Neil McLennan

Prion protein accumulation and neuroprotection in hypoxic brain damage

The function of the normal conformational isoform of prion protein, PrP(C), remains unclear although lines of research have suggested a role in the cellular response to oxidative stress. Here we investigate the expression of PrP(C) in hypoxic brain tissues to examine whether PrP(C) is in part regulated by neuronal stress. Cases of adult cerebral ischemia and perinatal hypoxic-ischemic injury in humans were compared with control tissues. PrP(C) immunoreactivity accumulates within neuronal processes in the penumbra of hypoxic damage in adult brain, and within neuronal soma in cases of perinatal hypoxic-ischemic injury, and in situ hybridization analysis suggests an up-regulation of PrP mRNA during hypoxia. Rodents also showed an accumulation of PrP(C) in neuronal soma within the penumbra of ischemic lesions. Furthermore, the infarct size in PrP-null mice was significantly greater than in the wild type, supporting the proposed role for PrP(C) in the neuroprotective adaptive cellular response to hypoxic injury.

GroE is vital for cell-wall synthesis.

Chaperone proteins help other proteins to fold. GroEL, the Escherichia coli form of the ubiquitous Cpn60 chaperonins, has a multimeric barrel-shaped structure with a central cavity, within which almost any protein can fold in vitro. But what does GroE (GroEL plus its co-chaperone GroES) fold in the cell? Why is it needed for cell survival? We report here the first definite identification of an essential, GroE-dependent E. coli protein, dihydropicolinate synthase (DapA), without which cell-wall synthesis fails.

The lytB Gene of Escherichia coli Is Essential and Specifies a Product Needed for Isoprenoid Biosynthesis

LytB and GcpE, because they are codistributed with other pathway enzymes, have been predicted to catalyze unknown steps in the nonmevalonate pathway for isoprenoid biosynthesis. We constructed a conditional Escherichia coli lytB mutant and found that LytB is essential for survival and that depletion of LytB results in cell lysis, which is consistent with a role for this protein in isoprenoid biosynthesis. Alcohols which can be converted to pathway intermediates beyond the hypothesized LytB step(s) support limited growth of E. coli lytB mutants. An informatic analysis of protein structure suggested that GcpE is a globular protein of the TIM barrel class and that LytB is also a globular protein. Possible biochemical roles for LytB and GcpE are suggested.

The strongly conserved carboxyl-terminus glycine-methionine motif of the Escherichia coli GroEL chaperonin is dispensable

The universally distributed heat‐shock proteins (HSPs) are divided into classes based on molecular weight and sequence conservation. The members of at least two of these classes, the HSP60s and the HSP70S, have chaperone activity. Most HSP60s and many HSP70s feature a striking motif at or near the carboxyl terminus which consists of a string of repeated glycine and methionine residues. We have altered the groEL gene (encoding the essential Escherichia coli HSP60 chaperonin) so that the protein produced lacks its 16 final (including nine gly, and five met) residues. This truncated product behaves like the intact protein in several in vitro tests, the only discernible difference between the two proteins being in the rate at which ATP is hydrolysed. GroELtr can substitute for GroEL in vivo although cells dependent for survival on the truncated protein survive slightly less well during the stationary phase of growth. Elevated levels of the wild‐type protein can suppress a number of temperature‐sensitive mutations; the truncated protein lacks this ability.

Male Infertility and DNA Damage in Doppel Knockout and Prion Protein/Doppel Double-Knockout Mice

The prion protein (PrP) and Doppel (Dpl) have many structural and biochemical properties in common, leading to the suggestion that the lack of an obvious phenotype in PrP-deficient mice maybe because of compensation by Dpl. To test this hypothesis and also investigate the function of Dpl we have generated Prnd(-/-) and Prnp(-/-)/Prnd(-/-) mouse lines. Both develop normally and display an identical male sterility phenotype that differs from that reported for another Prnd(-/-) mouse line. Sperm from both our mutant lines were present at normal concentrations, had normal motility, and no morphological abnormalities. Despite only rarely fertilizing oocytes in vivo, because of an inability to perform the acrosome reaction, mutant sperm were capable of fertilization in vitro, albeit at reduced rates compared to wild type. Elevated levels of oxidative DNA damage were found in both types of mutant sperm and resulting embryos failed at an early stage. Therefore we found no evidence that Dpl compensates for the loss of PrP function in mutant mouse lines, but it does have an important anti-oxidant function necessary for sperm integrity and male fertility.

In situ hybridization analysis of PrP mRNA in human CNS tissues.

