Current Protein and Peptide Science - Volume 10, Issue 5, 2009

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β-Lactamases are the greatest single source of resistance to β-lactam antibiotics. For over 60 years, clinicians have seen a pattern whereby useful new β-lactam analogues are introduced but then select for new β-lactamases that cause resistance. Thus, penicillin G was undermined by swift accumulation of staphylococcal penicillinase, ampicillin by TEM- 1 enzyme and modern oxymino cephalosporins by “extended-spectrum” β-lactamases. Tony Fink's work contributed greatly to our understanding of the mechanisms and active site function of β-lactamases and this knowledge now informs the search for new β-lactams and β-lactamase inhibitors.

The discovery that the mechanism of β-lactam hydrolysis catalyzed by the class A (active site serinedependent) β-lactamases proceeds via an acyl-enzyme intermediate was made thirty years ago. Since this discovery, the active site circumstance that enables acylation of the active site serine and further enables hydrolytic deacylation of the acyl-serine intermediate, has received extraordinary scrutiny. The justification for this scrutiny is the direct relevance of the β-lactamases to the manifestation of bacterial resistance to the β-lactam antibiotics, and the subsequent (to the discovery of the β-lactamase acyl-enzyme) recognition of the direct evolutionary relationship between the serine β-lactamase acyl-enzyme, and the penicillin binding protein acyl-enzyme that is key to β-lactam antibiotic activity. This short review describes the early events leading to the recognition that serine β-lactamase catalysis proceeds via an acyl-enzyme intermediate, and summarizes several of the key mechanistic studies—including infrared spectroscopy, cryoenzymology, β- lactam design, and x-ray crystallography—that have been exploited to understand this pivotal catalytic intermediate.

Knowledge of the existence and structure of intermediates on the reaction pathway is necessary before specific details of the mechanism may be successfully resolved. However, enzymatic catalysis is an extremely fast process. This rapidity of enzyme-catalyzed reactions and the short life times of intermediates represent a major problem in studying the dynamic processes which occur during catalysis, as they prevent the accumulation of intermediates under normal conditions for concentrations and time periods required by most high-resolution structural methods. Therefore, a method that would utilize specific substrates but would permit the detection and characterization of intermediates was highly desired. As one of the approaches to overcome this problem the use of cryoenzymology to allow the accumulation and stabilization of intermediates at very low temperatures was proposed. This review describes the contribution of Prof. Anthony L. Fink to cryoenzymology and shows how his work shaped this exciting area.

During the past thirty years significant contributions to our understanding of the structural origins of the catalytic power of enzymes have come from solution and crystallographic studies of enzyme-substrate and enzymeintermediate complexes trapped at subzero temperatures, a field that was pioneered in large part by Anthony L. Fink and Pierre Douzou. Here I review, from a personal perspective, the history of crystallographic cryoenzymology, with an emphasis on the contributions of Tony Fink. The story has a moral: if you choose your collaborators based not only on their scientific prowess but also on their human qualities, the resulting friendships will enrich your life.

In a Nature article published in 1993, Anthony L. Fink and colleagues reported how Hsp70 chaperones use ATP binding in the presence of K+, rather than hydrolysis, to accelerate substrate release. As of the summer of 2008, his article had received 297 citations. I discuss here the influence Tony's iconic article has had on the chaperone field.

Stress, molecular crowding and mutations may jeopardize the native folding of proteins. Misfolded and aggregated proteins not only loose their biological activity, but may also disturb protein homeostasis, damage membranes and induce apoptosis. Here, we review the role of molecular chaperones as a network of cellular defences against the formation of cytotoxic protein aggregates. Chaperones favour the native folding of proteins either as “holdases”, sequestering hydrophobic regions in misfolding polypeptides, and/or as “unfoldases”, forcibly unfolding and disentangling misfolded polypeptides from aggregates. Whereas in bacteria, plants and fungi Hsp70/40 acts in concert with the Hsp100 (ClpB) unfoldase, Hsp70/40 is the only known chaperone in the cytoplasm of mammalian cells that can forcibly unfold and neutralize cytotoxic protein conformers. Owing to its particular spatial configuration, the bulky 70 kDa Hsp70 molecule, when distally bound through a very tight molecular clamp onto a 50-fold smaller hydrophobic peptide loop extruding from an aggregate, can locally exert on the misfolded segment an unfolding force of entropic origin, thus destroying the misfolded structures that stabilize aggregates. ADP/ATP exchange triggers Hsp70 dissociation from the ensuing enlarged unfolded peptide loop, which is then allowed to spontaneously refold into a closer-to-native conformation devoid of affinity for the chaperone. Driven by ATP, the cooperative action of Hsp70 and its co-chaperone Hsp40 may thus gradually convert toxic misfolded protein substrates with high affinity for the chaperone, into non-toxic, natively refolded, low-affinity products. Stress- and mutation-induced protein damages in the cell, causing degenerative diseases and aging, may thus be effectively counteracted by a powerful network of molecular chaperones and of chaperone-related proteases.

