Structural and Functional Requirements for Activity of the Tim9–Tim10 Complex in Mitochondrial Protein Import

The Tim9–Tim10 complex plays an essential role in mitochondrial protein import by chaperoning select hydrophobic precursor proteins across the intermembrane space. How the complex interacts with precursors is not clear, although it has been proposed that Tim10 acts in substrate recognition, whereas Tim9 acts in complex stabilization. In this study, we report the structure of the yeast Tim9–Tim10 hexameric assembly determined to 2.5 Å and have performed mutational analysis in yeast to evaluate the specific roles of Tim9 and Tim10. Like the human counterparts, each Tim9 and Tim10 subunit contains a central loop flanked by disulfide bonds that separate two extended N- and C-terminal tentacle-like helices. Buried salt-bridges between highly conserved lysine and glutamate residues connect alternating subunits. Mutation of these residues destabilizes the complex, causes defective import of precursor substrates, and results in yeast growth defects. Truncation analysis revealed that in the absence of the N-terminal region of Tim9, the hexameric complex is no longer able to efficiently trap incoming substrates even though contacts with Tim10 are still made. We conclude that Tim9 plays an important functional role that includes facilitating the initial steps in translocating precursor substrates into the intermembrane space.     

X-ray structures of the signal recognition particle receptor reveal targeting cycle intermediates

The signal recognition particle (SRP) and its conjugate receptor (SR) mediate cotranslational targeting of a subclass of proteins destined for secretion to the endoplasmic reticulum membrane in eukaryotes or to the plasma membrane in prokaryotes. Conserved active site residues in the GTPase domains of both SRP and SR mediate discrete conformational changes during formation and dissociation of the SRP.SR complex. Here, we describe structures of the prokaryotic SR, FtsY, as an apo protein and in two different complexes with a non-hydrolysable GTP analog (GMPPNP). These structures reveal intermediate conformations of FtsY containing GMPPNP and explain how the conserved active site residues position the nucleotide into a non-catalytic conformation. The basis for the lower specificity of binding of nucleotide in FtsY prior to heterodimerization with the SRP conjugate Ffh is also shown. We propose that these structural changes represent discrete conformational states assumed by FtsY during targeting complex formation and dissociation.     

Evolution of mitochondrial protein biogenesis

Mitochondria and the nucleus are key features that distinguish eukaryotic cells from prokaryotic cells. Mitochondria originated from a bacterium that was endosymbiotically taken up by another cell more than a billion years ago. Subsequently, most mitochondrial genes were transferred and integrated into the host cell's genome, making the evolution of pathways for specific import of mitochondrial proteins necessary. The mitochondrial protein translocation machineries are composed of numerous subunits. Interestingly, many of these subunits are at least in part derived from bacterial proteins, although only few of them functioned in bacterial protein translocation. We propose that the primitive α-proteobacterium, which was once taken up by the eukaryote ancestor cell, contained a number of components that were utilized for the generation of mitochondrial import machineries. Many bacterial components of seemingly unrelated pathways were integrated to form the modern cooperative mitochondria-specific protein translocation system.     

The Caenorhabditis elegans bus-2 Mutant Reveals a New Class of O-Glycans Affecting Bacterial Resistance

Microbacterium nematophilum causes a deleterious infection of the C. elegans hindgut initiated by adhesion to rectal and anal cuticle. C. elegans bus-2 mutants, which are resistant to M. nematophilum and also to the formation of surface biofilms by Yersinia sp., carry genetic lesions in a putative glycosyltransferase containing conserved domains of core-1 β1,3-galactosyltransferases. bus-2 is predicted to act in the synthesis of core-1 type O-glycans. This observation implies that the infection requires the presence of host core-1 O-glycoconjugates and is therefore carbohydrate-dependent. Chemical analysis reported here reveals that bus-2 is indeed deficient in core-1 O-glycans. These mutants also exhibit a new subclass of O-glycans whose structures were determined by high performance tandem mass spectrometry; these are highly fucosylated and have a novel core that contains internally linked GlcA. Lectin studies showed that core-1 glycans and this novel class of O-glycans are both expressed in the tissue that is infected in the wild type worms. In worms having the bus-2 genetic background, core-1 glycans are decreased, whereas the novel fucosyl O-glycans are increased in abundance in this region. Expression analysis using a red fluorescent protein marker showed that bus-2 is expressed in the posterior gut, cuticle seam cells, and spermatheca, the first two of which are likely to be involved in secreting the carbohydrate-rich surface coat of the cuticle. Therefore, in the bus-2 background of reduced core-1 O-glycans, the novel fucosyl glycans likely replace or mask remaining core-1 ligands, leading to the resistance phenotype. There are more than 35 Microbacterium species, some of which are pathogenic in man. This study is the first to analyze the biochemistry of adhesion to a host tissue by a Microbacterium species.     

