X hits on this document

68 views

0 shares

0 downloads

0 comments

15 / 22

Ribonuclease A

YNFEVL, binds to S-protein to form a complex with a Kd of 5.5 × 10-6 M, which is comparable to that of RNase S. The S-proteinYNFEVL complex has no detectable enzymatic activity, so the phage display system has revealed a true antagonist.

5. Semisynthetic Transaminases

The architecture of an enzyme can be used to modulate an unrelated chemical reaction.347 The architecture of RNase S has been shown to enhance a transamination reaction. In a variety of semisyn- thetic enzymes, Phe8 of C-peptide (which encom- passes residues 1-14348) has been replaced with a coenzyme-amino acid chimera containing a pyri- doxal349,350 or pyridoxamine351 side chain. The result- ing complexes with S-protein increase (albeit mod- estly) the rate and enantioselectivity of the conversion of an R-amino acid to an R-keto acid, or vice versa.

6. Protein Ubiquitination

RNase S has been used to explore the specificity of protein ubiquitination.352 A fusion between S15 and the ubiquitin-conjugating enzyme E2 directs crude cell extracts to attach ubiquitin to S-protein. This result demonstrates that a target protein can be ubiquitinated (and thereby fated for degradation) simply by appending an appropriate interaction domain onto a ubiquitin-conjugating enzyme.

7. ProteinProtein Interactions

The RNase S system has been used to demonstrate the utility of green fluorescent protein (GFP) in the revelation and characterization of protein-protein interactions.353 Recombinant DNA technology has been used to produce a fusion protein in which S15 is attached covalently to a GFP variant that re- sembles fluorescein in its excitation and emission wavelengths. The interaction of this fusion protein with S-protein has been analyzed by two distinct methods: fluorescence gel retardation and fluores- cence polarization. The fluorescence gel retardation assay is a rapid method to reveal a protein-protein interaction and to estimate the Kd of the resulting complex. The fluorescence polarization assay is an accurate method to evaluate Kd in a homogeneous solution and can be adapted for the high throughput screening of protein or peptide libraries. 354

XI. Molecular Evolution

The amino acid sequences of proteins that are homologous (that is, have a common evolutionary origin355) often vary between different species of organisms. This variation results from both selective adaptation356 and neutral drift.357,358 The conserva- tion (or divergence) of particular amino acid residues in homologous proteins can lend support to experi- mental findings as well as provoke new questions about protein structure and protein function. In addition, sequence data enables the reconstruction of the evolutionary history of a protein. This recon- struction is done by applying parsimony analysis359 to the aligned amino acid sequences. The result is a

Chemical Reviews, 1998, Vol. 98, No. 3 1059

phylogenetic tree that predicts the amino acid se- quences in ancestral organisms. 360

The amino acid sequences of RNase A homologues have been obtained from over 40 different verte- brates. (For reviews, see refs 20, 22, and 361.) RNase A has thus become a model system for elaborating the consequences of molecular evolution in vertebrate taxa. From these sequences and their organismal distribution, it is apparent that RNase A is a modern protein that is evolving rapidly. Using site-directed mutagenesis, several putative ancestors of RNase A have been produced to address issues in the evolution of vertebrate physiology. The evolutionary reconstruction of artiodactyl homo- logues of RNase A has been the subject of a recent review. 362 363,364 365

XII. Unusual Homologues

The functions typically ascribed to ribonucleases are to process and turnover cellular RNA and to degrade dietary RNA. Yet, some homologues of RNase A appear to have quite different biological roles. (For general reviews, see refs 366 and 367.) These homologues were discovered on the basis of their unusual activities. Only later, sometimes much later, were the proteins identified as ribonucleases.

Ribonucleases can be cytotoxic because cleaving RNA renders indecipherable its encoded information (eq 1). The cytotoxicity of ribonucleases was discov- ered in the 1950s. RNase A was shown then to be toxic to tumor cells, both in vitro368 and in vivo.369,370 Large doses of RNase A were used in these early studiesseffects were observed only after milligrams of enzyme were injected into solid tumors. Subse- quently, smaller doses of RNase A were found to have no effect. 371

Over 20 years ago, a homologue of RNase A was discovered in bull seminal plasma that is cytotoxic at low levels.372,373 In the past decade, even more cytotoxic homologues were isolated from the eggs of

374,375 376 377 the bullfrog Rana catesbeiana, paddy frog Rana japonica, leopard frog Rana pipiens. the Japanese rice and the Northern All of these Rana

ribonucleases are toxic to tumor cells in vitro with IC50 values near 1 µM.378,379 The mechanism of cytotoxicity could involve binding to cell-surface glycolipids, retrograde transport to the Golgi ap- paratus380 and the endoplasmic reticulum, translo- cation into the cytosol, and degradation of cellular RNA.381 A key to this last step is the evasion of RI (see section VIII), which binds tightly to RNase A but not to its cytotoxic homologues.378,382-384 Bovine seminal ribonuclease385,386 and the Rana ribonu- cleases387 have been the objects of recent reviews. Humans contain at least five homologues of RNase A (Figure 10).395 RNase 1 (which is from human p a n c r e a s ) 3 8 8 , 3 9 6 a n d R N a s e 4 ( w h i c h i s f r o m h u m a n l i v e r ) 3 9 1 , 3 9 7 a r e d i s t i n c t e n z y m e s plasma enzyme that promotes neovasculariza- . A n g i o g e n i n i s a

tion.389,398,399

Eosinophilic leukocytes contain RNase

2 (eosinophil-derived neuorotoxin; EDN), which is neurotoxic, and RNase 3 (eosinophil cationic protein; ECP) which has helminthotoxic and antibacterial activities.400 Human urine and human erythrocytes

Document info
Document views68
Page views68
Page last viewedMon Dec 05 04:54:10 UTC 2016
Pages22
Paragraphs1374
Words20315

Comments