Trapping of a spiral-like intermediate of the bacterial cytokinetic protein FtsZ

The earliest stage in bacterial cell division is the formation of a ring, composed of the tubulin-like protein FtsZ, at the division site. Tight spatial and temporal regulation of Z-ring formation is required to ensure that division occurs precisely at midcell between two replicated chromosomes. However, the mechanism of Z-ring formation and its regulation in vivo remain unresolved. Here we identify the defect of an interesting temperature-sensitive ftsZ mutant (ts1) of Bacillus subtilis. At the nonpermissive temperature, the mutant protein, FtsZ(Ts1), assembles into spiral-like structures between chromosomes. When shifted back down to the permissive temperature, functional Z rings form and division resumes. Our observations support a model in which Z-ring formation at the division site arises from reorganization of a long cytoskeletal spiral form of FtsZ and suggest that the FtsZ(Ts1) protein is captured as a shorter spiral-forming intermediate that is unable to complete this reorganization step. The ts1 mutant is likely to be very valuable in revealing how FtsZ assembles into a ring and how this occurs precisely at the division site.

Nanoformulated cell-penetrating survivin mutant and its dual actions

In this study, we investigated the differential actions of a dominant-negative survivin mutant (SurR9-C84A) against cancerous SK-N-SH neuroblastoma cell lines and differentiated SK-N-SH neurons. In both the cases, the mutant protein displayed dual actions, where its effects were cytotoxic toward cancerous cells and proliferative toward the differentiated neurons. This can be explained by the fact that tumorous (undifferentiated SK-N-SH) cells have a high endogenous survivin pool and upon treatment with mutant SuR9-C84A causes forceful survivin expression. These events significantly lowered the microtubule dynamics and stability, eventually leading to apoptosis. In the case of differentiated SK-N-SH neurons that express negligible levels of wild-type survivin, the mutant indistinguishably behaved in a wild-type fashion. It also favored cell-cycle progression, forming the chromosome-passenger complex, and stabilized the microtubule-organizing center. Therefore, mutant SurR9-C84A represents a novel therapeutic with its dual actions (cytotoxic toward tumor cells and protective and proliferative toward neuronal cells), and hence finds potential applications against a variety of neurological disorders. In this study, we also developed a novel poly(lactic-co-glycolic acid) nanoparticulate formulation to surmount the hurdles associated with the delivery of SurR9-C84A, thus enhancing its effective therapeutic outcome.

The role of the yeast cleavage and polyadenylation factor subunit Ydh1p/Cft2p in pre-mRNA 3' end formation

Cleavage and polyadenylation factor (CPF) is a multi‐protein complex that functions in pre‐mRNA 3′‐end formation and in the RNA polymerase II (RNAP II) transcription cycle. Ydh1p/Cft2p is an essential component of CPF but its precise role in 3′‐end processing remained unclear. We found that mutations in YDH1 inhibited both the cleavage and the polyadenylation steps of the 3′‐end formation reaction in vitro. Recently, we demonstrated that an important function of CPF lies in the recognition of poly(A) site sequences and RNA binding analyses suggesting that Ydh1p/Cft2p interacts with the poly(A) site region. Here we show that mutant ydh1 strains are deficient in the recognition of the ACT1 cleavage site in vivo. The C‐terminal domain (CTD) of RNAP II plays a major role in coupling 3′‐end processing and transcription. We provide evidence that Ydh1p/Cft2p interacts with the CTD of RNAP II, several other subunits of CPF and with Pcf11p, a component of CF IA. We propose that Ydh1p/Cft2p contributes to the formation of important interaction surfaces that mediate the dynamic association of CPF with RNAP II, the recognition of poly(A) site sequences and the assembly of the polyadenylation machinery on the RNA substrate.

Tissue specific roles for the Ribosome Biogenesis Factor Wdr43 in zebrafish development

During vertebrate craniofacial development, neural crest cells (NCCs) contribute to most of the craniofacial pharyngeal skeleton. Defects in NCC specification, migration and differentiation resulting in malformations in the craniofacial complex are associated with human craniofacial disorders including Treacher-Collins Syndrome, caused by mutations in TCOF1. It has been hypothesized that perturbed ribosome biogenesis and resulting p53 mediated neuroepithelial apoptosis results in NCC hypoplasia in mouse Tcof1 mutants. However, the underlying mechanisms linking ribosome biogenesis and NCC development remain poorly understood. Here we report a new zebrafish mutant, fantome (fan), which harbors a point mutation and predicted premature stop codon in zebrafish wdr43, the ortholog to yeast UTP5. Although wdr43 mRNA is widely expressed during early zebrafish development, and its deficiency triggers early neural, eye, heart and pharyngeal arch defects, later defects appear fairly restricted to NCC derived craniofacial cartilages. Here we show that the C-terminus of Wdr43, which is absent in fan mutant protein, is both necessary and sufficient to mediate its nucleolar localization and protein interactions in metazoans. We demonstrate that Wdr43 functions in ribosome biogenesis, and that defects observed in fan mutants are mediated by a p53 dependent pathway. Finally, we show that proper localization of a variety of nucleolar proteins, including TCOF1, is dependent on that of WDR43. Together, our findings provide new insight into roles for Wdr43 in development, ribosome biogenesis, and also ribosomopathy-induced craniofacial phenotypes including Treacher-Collins Syndrome.

SurR9-C84A mutant nanocarrier and milk protein therapeutics for neurological disorders

 The current project revealed that the novel nanoformulation of SurR9-C84A was able to rescue the neurons following the Alzheimer’s related ß-amyloid toxicity and inflammation. In addition, bovine lactoferrin was found to have potential differentiating effect in the tumor cells and hence is a valuable therapeutic for strengthening the degenerating neurons.

