, 66 of the 104 C-terminal AID amino acid residues were computer predicted to exist in structurally disordered peptide regions, begetting interest as to how such dynamics could be coupled to autoregulation. To begin addressing this issue, we developed and optimized protocols for efficient AID expression and purification. Consistent with computer predictions, aberrant mobilities in both SDS-PAGE and size-exclusion chromatography, as well as low chemical shift dispersion in (1)H-(15)N HSQC NMR spectra, indicated purified recombinant AID to be largely unfolded. Yet, trans-addition of purified AID effectively inhibited PDK1-catalyzed T-loop phosphorylation of a catalytic kinase domain construct of S6K1. Using an identical purification protocol, similar protein yields of a tetraphospho-mimic mutant AID(D(2)ED) construct were obtained; and this construct displayed only weak inhibition of PDK1-catalyzed T229 phosphorylation. Purification of the structurally 'disordered' and functional C-terminal AID and AID(D(2)ED) constructs will facilitate studies aimed to understand the role of conformational plasticity and protein phosphorylation in modulating autoregulatory domain-domain interactions.     

Comparative proteomics analysis of silkworm hemolymph during the stages of metamorphosis via liquid chromatography and mass spectrometry

The silkworm is a lepidopteran insect that has an open circulatory system with hemolymph consisting of blood and lymph fluid. Hemolymph is not only considered as a depository of nutrients and energy, but it also plays a key role in substance transportation, immunity response, and proteolysis. In this study, we used LC‐MS/MS to analyze the hemolymph proteins of four developmental stages during metamorphosis. A total of 728 proteins were identified from the hemolymph of the second day of wandering stage, first day of pupation, ninth day of pupation, and first day as an adult moth. GO annotations and categories showed that silkworm hemolymph proteins were enriched in carbohydrate metabolism, proteolysis, protein binding, and antibacterial humoral response. The levels of nutrient, immunity‐related, and structural proteins changed significantly during development and metamorphosis. Some, such as cuticle, odorant‐binding, and chemosensory proteins, showed stage‐specific expression in the hemolymph. In addition, the expression of several antimicrobial peptides exhibited their highest level of abundance in the hemolymph of the early pupal stage. These findings provide a comprehensive proteomic insight of the silkworm hemolymph and suggest additional molecular targets for studying insect metamorphosis.     

Mapping the eosinophil cationic protein antimicrobial activity by chemical and enzymatic cleavage

The eosinophil cationic protein (ECP) is a human antimicrobial protein involved in the host immune defense that belongs to the pancreatic RNase A family. ECP displays a wide range of antipathogen activities. The protein is highly cationic and its bactericidal activity is dependant on both cationic and hydrophobic surface exposed residues. Previous studies on ECP by site-directed mutagenesis indicated that the RNase activity is not essential for its bactericidal activity. To further understand the ECP bactericidal mechanism, we have applied enzymatic and chemical limited cleavage to search for active sequence determinants.Following a search for potential peptidases we selected the Lys-endoproteinase, which cleaves the ECP polypeptide at the carboxyl side of its unique Lys residue, releasing the N-terminal fragment (0-38).Chemical digestion using cyanogen bromide released several complementary peptides at the protein N-terminus. Interestingly, ECP treatment with cyanogen bromide represents a new example of selective chemical cleavage at the carboxyl side of not only Met but also Trp residues. Recombinant ECP was denatured and carboxyamidomethylated prior to enzymatic and chemical cleavage. Irreversible denaturation abolishes the protein bactericidal activity.The characterization of the digestion products by both enzymatic and chemical approaches identifies a region at the protein N-terminus, from residues 11 to 35, that retains the bactericidal activity. The most active fragment, ECP(0-38), is further compared to ECP derived synthetic peptides. The region includes previously identified stretches related to lipopolysaccharide binding and bacteria agglutination. The results contribute to define the shortest ECP minimized version that would retain its antimicrobial properties. The data suggest that the antimicrobial RNase can provide a scaffold for the selective release of cytotoxic peptides.     

Protein degradation machinery is present broadly during early development in the sea urchin

Ubiquitin-dependent proteosome-mediated proteolysis is an important pathway of degradation that controls the timed destruction of cellular proteins in all tissues. All intracellular proteins and many extracellular proteins are continually being hydrolyzed to their constituent amino acids as a result of their recognition by E3 ligases for specific targeting of ubiquitination. Gustavus is a member of an ECS-type E3 ligase which interacts with Vasa, a DEAD-box RNA helicase, to regulate its localization during sea urchin embryonic development, and Gustavus mRNA accumulation is highly localized and dynamic during development. We tested if the core complex for Gustavus function was present in the embryo and if other SOCS box proteins also had restricted expression profiles that would inform future research. Expression patterns of the key members of the proteasomal function, such as the E3 core complex which interacts with Gustavus, and other E3-SOCS box proteins, are widely spread and dynamic in early development of the embryo suggesting broad core complex availability in the proteasome degradation pathway and temporal/spatial enrichments of various E3 ligase dependent targeting mechanisms.