Computational Design of a Protein-Based Enzyme Inhibitor

While there has been considerable progress in designing protein–protein interactions, the design of proteins that bind polar surfaces is an unmet challenge. We describe the computational design of a protein that binds the acidic active site of hen egg lysozyme and inhibits the enzyme. The design process starts with two polar amino acids that fit deep into the enzyme active site, identifies a protein scaffold that supports these residues and is complementary in shape to the lysozyme active-site region, and finally optimizes the surrounding contact surface for high-affinity binding. Following affinity maturation, a protein designed using this method bound lysozyme with low nanomolar affinity, and a combination of NMR studies, crystallography, and knockout mutagenesis confirmed the designed binding surface and orientation. Saturation mutagenesis with selection and deep sequencing demonstrated that specific designed interactions extending well beyond the centrally grafted polar residues are critical for high-affinity binding.     

Eradication of Helicobacter pylori infection in the management of patients with dyspepsia and non-ulcer dyspepsia.

Although H. pylori infection has been recognized as a major etiological agent for the development of chronic active gastritis, duodenal ulcer and benign non-NSAID related gastric ulcer, its role in the development of symptoms in patients with dyspepsia remains uncertain. Results from population-based epidemiological studies have been conflicting regarding a causal link between H. pylori infection and dyspepsia. Abnormalities in gastric acid secretion may exist in some dyspeptic patients. Whether disordered gastric motility seen in dyspeptic patients is related to the infection is not clear based on the results in the literature. Numerous clinical trials have been undertaken to eradicate H. pylori infection and improve the symptoms in dyspeptic patients; however, the results have been discrepant between studies. Many published studies suffer from methodological problems that have made interpretation difficult. Large, well-conducted, randomized, placebo-controlled, clinical trials with long-term follow-up are needed to justify the beneficial effect of H. pylori eradication treatment in dyspeptic patients seen in some small studies. H. pylori eradication therapy is cost-effective in H. pylori-infected dyspeptic patients although this benefit may take a long time to accrue, especially in younger patients.     

NMR-based metabolomics associated with chronic kidney disease in humans and animals: a one health perspective

Molecular and Cellular Biochemistry - Chronic kidney disease (CKD) is a renal dysfunction that can lead to high rates of mortality and morbidity, particularly when coupled with late diagnosis. CKD...     

Biochemical and physiological characterization of the efrotomycin fermentation

Summary An efrotomycin fermentation was characterized through physical, chemical and biochemical studies. Growth of the actinomycete,Nocardia lactamdurans occurred during the first 50 h of the fermentation cycle at the expense of glucose, protein, and triglycerides. The initiation of efrotomycin biosynthesis was observed when glucose dropped to a low concentration. Upon glucose depletion, cell growth ceased and a switch in the respiratory quotient occurred. Efrotomycin biosynthesis was supported by the utilization of soybean oil and starch. Analysis of triglyceride metabolism showed that no diglycerides or monoglycerides accumulated during the fermentation. The activity of extracellular enzymes (lipase, protease, and amylase) increase during the cell growth phase and decreased significantly after 150 h. The concentrations of DNA, tetrahydro-vitamin K₂ (a membrane component), and free amino acids in the supernatant increased dramatically late in the fermentation cycle (225 h), indicating massive cell lysis. During this same time period, a reduction in cellular respiratory activity and efrotomycin biosynthesis were observed.     

The 'human revolution' in lowland tropical Southeast Asia: the antiquity and behavior of anatomically modern humans at Niah Cave (Sarawak, Borneo)