The structure of a globular protein is known to be affected by addition of acids or alkalis. The fact that a protein unfolds or at least denatures in solutions with extreme pH values was known for a long time. Prof. Anthony L. Fink (Tony) brought this field to a new level by showing that acid-unfolded proteins can partially regain their ordered structures in the presence of various anions. This review analyses his contributions to this branch of protein science. His studies provided an explanation for the molecular mechanisms underlying anion-induced refolding of acid-unfolded proteins. He was the first who clearly showed that different anions differed dramatically in their efficiency to bring about this refolding. This difference was shown to be manifested in the amounts of anions needed to complete a structural transition, in the degree of cooperativity of these transitions, and in the amounts of ordered structure induced in the acid-unfolded proteins at the completion of the corresponding transitions. He was also first who undoubtedly demonstrated that, at low pH, proteins can populate discrete partially folded conformations in the presence of different anions. His papers are highly cited, clearly showing that the work of Tony Fink on anion-induced folding of globular proteins made a great impact to the protein folding field.

Protein aggregation, being one of the hottest topics of modern protein science, is recognized now as a serious biomedical and biotechnological problem. Protein aggregation is considered as a causative factor (or at least an associated symptom) of a wide spectrum of human pathologies. Furthermore, aggregation and precipitation are known to trammel recombinant protein production, as well as to affect the manufacture, storage and delivery of proteinaceous drugs. Therefore, this topic attracts the serious attention of many researchers, a conclusion that follows from the average daily publication of 7-8 scientific papers dedicated to the various aspects of protein aggregation. However, the situation was rather different 15-20 years ago when it was believed that the formation of protein aggregates causing the irreversibility of unfolding or denaturation of some proteins was nothing more than an annoying experimental artifact, hampering the detailed characterization of the unfolding/denaturation processes. At that time, only a few laboratories (including the laboratory of Prof. Anthony L. Fink) seriously worked on understanding the molecular mechanisms of this “artifact”. In this review, we summarize some of the early work of Tony Fink on aggregation, of protein folding intermediates and on the analysis of the structural consequences of this process.

In a 1998 collaboration with Tony Fink, we coupled nanosecond circular dichroism methods (TRCD) with a CO-photolysis system for quickly triggering folding in cytochrome c (cyt c) in order to make the first time-resolved far- UV CD measurement of early secondary structure formation in a protein. The small signal observed in that initial study, ∼10% of native helicity, became the seed for increasingly robust results from subsequent studies bringing additional natural and magnetic circular polarization dichroism and optical rotatory dispersion detection methods (e.g., TRORD, TRMCD, and TRMORD), coupled to fast photolysis and photoreduction triggers, to the study of early folding events. Nanosecond polarization methods are reviewed here in the context of the range of initiation methods and structuresensitive probes currently available for fast folding studies. We also review the impact of experimental results from fast polarization studies on questions in folding dynamics such as the possibility of multiple folding pathways implied by energy landscape models, the sequence dependence of ultrafast helix formation, and the simultaneity of chain collapse and secondary structure formation implicit in molten globule models for kinetic folding intermediates.

When caused by multiplication mutations of its gene, increased expression of α-synuclein is associated with familial parkinsonism. Here we discuss the possibility that other mechanisms of α-synuclein elevation contribute to the pathogenesis of idiopathic, sporadic Parkinson's disease and other human synucleinopathies. Environmental (e.g. toxic exposures) and genetic (e.g. gene polymorphisms) risk factors, on the background of normal aging, are likely to enhance vulnerability to neurodegenerative processes. Current evidence suggests that an increased level of neuronal α-synuclein may represent a key pathogenetic event common to these risk factors. Higher protein expression could underlie a gain of toxic properties of α-synuclein (e.g. enhanced tendency to aggregate) that predispose and/or directly contribute to neuronal demise. An important corollary to this latter concept is that a promising therapeutic approach against Parkinson's and other neurodegenerative diseases is to prevent α-synuclein accumulation. Means to achieve this include (i) the use of RNA interference to suppress α-synuclein expression, (ii) the induction of neuronal pathways of protein degradation (e.g. macroautophagy) involved in α-synuclein clearance, and (iii) the development of anti-aggregation agents counteracting the formation of toxic oligomeric or fibrillar forms of the protein.

