M14D subfamily or cytosolic carboxypeptidases (CCPs) are distributed throughout the phylogenetic tree. CCPs common domains architecture consists in the amino-terminal and the carboxypeptidase domains, both conserved in the subfamily. Eukaryotic CCPs process the C-terminal posttranslational modifications of tubulins and possibly of other proteins. Its cellular function could be primarily related to cilia and basal bodies. On the other hand, prokaryotic CCPs are poorly characterized from the biochemical and functional point of view. The present work aimed to provide more information on key structural and functional features of M14D subfamily using the CCP from Pseudomonas aeruginosa (PaCCP) and the human CCP6 (hCCP6) as models. The crystal 3D structure of recombinant PaCCP was solved and provides a first detailed structural insight into mammalian CCPs. The functional processes involving PaCCP and hCCP6 were explore using biochemical, microbiological and proteomics methods.
A model plant, Arabidopsis thaliana, duplicates its chromosomes without undergoing cellular division, in a process known as endoreplication. The primary objective of this study was to identify genes and proteins that specifically accumulate in endoreplicated nuclei in Arabidopsis thaliana. The secondary objective was to identify the biological function of unique domains found in Arabidopsis topoisomerase VI subunit B (AtTopVIB) that contributes to endoreplication. Using the AtTopVIB amino acid sequence and protein database search engine, I identified two unique domains to which I designated the insertion of the N-terminal domain (IND) and the extension in the C-terminal domain (ECD). These domains are well conserved between Arabidopsis and Oryza sativa (rice) but very unique in the entire family. I analyzed the localization of AtTopVIB in Arabidopsis protoplasts with yellow-fluorescent protein (YFP) in Arabidopsis protoplasts using split-luciferase.
Serine Protease inhibitors like antitrypsin, antichymotrypsin, C1-inhibitor, antithrombin and plasminogen activator inhibitor, play absolutely critical role in the control of proteinases, involved in the inflammatory, complement, coagulation and fibrinolytic pathways respectively, and are associated with diseases like emphysema/cirrhosis, angioedema, familial dementia, chronic obstructive bronchitis and thrombosis. The mechanism of inhibition of serpin requires large scale conformation change and native state of serpin is in a metastable state which transforms into a stable state when they inhibit target proteases. Serpins are prone to conformational diseases due to their susceptibility to undergo point mutations especially in mobile domains that can results in aberrant intermolecular linkage and polymer formation. The effects of such protein aggregation are cumulative, with a progressive loss of cellular function. Serpin polymerization is a significant problem and devising a cure has been cumbersome owing to their complex mechanism of inhibition, metastable nature, cofactor binding ability and large scale conformational change. Critical understanding of the factors and mechanisms
Endocytosis is an essential cellular process requiredfor functions including nutrient uptake, membranerecycling, and signal transduction. In comparison tothe clathrin-mediated pathway, clathrin-independentpathways are poorly understood. New work is nowbeginning to reveal a picture of multiplenon-clathrin pathways. Findings presented in thisbook identify that ErbB2 is internalized through anon-clathrin pathway in geldanamycin-treated SKBr3human breast cancer cells. ErbB2 internalizationresembled a newly described non-clathrin pathwaytermed the GEEC pathway, a pathway thought to bespecific for internalization of GPI-anchoredproteins. Suprisingly, ErbB2 and GPI-anchoredproteins also co-localized with chimeric fusionproteins containing transmembrane domains, proteinsexpected to be excluded from the GEEC pathway. Combined with other data from the lab, these resultssuggest that this pathway is not specific forGPI-anchored proteins and may instead represent abulk internalization pathway.
Post-translational phosphorylation is one of the most common protein modifications that occur in animal cells. The vast majority of phosphorylation occurs as a mechanism of acute and reversible regulation of protein function. Studies of mammalian cells metabolically labeled with p32 orthophosphate suggest that as many as one-third of all cellular proteins are covalently modified by protein phosphorylation. Covalent attachment of a phosphate group to an amino acid side chain of a protein can cause a structural change, for example, by attracting a cluster of positively charged side chains. Such a change occurring at one site in a protein can in turn alter the protein s conformation elsewhere. Reversible protein phosphorylation is the predominant strategy used to control the activity of proteins in eukaryotic cells. The phosphates are transferred from ATP molecules by protein kinases and are taken off by protein phosphatases. In animal cells, serine, threonine and tyrosine are the amino acids subjected to phosphorylation. The protein kinases belong to a large family of enzymes, which contain a similar amino acid catalytic (kinase) domains.
RNA helicases are essential in all steps of RNA maturation, beginning with transcription and ending with RNA decay. They catalyze the separation of nucleic acid double strands and thereby facilitate structural remodeling. Their cellular importance is reflected in severe diseases caused by RNA helicase dysregulation. The largest group of RNA helicases is confined by the so called DEAD-box helicases of superfamily 2, characterized by the signature sequence D-E-A-D. DEAD-box helicases share a structurally conserved core of two RecA-like domains that carry signature motifs involved in ATP-binding, ATP-hydrolysis, RNA-binding and RNA-remodeling. An exceptional member of the DEAD-box protein family is the human RNA helicase DDX1(DEAD-box helicase1). In contrast to all other family members, DDX1 harbors a SPRY domain insertion in between the signature motifs of the helicase core. DDX1 is involved in a plethora of different RNA maturation processes and has been associated with tumor progression. Moreover due to its versatile function in RNA processing, it is hijacked by viruses for their replication. This medical relevance makes DDX1 a potential target for the development of pharmaceutics.