Recent research in Europe, Africa, and Southeast Asia suggests that we can no longer assume a direct and exclusive link between anatomically modern humans and behavioral modernity (the 'human revolution'), and assume that the presence of either one implies the presence of the other: discussions of the emergence of cultural complexity have to proceed with greater scrutiny of the evidence on a site-by-site basis to establish secure associations between the archaeology present there and the hominins who created it. This paper presents one such case study: Niah Cave in Sarawak on the island of Borneo, famous for the discovery in 1958 in the West Mouth of the Great Cave of a modern human skull, the 'Deep Skull,' controversially associated with radiocarbon dates of ca. 40,000 years before the present. A new chronostratigraphy has been developed through a re-investigation of the lithostratigraphy left by the earlier excavations, AMS-dating using three different comparative pre-treatments including ABOX of charcoal, and U-series using the Diffusion-Absorption model applied to fragments of bones from the Deep Skull itself. Stratigraphic reasons for earlier uncertainties about the antiquity of the skull are examined, and it is shown not to be an 'intrusive' artifact. It was probably excavated from fluvial-pond-desiccation deposits that accumulated episodically in a shallow basin immediately behind the cave entrance lip, in a climate that ranged from times of comparative aridity with complete desiccation, to episodes of greater surface wetness, changes attributed to regional climatic fluctuations. Vegetation outside the cave varied significantly over time, including wet lowland forest, montane forest, savannah, and grassland. The new dates and the lithostratigraphy relate the Deep Skull to evidence of episodes of human activity that range in date from ca. 46,000 to ca. 34,000 years ago. Initial investigations of sediment scorching, pollen, palynomorphs, phytoliths, plant macrofossils, and starch grains recovered from existing exposures, and of vertebrates from the current and the earlier excavations, suggest that human foraging during these times was marked by habitat-tailored hunting technologies, the collection and processing of toxic plants for consumption, and, perhaps, the use of fire at some forest-edges. The Niah evidence demonstrates the sophisticated nature of the subsistence behavior developed by modern humans to exploit the tropical environments that they encountered in Southeast Asia, including rainforest.     

The molecular evolution of avian ultraviolet- and violet-sensitive visual pigments

The shortwave-sensitive SWS1 class of vertebrate visual pigments range in lambda(max) from the violet (385-445 nm) to the ultraviolet (UV) (365-355 nm), with UV-sensitivity almost certainly ancestral. In birds, however, the UV-sensitive pigments present in a number of species have evolved secondarily from an avian violet-sensitive (VS) pigment. All avian VS pigments expressed in vitro to date encode Ser86 whereas Phe86 is present in all non-avian ultraviolet sensitive (UVS) pigments. In this paper, we show by site directed mutagenesis of avian VS pigments that Ser86 is required in an avian VS pigment to maintain violet-sensitivity and therefore underlies the evolution of avian VS pigments. The major mechanism for the evolution of avian UVS pigments from an ancestral avian VS pigment is undoubtedly a Ser90Cys substitution. However, Phe86, as found in the Blue-crowned trogon, will also short-wave shift the pigeon VS pigment into the UV whereas Ala86 and Cys86 which are also found in natural avian pigments do not generate short-wave shifts when substituted into the pigeon pigment. From available data on avian SWS1 pigments, it would appear that UVS pigments have evolved on at least 5 separate occasions and utilize 2 different mechanisms for the short-wave shift.     

ULK1 inhibits mTORC1 signaling, promotes multisite Raptor phosphorylation and hinders substrate binding

Protein synthesis and autophagy work as two opposing processes to control cell growth in response to nutrient supply. The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) pathway, which acts as a master regulator to control protein synthesis, has recently been shown to inhibit autophagy by phosphorylating and inactivating ULK1, an autophagy regulatory protein. ULK1 also inhibits phosphorylation of a mTORC1 substrate, S6K1, indicating that a complex signaling interplay exists between mTORC1 and ULK1. Here, we demonstrate that ULK1 induces multisite phosphorylation of Raptor in vivo and in vitro. Using phospho-specific antibodies we identify Ser855 and Ser859 as being strongly phosphorylated by ULK1, with moderate phosphorylation of Ser792 also observed. Interestingly, ULK1 overexpression also increases phosphorylation of Raptor Ser863 and the mTOR autophosphorylation site, Ser2481 in a mTORC1-dependent manner. Despite this evidence for heightened mTORC1 kinase activity following ULK1 overexpresssion, mTORC1-mediated phosphorylation of S6K1 and 4E-BP1 is significantly inhibited. ULK1 expression has no effect on protein-protein interactions between the components of mTORC1, but does reduce the ability of Raptor to bind to the substrate 4E-BP1. Furthermore, shRNA knockdown of ULK1 leads to increased phosphorylation of mTORC1 substrates and decreased phosphorylation of Raptor at Ser859 and Ser792. We propose a new mechanism whereby ULK1 contributes to mTORC1 inhibition through hindrance of substrate docking to Raptor. This is a novel negative feedback loop that occurs upon activation of autophagy to maintain mTORC1 inhibition when nutrient supplies are limiting.     