Expression of the prion protein gene (Prnp) and production of the PrP protein are essential requirements for acquisition and spread of transmissible spongiform encephalopathies such as Creutzfeldt–Jakob disease (CJD) in humans. Here we have developed an in situ hybridization method for use on human post‐mortem central nervous system (CNS) tissues in order to determine those cell which are transcribing the Prnp gene and thus expressing PrP mRNA. Tissues from 11 adult individuals (age range 21–79 years) were analysed. Similar to previous studies in other animal systems, it was shown that PrP production occurs primarily in neuronal populations throughout the human brain. Neurones of the hippocampus, cortex, thalamus, cerebellum and medulla all synthesize PrP mRNA at readily detectable levels. No age‐related differences were observed between the cases studied. It was also found that the ependymal cells produced PrP mRNA; these were the only non‐neuronal cell type expressing the Prnp gene in the CNS. It is hoped that the information produced here will be helpful in understanding the pathology associated with CJD and other prion diseases in humans.

Protein folding in Escherichia coli: the chaperonin GroE and its substrates

A brief summary of the role of DnaK and GroE chaperones in protein folding precedes a discussion of the role of GroE in Escherichia coli. We consider its obligate substrates, the 8 that are both obligate and essential, and the prospects for constructing a mutant that could survive without it. Structural features of GroE-dependent polypeptides are also considered.

The tail of a chaperonin: the C‐terminal region of Esctierichia coli GroEL protein

The active form of the KSP60 molecular chaperone of Escherichia coli, GroEL, is a pair of seven‐membered rings. We have used site‐directed mutagenesis to construct forms of the 547‐amino‐acid monomer truncated at the C‐terminus. We show here that forms that are 520 amino acids long or longer are close to being fully functional. Removing one further amino acid, however, results in a protein, GroEL519, which retains little function. This truncated form is metabolically stable but is not recovered from the cell in particle form. When synthesized at high levels, it prevents the normal assembly of GroEL547 present in the same cell. When synthesized at low levels, it can be included, probably at low molar ratios, in particles formed by assembly‐competent forms of GroEL. This can be seen as partial complementation of the temperature‐sensitive mutant groEL44. We conclude that amino acid 520 is cruical for particle assembly. GroEL516 has in vivo properties similar to those of GroEL519, but the still shorter form, GroEL504, appears to be inactive.

Isolation and biochemical characterization of highly purified Escherichia coli molecular chaperone Cpn60 (GroEL) by affinity chromatography and urea-induced monomerization.

Isolated Escherichia coli molecular chaperone Cpn60 (GroEL) has been further purified from tightly bound substrate polypeptides by two different procedures: (i) group-specific affinity chromatography by using the triazine dye Procion yellow HE-3G as affinity ligand, and (ii) urea-induced monomerization and subsequent chromatography. Procion yellow binds specifically to aromatic amino-acid side chains present in the majority of proteins, but has no affinity to GroEL because of its low content of aromatic residues. Some GroEL-bound polypeptides are buried within the aqueous cavity of the GroEL oligomer, whereas others are exposed on its surface and available for affinity-ligand interactions and the complex is thereby retarded on Procion yellow columns. Pure substrate-free GroEL was obtained after ion-exchange chromatography of GroEL monomers followed by reassembly of the purified monomers into functional GroEL oligomers. The final preparation contained no substrate polypeptides bound to GroEL as judged by electrophoretic analysis and lack of tryptophan fluorescence. GroEL preparations also displayed two equally strong bands on native electrophoresis suggesting the presence of two conformers. Monomers of GroEL showed heterogeneity with respect to isoelectric point and molecular mass when analysed by MALDI-MS and electrophoresis under native and denaturing conditions respectively. By use of MALDI-MS, highly accurate molecular masses of wild-type and a truncated form of GroEL were determined and verified, by comparison with their respective gene sequences.

Properties of the cellular prion protein expressed in Xenopus oocytes.

The cellular prion protein (PrPC) from different species can be reproducibly expressed in Xenopus oocytes following injection of in vitro transcribed mRNAs. The level of PrPC accumulation increases with the amount of RNA injected and with the duration of incubation. PrPC expressed in oocytes is similar in size and abundance to PrPC protein in mouse brain and >100 ng of PrPC is expressed per oocyte allowing complete experiments to be carried out in single living cells. The protein is glycosylated, fully protease sensitive and expressed on the cell surface. Xenopus oocytes therefore provide a useful model system for the study of prion proteins and their associated disease processes.