Arwa Kurabi

Weak lignin-binding enzymes: a novel approach to improve activity of cellulases for hydrolysis of lignocellulosics.

Economic barriers preventing commercialization of lignocellulose-to-ethanol bioconversion processes include the high cost of hydrolytic enzymes. One strategy for cost reduction is to improve the specific activities of cellulases by genetic engineering. However, screening for improved activity typically uses “ideal” cellulosic substrates, and results are not necessarily applicable to more realistic substrates such as pretreated hardwoods and softwoods. For lignocellulosic substrates, nonproductive binding and inactivation of enzymes by the lignin component appear to be important factors limiting catalytic efficiency. A better understanding of these factors could allow engineering of cellulases with improved activity based on reduced enzyme-lignin interaction (“weak lignin-binding cellulases”). To prove this concept, we have shown that naturally occurring cellulases with similar catalytic activity on a model cellulosic substrate can differ significantly in their affinities for lignin. Moreover, although cellulose-binding domains (CBDs) are hydrophobic and probably participate in lignin binding, we show that cellulases lacking CBDs also have a high affinity for lignin, indicating the presence of lignin-binding sites on the catalytic domain.

Weak Lignin-Binding Enzymes

Economic barriers preventing commercialization of lignocellulose-to-ethanol bioconversion processes include the high cost of hydrolytic enzymes. One strategy for cost reduction is to improve the specific activities of cellulases by genetic engineering. However, screening for improved activity typically uses “ideal” cellulosic substrates, and results are not necessarily applicable to more realistic substrates such as pretreated hardwoods and softwoods. For lignocellulosic substrates, nonproductive binding and inactivation of enzymes by the lignin component appear to be important factors limiting catalytic efficiency. A better understanding of these factors could allow engineering of cellulases with improved activity based on reduced enzyme-lignin interaction (“weak lignin-binding cellulases”). To prove this concept, we have shown that naturally occurring cellulases with similar catalytic activity on a model cellulosic substrate can differ significantly in their affinities for lignin. Moreover, although cellulose-binding domains (CBDs) are hydrophobic and probably participate in lignin binding, we show that cellulases lacking CBDs also have a high affinity for lignin, indicating the presence of lignin-binding sites on the catalytic domain.

Esophageal cancer related gene-4 is a choroid plexus-derived injury response gene: evidence for a biphasic response in early and late brain injury.

By virtue of its ability to regulate the composition of cerebrospinal fluid (CSF), the choroid plexus (CP) is ideally suited to instigate a rapid response to traumatic brain injury (TBI) by producing growth regulatory proteins. For example, Esophageal Cancer Related Gene-4 (Ecrg4) is a tumor suppressor gene that encodes a hormone-like peptide called augurin that is present in large concentrations in CP epithelia (CPe). Because augurin is thought to regulate senescence, neuroprogenitor cell growth and differentiation in the CNS, we evaluated the kinetics of Ecrg4 expression and augurin immunoreactivity in CPe after CNS injury. Adult rats were injured with a penetrating cortical lesion and alterations in augurin immunoreactivity were examined by immunohistochemistry. Ecrg4 gene expression was characterized by in situ hybridization. Cell surface augurin was identified histologically by confocal microscopy and biochemically by sub-cellular fractionation. Both Ecrg4 gene expression and augurin protein levels were decreased 24–72 hrs post-injury but restored to uninjured levels by day 7 post-injury. Protein staining in the supraoptic nucleus of the hypothalamus, used as a control brain region, did not show a decrease of auguin immunoreactivity. Ecrg4 gene expression localized to CPe cells, and augurin protein to the CPe ventricular face. Extracellular cell surface tethering of 14 kDa augurin was confirmed by cell surface fractionation of primary human CPe cells in vitro while a 6–8 kDa fragment of augurin was detected in conditioned media, indicating release from the cell surface by proteolytic processing. In rat CSF however, 14 kDa augurin was detected. We hypothesize the initial release and proteolytic processing of augurin participates in the activation phase of injury while sustained Ecrg4 down-regulation is dysinhibitory during the proliferative phase. Accordingly, augurin would play a constitutive inhibitory function in normal CNS while down regulation of Ecrg4 gene expression in injury, like in cancer, dysinhibits proliferation.