Identification of blottin: a novel gastric trefoil factor family-2 binding protein

The trefoil factor family (TFF) peptides are important in gastro-intestinal mucosal protection and repair. Their mechanism of action remains unclear and receptors are sought. We aimed to identify and characterise proteins binding to TFF2. A fusion protein of mouse TFF2 with alkaline phosphatase was generated and used to probe 2-D protein blots of mouse stomach. The resulting spots were analysed by MS. The protein identified was characterised by bioinformatics, rapid amplification of cDNA ends, in situ hybridisation (ISH) and immunohistochemistry (IHC). Functional assays were performed in gastrointestinal cell lines. A single major murine protein was identified and named blottin. It was previously unknown as a translated product. Blottin is also present in rat and human; the latter gene is also known as GDDR. The predicted full-length proteins are 184 amino acids long (20 kDa), reducing to 164 amino acids (18 kDa) after signal peptide cleavage. ISH of gastrointestinal tissues shows abundant blottin mRNA in gastric surface and foveolar epithelium. IHC shows cytoplasmic staining for blottin protein, and by immunoelectron microscopy in mucus granules and Golgi stacks. Previous work showed that blottin is down-regulated in gastric cancers. Blottin contains a BRICHOS domain, and has 56% similarity with gastrokine-1. Cultured HT-29 cells express blottin and show increased DNA synthesis with antiblottin antibody; however, this effect is reversed by the immunising peptide. We have identified and characterised a TFF2-binding protein produced by gastric epithelium. Blottin may play a role in gastrointestinal mucosal protection and modulate gut epithelial cell proliferation.     

Sex-specific effects of <Emphasis Type="Italic">ACE</Emphasis> I/D and <Emphasis Type="Italic">AGT</Emphasis>-M235T on pulse pressure: the HyperGEN Study

Evidence shows that an elevated pulse pressure (PP) may lead to an increased risk of cardiovascular morbidity and mortality. There is also evidence that PP is a sexually dimorphic trait, and that genetic factors influence inter-individual variation in PP. The aim of this project was to assess the genotype-by-sex interaction on PP in a sample of mostly hypertensive African American and White participants using candidate genes involved in the renin–angiotensin–aldosterone system. Subjects were participants in the HyperGEN Study, including men (43%) and women (57%) over the age of 55 years (mean age = 65). Candidate gene polymorphisms used were ACE insertion/deletion (1,789 subjects genotyped) and AGT-M235T (1,800 subjects genotyped). We employed linear regression methods to assess the genotype-by-sex interaction. For ACE, genotype-by-sex interaction on PP was detected (P = 0.04): the “D/D” genotype predicted a 2.2 mmHg higher pulse pressure among women, but a 1.2 mmHg lower PP among men, compared to those with an “I” allele, after adjusting for age, weight, height, ethnicity, and antihypertension medication use. A similar interaction was found for systolic blood pressure. The genotype-by-sex interaction was consistent across ethnicity. The interaction was evident among those on antihypertensive medications (P = 0.05), but not among those not taking such medications (P = 0.55). In our analysis of AGT, no evidence of a genotype-by-sex interaction affecting PP, SBP, or DBP was detected. This evidence for a genotype-by-sex interaction helps our understanding of the complex genetic underpinnings of blood pressure phenotypes.