The post-registration monitoring of glyphosate-treated plants using anecic earthworms

Glyphosate N-(phosphonomethyl) glycine is a widely-used herbicide in agriculture. The anecic earthworm, Lumbricus terrestris feeds and forages for surface plant materials meaning that this species has a unique and direct exposure to agrichemicals. At the recommended product rates, significantly (F_1,44 = 8.67, p = .005) higher numbers of L. terrestris middens were found in the glyphosate treated areas of an arable crop field. Laboratory feeding assays using field aged plant materials indicated that previous glyphosate treatment was a statistically significant factor affecting earthworm L. terrestris biomass (F₁,₁₂ = 5.75, p = .03). Negligible glyphosate residues were detectable, and the field aged plant materials were encrusted with fungal hyphae. This suggests that glyphosate influences the colonisation of plant material by a litter-fungus complex which improves the food quality to earthworms. Concentrations of epoxiconazole, a fungicide, were detected in some plant materials and may influence overall food quality to earthworms. Glyphosate treatment on fresh volunteer plant leaves (unwanted crop seedlings) was not a statistically significant factor affecting earthworm L. terrestris biomass (F₁,₆ = 0.16, p = .92). These results indicate fungal communities influence feeding behaviours, and plant materials are a direct source of agrichemicals to anecic earthworms.     

Fabs enable single particle cryoEM studies of small proteins

In spite of its recent achievements, the technique of single particle electron cryomicroscopy (cryoEM) has not been widely used to study proteins smaller than 100 kDa, although it is a highly desirable application of this technique. One fundamental limitation is that images of small proteins embedded in vitreous ice do not contain adequate features for accurate image alignment. We describe a general strategy to overcome this limitation by selecting a fragment antigen binding (Fab) to form a stable and rigid complex with a target protein, thus providing a defined feature for accurate image alignment. Using this approach, we determined a three-dimensional structure of an ～65 kDa protein by single particle cryoEM. Because Fabs can be readily generated against a wide range of proteins by phage display, this approach is generally applicable to study many small proteins by single particle cryoEM.     

Systems biological approach to investigate the lack of familial link between Down's Syndrome & Neural Tube Disorders

Systems Biology involves the study of the interactions of biological systems and ultimately their functions. Down''s syndrome (DS) is one of the most common genetic disorders which are caused by complete, or occasionally partial, triplication of chromosome 21, characterized by cognitive and language dysfunction coupled with sensory and neuromotor deficits. Neural Tube Disorders (NTDs) are a group of congenital malformations of the central nervous system and neighboring structures related to defective neural tube closure during the first trimester of pregnancy usually occurring between days 18-29 of gestation. Several studies in the past have provided considerable evidence that abnormal folate and methyl metabolism are associated with onset of DS & NTDs. There is a possible common etiological pathway for both NTDs and Down''s syndrome. But, various research studies over the years have indicated very little evidence for familial link between the two disorders. Our research aimed at the gene expression profiling of microarray datasets pertaining to the two disorders to identify genes whose expression levels are significantly altered in these conditions. The genes which were 1.5 fold unregulated and having a p-value <0.05 were filtered out and gene interaction network were constructed for both NTDs and DS. The top ranked dense clique for both the disorders were recognized and over representation analysis was carried out for each of the constituent genes. The comprehensive manual analysis of these genes yields a hypothetical understanding of the lack of familial link between DS and NTDs. There were no genes involved with folic acid present in the dense cliques. Only – CBL, EGFR genes were commonly present, which makes the allelic variants of these genes – good candidates for future studies regarding the familial link between DS and NTDs.

Abbreviations

NTD - Neural Tube Disorders, DS - Down''s Syndrome, MTHFR - Methylenetetrahydrofolate reductase, MTRR– 5 - methyltetrahydrofolate-homocysteine methyltransferase reductase.     

Protein Structure Prediction with a Combined Solvation Free Energy-Molecular Mechanics Force Field

Abstract

Models of protein structure are frequently used to determine the physical characteristics of a protein when the crystal structure is not available. We developed a procedure to optimize such models, by use of a combined solvation free energy and molecular mechanics force field. Appropriately chosen atomic solvation parameters were defined using the criterion that the resulting protein model should deviate least from the crystal structure upon a forty picosecond molecular dynamics simulation carried out using the combined force field. Several tests were performed to refine the set of atomic solvation parameters which best complement the molecular mechanics forces. Four sets of parameters from the literature were tested and an empirically optimized set is proposed. The parameters are defined on a well characterized small molecule (alanyl dipeptide) and on the highly refined crystal structure of rat trypsin, and then tested on a second highly refined crystal structure of α-lytic protease. The new set of atomic solvation parameters provides a significant improvement over molecular mechanics alone in energy minimization of protein structures. This combined force field also has advantages over the use of explicit solvent as it is possible to take solvent effects into account during energetic conformational searching when modeling a homologous protein structure from a known crystal structure.     