Parkinson's disease (PD) is a slowly progressive movement disorder that results from the loss of dopaminergic neurons in the substantia nigra, a small area of cells in the mid-brain. PD is a multifactorial disorder with unknown etiology, in which both genetic and environmental factors play important roles. Substantial evidence links α-synuclein, a small highly conserved presynaptic protein with unknown function, to both familial and sporadic PD. Rare familial cases of PD are associated with missense point mutations in α-synuclein, or with the hyper-expression of the wild type protein due to its gene duplication/triplication. Furthermore, α-synuclein was identified as the major component of amyloid fibrils found in Lewy body and Lewy neurites, the characteristic proteinaceous deposits that are the diagnostic hallmarks of PD. α- Synuclein is abundant in various regions of the brain and has two closely related homologs, β-synuclein and γ-synuclein. When isolated in solution, the protein is intrinsically disordered, but in the presence of lipid surfaces α-synuclein adopts a highly helical structure that is believed to mediate its normal function(s). A number of different conformational states of α-synuclein have been observed. Besides the membrane-bound form, other critical conformations include a partiallyfolded state that is a key intermediate in aggregation and fibrillation, various oligomeric species, and fibrillar and amorphous aggregates. A number of intrinsic and extrinsic factors that either accelerate or inhibit the rate of α-synuclein aggregation and fibrillation in vitro are known. There is a strong correlation between the conformation of α-synuclein (induced by various factors) and its rate of fibrillation. The aggregation process appears to be branched, with one pathway leading to fibrils and another to oligomeric intermediates that may ultimately form amorphous deposits. The molecular basis of Parkinson's disease appears to be tightly coupled to the aggregation of α-synuclein and the factors that affect its conformation. This review focuses on the contributions of Prof. Anthony L. Fink to the field and presents some recent developments in this exciting area.

Systemic light chain amyloidosis (AL) is one of several protein misfolding diseases and is characterized by extracellular deposition of immunoglobulin light chains in the form of amyloid fibrils [1]. Immunoglobulin (Ig) proteins consist of two light chains (LCs) and two heavy chains (HCs) that ordinarily form a heterotetramer which is secreted by a plasma cell. In AL, however, a monoclonal plasma cell population produces an abundance of a pathogenic LC protein. In this case, not all of the LCs pair with the HCs, and free LCs are secreted into circulation. The LC-HC dimer is very stable, and losing this interaction may result in an unstable LC protein [2]. Additionally, somatic mutations are thought to cause amyloidogenic proteins to be less stable compared to non-amyloidogenic proteins [3-5], leading to protein misfolding and amyloid fibril formation. The amyloid fibrils cause tissue damage and cell death, leading to patient death within 12-18 months if left untreated [6]. Current therapies are harsh and not curative, including chemotherapy and autologous stem cell transplants. Studies of protein pathogenesis and fibril formation mechanisms may lead to better therapies with an improved outlook for patient survival. Much has been done to determine the molecular factors that make a particular LC protein amyloidogenic and to elucidate the mechanism of amyloid fibril formation. Anthony Fink's work, particularly with discerning the role of intermediates in the fibril formation pathway, has made a remarkable impact in the field of amyloidosis research. This review provides a general overview of the current state of AL research and also attempts to capture the most recent ideas and knowledge generated from the Fink laboratory.

The therapeutic importance of gaining a thorough knowledge on insulin fibrillation in relation to type I diabetes has lead to six decades of studies focusing on its formation kinetics and structural characteristics. Insulin fibrils feature characteristics common to amyloid fibrils such as an elongated morphology, characteristic cross-β diffraction pattern, Thioflavin T fluorescence, and Congo Red birefringence. A full understanding of the fibrillation process requires structural elucidation of every species and determination of the kinetics of interconversion between species on the reaction pathway. Therefore, describing the underlying mechanism is complicated and different mechanisms have been proposed. In the recent years increased knowledge has been obtained on the importance of prefibrillar oligomeric species present during the process. A solution structure of such a species and also low-resolution structures of mature insulin fibrils have been obtained as well as high-resolution structures of two insulin hexa-peptide segments forming cross-β sheet structures. However, it still remains to be elucidated whether these cross-β structures corresponds to the structure in the fibril formed from the full-length protein. Different morphologies of insulin fibrils are observed depending on the arrangement of the protofilament, and even circular amyloids and spherulites composed of a core with many fibrils extending from it have been observed. This review will mainly focus on the structure of the species present during the insulin fibrillation process such as the partially unfolded monomeric intermediate, prefibrillar oligomeric species, and the insulin fibrils. Furthermore, it will address the formation mechanism and potential inhibition of the fibrillation process.

Current research suggests that the function of the prion protein (PrP) is linked to its ability to bind copper. PrP is implicated in copper regulation, copper buffering and copper-dependent signaling. Moreover, in the development of prion disease, copper may modulate the rate of protein misfolding. PrP possesses a number of copper sites, each with distinct chemical characteristics. Most studies thus far have concentrated on elucidating chemical features of the octarepeat region (residues 60-91, hamster sequence), which can take up to four equivalents of copper, depending on the ratio of Cu2+ to protein. However, other sites have been proposed, including those at histidines 96 and 111, which are adjacent to the octarepeats, and also at histidines within PrP's folded C-terminal domain. Here, we review the literature of these copper sites extrinsic to the octarepeat region and add new findings and insights from recent experiments.