Ecrg4 Attenuates the Inflammatory Proliferative Response of Mucosal Epithelial Cells to Infection

We report an inverse relationship between expression of the orphan candidate tumor suppressor gene esophageal cancer related gene 4 (Ecrg4), and the mucosal epithelial cell response to infection in the middle ear (ME). First, we found constitutive Ecrg4 mRNA expression in normal, quiescent ME mucosa that was confirmed by immunostainning of mucosal epithelial cells and immunoblotting of tissue lysates for the 14 kDa Ecrg4 protein. Upon experimental ME infection, Ecrg4 gene expression rapidly decreased by over 80%, between 3 to 48 hrs, post infection. When explants of this infected mucosa were placed in culture and transduced with an adenovirus (AD) encoding Ecrg4 gene (ADEcrg4), the proliferative and migratory responses of mucosal cells were significantly inhibited. ADEcrg4 transduction of control explants from uninfected MEs had no effect on basal growth and migration. Over-expression of Ecrg4 in vivo, by pre-injecting MEs with ADEcrg4 48 hrs prior to infection, prevented the natural down-regulation of Ecrg4, reduced mucosal proliferation and prevented inflammatory cell infiltration normally observed after infection. Taken together, these data support a hypothesis that Ecrg4 plays a role in coordinating the inflammatory and proliferative response to infection of mucosal epithelium suggesting a possible mechanism for its putative anti-tumor activity.

A nuclear localization signal at the SAM-SAM domain interface of AIDA-1 suggests a requirement for domain uncoupling prior to nuclear import.

The neuronal scaffolding protein AIDA-1 is believed to act as a convener of signals arising at postsynaptic densities. Among the readily identifiable domains in AIDA-1, two closely juxtaposed sterile alpha motif (SAM) domains and a phosphotyrosine binding domain are located within the C-terminus of the longest splice variant and exclusively in four shorter splice variants. As a first step towards understanding the possible emergent properties arising from this assembly of ligand binding domains, we have used NMR methods to solve the first structure of a SAM domain tandem. Separated by a 15-aa linker, the two SAM domains are fused in a head-to-tail orientation that has been observed in other hetero- and homotypic SAM domain structures. The basic nuclear import signal for AIDA-1 is buried at the interface between the two SAM domains. An observed disparity between the thermal stabilities of the two SAM domains suggests a mechanism whereby the second SAM domain decouples from the first SAM domain to facilitate translocation of AIDA-1 to the nucleus.

Cellular mechanisms of noise-induced hearing loss

Exposure to intense sound or noise can result in purely temporary threshold shift (TTS), or leave a residual permanent threshold shift (PTS) along with alterations in growth functions of auditory nerve output. Recent research has revealed a number of mechanisms that contribute to noise-induced hearing loss (NIHL). The principle cause of NIHL is damage to cochlear hair cells and associated synaptopathy. Contributions to TTS include reversible damage to hair cell (HC) stereocilia or synapses, while moderate TTS reflects protective purinergic hearing adaptation. PTS represents permanent damage to or loss of HCs and synapses. While the substrates of HC damage are complex, they include the accumulation of reactive oxygen species and the active stimulation of intracellular stress pathways, leading to programmed and/or necrotic cell death. Permanent damage to cochlear neurons can also contribute to the effects of NIHL, in addition to HC damage. These mechanisms have translational potential for pharmacological intervention and provide multiple opportunities to prevent HC damage or to rescue HCs and spiral ganglion neurons that have suffered injury. This paper reviews advances in our understanding of cellular mechanisms that contribute to NIHL and their potential for therapeutic manipulation.

Solution structure of the carboxy-terminal Tudor domain from human Coilin

The Cajal body is a dynamic eukaryotic nuclear organelle that is known primarily as an organizational center for the assembly of snRNAs involved in transcript splicing. One of the most critical components of the Cajal body is the scaffolding protein, Coilin. Here, we demonstrate by NMR methods that the carboxy‐terminal region contains a Tudor domain. The Tudor domain is atypical due to the presence of several unstructured loops, one greater than thirty amino acids in length. Tudor domains have been noted previously to bind DNA, RNA and modified amino acids. The absence of these sequence and structural signatures in the Coilin Tudor domain supporting these established functions suggests an alternative role.