Motion analysis of the glenohumeral joint during activities of daily living

The shoulder complex has a larger range of motion (ROM) than any other joint complex in the human body, leaving it prone to numerous injuries. Objective kinematic analysis could yield useful functional insights that may assist clinical practice. Non-invasive optoelectronic motion analysis techniques have been used to assess the shoulders of five healthy subjects performing ROM tasks and 10 functional tasks of daily living. The four most demanding tasks – touching the side and back of the head, brushing the opposite side of the head, lifting an object to shoulder height and lifting an object to head height, required 78%, 60%, 61% and 71%, respectively, of the glenohumeral elevation necessary for full abduction in the scapular plane for the 10 shoulders. This has implications for clinical practice where maximum arm elevation is commonly used to determine a patient's ability to return to work and other everyday activities.     

Cerebrovascular Accident (Stroke) in Captive,Group-Housed,Female Chimpanzees

Over a 5-y period, 3 chimpanzees at our institution experienced cerebrovascular accidents (strokes). In light of the increasing population of aged captive chimpanzees and lack of literature documenting the prevalence and effectiveness of various treatments for stroke in chimpanzees, we performed a retrospective review of the medical records and necropsy reports from our institution. A survey was sent to other facilities housing chimpanzees that participate in the Chimpanzee Species Survival Plan to inquire about their experience with diagnosing and treating stroke. This case report describes the presentation, clinical signs, and diagnosis of stroke in 3 recent cases and in historical cases at our institution. Predisposing factors, diagnosis, and treatment options of cerebral vascular accident in the captive chimpanzee population are discussed also.Abbreviations: CVA, cerebrovascular accidentCerebrovascular accident (CVA; stroke) is a disturbance in brain function due to insufficient or complete loss of blood supply to an area of the brain. The lesion and clinical signs depend on the severity and location of the blockage. The 2 main categories of stroke—ischemic and hemorrhagic—both result in a loss of blood flow to an associated area of the brain. Ischemic strokes are due to either insufficient or direct loss of blood flow to the affected area of the brain from either temporary or permanent arterial occlusion of vessels supplying that area. Hemorrhagic strokes occur from rupture of a blood vessel and subsequent leakage of blood intracranially or into the subarachnoid space which results in clotting and decreased blood flow within that vessel and compression of the brain.^8,14,16,19 Loss of blood supply to a part of the brain, which can occur with ischemic or hemorrhagic stroke, initiates an ischemic cascade. The ischemic cascade is the result of secondary lack of oxygen and glucose; this lack consequently changes the intracellular metabolism from aerobic to anaerobic. This process ultimately leads to cell death and resultant disruption of cell membranes, thereby releasing toxins into the surrounding area and leading to increased cell death. This process results in a centrifugal progression of irreversible tissue damage and cell death.^1,11Brain tissue ceases to function when deprived of oxygen for more than 60 to 90 s, and irreversible tissue necrosis and brain damage can occur after a few hours. Ischemic strokes can result in varying degrees of damage to the tissue; consequently, clinical signs depend upon the amount of collateral circulation supplying the affected region of the brain. Part of the tissue may die immediately, whereas other parts may be injured only temporarily and ultimately recover.⁸ Clinical signs that are typical of stroke victims consist of abnormal sensations, hemiparesis (that is, paralysis in one arm or leg or on one side of the body), aphasia, ataxia, and urinary incontinence. Severe strokes can result in stupor or coma. The defect in the brain usually is manifested as clinical signs on the opposite side of the body, depending on the part of brain that is affected.^8,9 Diagnosis typically is based initially on clinical signs and confirmed with imaging techniques such as CT and MRI, or the lesion is noted at necropsy.^6,16Whereas strokes are common in humans, only one report to date has discussed and documented spontaneous stroke in a chimpanzee.⁶ In 2004, a 29-y-old male chimpanzee at a zoo experienced an ischemic stroke that most likely was due to occlusion of the middle cerebral artery.^2,6 The area of the brain supplied by the middle cerebral artery is the area most often affected in ischemic stroke in humans.^2,10 Although stroke has not been thoroughly researched in chimpanzees, studies in other species of nonhuman primates suggest that the predisposing factors and pathology are similar to those in humans.^3,18,20This case report describes the presentation, clinical signs, and diagnosis of CVA that occurred in 3 chimpanzees over a 5-y period at our institution and in an additional 3 animals identified during a retrospective review of the health records from the last 30 y. We also discuss predisposing factors, diagnosis, treatment options, and statistics of CVA in the captive chimpanzee population.