Core principles of intramembrane proteolysis: comparison of rhomboid and site-2 family proteases

Cleavage of proteins within their membrane-spanning segments is an ancient regulatory mechanism that has evolved to control a myriad of cellular processes in all forms of life. Although three mechanistic families of enzymes have been discovered that catalyze hydrolysis within the water-excluding environment of the membrane, how they achieve this improbable reaction has been both a point of controversy and skepticism. The crystal structures of rhomboid and site-2 protease, two different classes of intramembrane proteases, have been solved recently. Combined with current biochemical analyses, this advance provides an unprecedented view of how nature has solved the problem of facilitating hydrolysis within membranes in two independent instances. We focus on detailing the similarities between these unrelated enzymes to define core biochemical principles that govern this conserved regulatory mechanism.     

A family of Rhomboid intramembrane proteases activates all Drosophila membrane-tethered EGF ligands

Drosophila has three membrane-tethered epidermal growth factor (EGF)-like proteins: Spitz, Gurken and Keren. Spitz and Gurken have been genetically confirmed to activate the EGF receptor, but Keren is uncharacterized. Spitz is activated by regulated intracellular translocation and cleavage by the transmembrane proteins Star and the protease Rhomboid-1, respectively. Rhomboid-1 is a member of a family of seven similar proteins in Drosophila. We have analysed four of these: all are proteases that can cleave Spitz, Gurken and Keren, and all activate only EGF receptor signalling in vivo. Star acts as an endoplasmic reticulum (ER) export factor for all three. The importance of this translocation is highlighted by the fact that when Spitz is cleaved by Rhomboids in the ER it cannot be secreted. Keren activates the EGF receptor in vivo, providing strong evidence that it is a true ligand. Our data demonstrate that all membrane-tethered EGF ligands in Drosophila are activated by the same strategy of cleavage by Rhomboids, which are ancient and widespread intramembrane proteases. This is distinct from the metalloprotease-induced activation of mammalian EGF-like ligands.     

Computational prediction of structure, substrate binding mode, mechanism, and rate for a malaria protease with a novel type of active site

The histo-aspartic protease (HAP) from the malaria parasite P. falciparum is one of several new promising targets for drug intervention. The enzyme possesses a novel type of active site, but its 3D structure and mechanism of action are still unknown. Here we use a combination of homology modeling, automated docking searches, and molecular dynamics/reaction free energy profile simulations to predict the enzyme structure, conformation of bound substrate, catalytic mechanism, and rate of the peptide cleavage reaction. We find that the computational tools are sufficiently reliable both for identifying substrate binding modes and for distinguishing between different possible reaction mechanisms. It is found that the favored pathway only involves direct participation by the catalytic aspartate, with the neighboring histidine providing critical stabilization (by a factor of approximately 10000) along the reaction. The calculated catalytic rate constant of about 0.1 s(-1) for a hexapeptide substrate derived from the alpha chain of human hemoglobin is in excellent agreement with experimental kinetic data for a similar peptide fragment.     

An internal water-retention site in the rhomboid intramembrane protease GlpG ensures catalytic efficiency

Rhomboid proteases regulate key cellular pathways, but their biochemical mechanism including how water is made available to the membrane-immersed active site remains ambiguous. We performed four prolonged molecular dynamics simulations initiated from both gate-open and gate-closed states of Escherichia coli rhomboid GlpG in a phospholipid bilayer. GlpG was notably stable in both gating states, experiencing similar tilt and local membrane thinning, with no observable gating transitions, highlighting that gating is rate-limiting. Analysis of dynamics revealed rapid loss of crystallographic waters from the active site, but retention of a water cluster within a site formed by His141, Ser181, Ser185, and/or Gln189. Experimental interrogation of 14 engineered mutants revealed an essential role for at least Gln189 and Ser185 in catalysis with no effect on structural stability. Our studies indicate that spontaneous water supply to the intramembrane active site of rhomboid proteases is rare, but its availability for catalysis is ensured by an unanticipated active site element, the water-retention site.     

Automating human intuition for protein design

In the design of new enzymes and binding proteins, human intuition is often used to modify computationally designed amino acid sequences prior to experimental characterization. The manual sequence changes involve both reversions of amino acid mutations back to the identity present in the parent scaffold and the introduction of residues making additional interactions with the binding partner or backing up first shell interactions. Automation of this manual sequence refinement process would allow more systematic evaluation and considerably reduce the amount of human designer effort involved. Here we introduce a benchmark for evaluating the ability of automated methods to recapitulate the sequence changes made to computer‐generated models by human designers, and use it to assess alternative computational methods. We find the best performance for a greedy one‐position‐at‐a‐time optimization protocol that utilizes metrics (such as shape complementarity) and local refinement methods too computationally expensive for global Monte Carlo (MC) sequence optimization. This protocol should be broadly useful for improving the stability and function of designed binding proteins. Proteins 2014; 82:858–866. © 2013 Wiley Periodicals, Inc.     

Combined action of virus injection and local tumor irradiation on tumor growth in mice

The dynamics of SCCVII transplantable tumor growth in C3H/H mice was determined after local tumor irradiation and/or virus (NDV LaSota) i.p. injection. The virus applied alone significantly suppressed tumor growth, particularly until the 19th day after tumor transplantation. Local irradiation with 30 Gy resulted in tumor disappearance followed with its regrowth about 15 days later. However, if the virus was injected after the irradiation, there was no tumor growth until the end of the 31 day observation period. It should be noted that virus application prior to local irradiation did not have any additional influence on tumor growth. Thus, the pronounced efficacy of virus applied after tumor irradiation deserves attention. It is possible that the virus injected after irradiation induced a chain of cytokine production joining the action of tumor destruction induced by irradiation. This should be further studied in clarifying the approaches to combined tumor therapy with possible cell-free vaccine production.     

In vitro and in vivo antitumour effects of phenylboronic acid against mouse mammary adenocarcinoma 4T1 and squamous carcinoma SCCVII cells

The cytotoxic activity of phenylboroxine acid was evaluated in vitro on mouse mammary adenocarcinoma 4T1, mouse squamous cell carcinoma SCCVII, hamster lung fibroblast V79 and mouse dermal fibroblasts L929 cell lines. The cytotoxic effects were dose dependent for all tested tumour and non-tumour cell lines. Under in vivo conditions, three application routes of phenylboronic acid were studied: intra-peritoneal (i.p.), intra-tumour (i.t.) and per-oral. After tumour transplantation in syngeneic mice, phenylboronic acid was shown to slow the growth of both tumour cell lines (4T1 and SCCVII) compared with the control. The inhibitory effects were pronounced during the application of phenylboronic acid. For both tested tumour cell lines, the most prominent antitumour effect was obtained by intraperitoneal administration, followed significantly by oral administration.