The inflammasome adaptor ASC contributes to multiple innate immune processes in the resolution of otitis media

This study was designed to understand the contribution of the inflammasome and IL-1β activation in otitis media (OM). We examined the middle ear (ME) response to non-typeable Haemophilus influenzae (NTHi) in wild type (WT) mice using gene microarrays and a murine model of acute OM. Expression of members of the NOD domain-like receptor family of inflammasome genes was significantly up-regulated early in NTHi infection of the ME, potentially activating specific downstream regulatory cascades that contribute to the proliferative inflammatory response observed during OM. Expression of the pro-forms of the inflammasome targets IL-1β and IL-18 were also up-regulated. To evaluate the role of inflammasome-mediated cytokine maturation, NTHi-induced OM was examined in Asc−/−-deficient mice and compared with that seen in WT mice. Mice lacking the Asc gene showed near absence of IL-1β maturation in the ME and a reduction in leukocyte recruitment and infiltration to the cavity, and their macrophages exhibited reduced phagocytosis of NTHi. These inflammatory defects were linked to an increase in the degree and duration of mucosal epithelial hyperplasia in the ME of Asc−/− mice, as well as a delay in bacterial clearance from their MEs. These data demonstrate an important role for the inflammasome and cytokine processing in the course and resolution of OM.

Esophageal cancer-related gene 4 at the interface of injury, inflammation, infection, and malignancy.

In humans, esophageal cancer-related gene 4 (ECRG4) is encoded by four exons in the c2orf40 locus of chromosome 2. Translation of ECRG4 messenger ribonucleic acid produces a 148 amino acid-secreted 17 KDa protein that is then processed to 14, ten, eight, six, four, and two KDa peptides, depending on the cell in which the gene is expressed. As hypermethylation at the c2orf40 locus inhibits ECRG4 gene expression in many epithelial cancers, several investigators have speculated that ECRG4 is a candidate tumor suppressor. Indeed, overexpression of ECRG4 inhibits cell proliferation in vitro, but it also has a wide range of effects in vivo beyond its antitumor activity. ECRG4 overexpression affects apoptosis, senescence, cell migration, inflammation, injury, and infection responsiveness. ECRG4 activities also depend on its cellular localization, secretion, and post-translational processing. These cytokine/chemokine-like characteristics argue that ECRG4 is not a traditional candidate tumor suppressor gene, as originally predicted by its downregulation in cancer. We review how insights into the regulation of ECRG4 gene expression, knowledge of its primary structure, and the study of its emerging physiological functions come together to support a much more complex role for ECRG4 at the interface of inflammation, infection, and malignancy.

Discovery of a Biological Mechanism of Active Transport through the Tympanic Membrane to the Middle Ear

Otitis media (OM) is a common pediatric disease for which systemic antibiotics are often prescribed. While local treatment would avoid the systemic treatment side-effects, the tympanic membrane (TM) represents an impenetrable barrier unless surgically breached. We hypothesized that the TM might harbor innate biological mechanisms that could mediate trans-TM transport. We used two M13-bacteriophage display biopanning strategies to search for mediators of trans-TM transport. First, aliquots of linear phage library displaying 1010th 12mer peptides were applied on the TM of rats with active bacterial OM. The middle ear (ME) contents were then harvested, amplified and the preparation re-applied for additional rounds. Second, the same naïve library was sequentially screened for phage exhibiting TM binding, internalization and then transit. Results revealed a novel set of peptides that transit across the TM to the ME in a time and temperature dependent manner. The peptides with highest transport capacities shared sequence similarities. Historically, the TM was viewed as an impermeable barrier. However, our studies reveal that it is possible to translocate peptide-linked small particles across the TM. This is the first comprehensive biopanning for the isolation of TM transiting peptidic ligands. The identified mechanism offers a new drug delivery platform into the ME.