Detection of Arabidopsis thaliana AtRAD1 cDNA variants and assessment of function by expression in a yeast rad1 mutant

The Saccharomyces cerevisiae RAD1 and human XPF genes encode a subunit of a nucleotide excision repair endonuclease that also is implicated in some forms of homologous recombination. An Arabidopsis thaliana gene (AtRAD1) encoding the orthologous plant protein has been identified recently. Here we report the isolation of three structurally distinct AtRAD1 cDNAs from A. thaliana leaf tissue RNA. One of the isolates (AtRAD1-1) corresponds to the cDNA previously shown to encode the full-length AtRad1 protein, whereas the other two (AtRAD1-2, AtRAD1-3) differ slightly in size due to variations at the 5′ end of exon 6 or the 3′ end of exon 7, respectively. The sequence differences argue that these cDNAs were probably templated by mRNAs generated via alternative splicing. Diagnostic polymerase chain reaction pointed to the presence of the AtRAD1-1 and AtRAD1-2 but not AtRAD1-3 transcripts in bud and root tissue, and to a fourth transcript (AtRAD1-4), having both alterations identified in AtRAD1-2 and AtRAD1-3, in root tissue. However, the low frequency of detection of AtRAD1-3 and AtRAD1-4 makes the significance of these tissue-specific patterns unclear. The predicted AtRad1-2, AtRad1-3 and AtRad1-4 proteins lack part of the region likely required for endonuclease complex formation. Expression of AtRAD1-2 and AtRAD1-3 in a yeast rad1 mutant did not complement the sensitivity to ultraviolet radiation or the recombination defect associated with the rad1 mutation. These results suggest that alternative splicing may modulate the levels of functional AtRad1 protein.

The last 10 amino acid residues beyond the hydrophobic motif are critical for the catalytic competence and function of protein kinase Cá*

The segment C-terminal to the hydrophobic motif at the V5 domain of protein kinase C (PKC) is the least conserved both in length and in amino acid identity among all PKC isozymes. By generating serial truncation mutants followed by biochemical and functional analyses, we show here that the very C terminus of PKCα is critical in conferring the full catalytic competence to the kinase and for transducing signals in cells. Deletion of one C-terminal amino acid residue caused the loss of ~60% of the catalytic activity of the mutant PKCα, whereas deletion of 10 C-terminal amino acid residues abrogated the catalytic activity of PKCα in immune complex kinase assays. The PKCα C-terminal truncation mutants were found to lose their ability to activate mitogen-activated protein kinase, to rescue apoptosis induced by the inhibition of endogenous PKC in COS cells, and to augment melatonin-stimulated neurite outgrowth. Furthermore, molecular dynamics simulations revealed that the deletion of 1 or 10 C-terminal residues results in the deformation of the V5 domain and the ATP-binding pocket, respectively. Finally, PKCα immunoprecipitated using an antibody against its C terminus had only marginal catalytic activity compared with that of the PKCα immunoprecipitated by an antibody against its N terminus. Therefore, the very C-terminal tail of PKCα is a novel determinant of the catalytic activity of PKC and a promising target for selective modulation of PKCα function. Molecules that bind preferentially to the very C terminus of distinct PKC isozymes and suppress their catalytic activity may constitute a new class of selective inhibitors of PKC.

The PKCÁ-D294G mutant found in pituitary and thyroid tumors fails to transduce extracellular signals

Protein kinase C (PKC) is a key regulator of cell proliferation, differentiation, and apoptosis and is one of the drug targets of anticancer therapy. Recently, a single point mutation (D294G) in PKCα has been found in pituitary and thyroid tumors with more invasive phenotype. Although the PKCα-D294G mutant is implicated in the progression of endocrine tumors, no apparent biochemical/cell biological abnormalities underlying tumorigenesis with this mutant have been found. We report here that the PKCα-D294G mutant is unable to bind to cellular membranes tightly despite the fact that it translocates to the membrane as efficiently as the wild-type PKCα upon treatment of phorbol ester. The impaired membrane binding is associated with this mutant's inability to transduce several antitumorigenic signals as it fails to mediate phorbol ester–stimulated translocation of myristoylated alanine–rich protein kinase C substrate (MARCKS), to activate mitogen-activated protein kinase and to augment melatonin-stimulated neurite outgrowth. Thus, the PKCα-D294G is a loss-of-function mutation. We propose that the wild-type PKCα may play important antitumorigenic roles in the progression of endocrine tumors. Therefore, developing selective activators instead of inhibitors of PKCα might provide effective pharmacological interventions for the treatment of certain endocrine tumors.

The very C-terminus of protein kinase CĹ is critical for the full catalytic competence but its hydrophobic motif is dispensable for the interaction with 3-phosphoinositide-dependent kinase-1

In this article, we explore the role of the C-terminus (V5 domain) of PKCvar epsilon plays in the catalytic competence of the kinase using serial truncations followed by immune-complex kinase assays. Surprisingly, removal of the last seven amino acid residues at the C-terminus of PKCvar epsilon resulted in a PKCvar epsilon-Δ731 mutant with greatly reduced intrinsic catalytic activity while truncation of eight amino acid residues at the C-terminus resulted in a catalytically inactive PKCvar epsilon mutant. Computer modeling and molecular dynamics simulations showed that the last seven and/or eight amino acid residues of PKCvar epsilon were involved in interactions with residues in the catalytic core. Further truncation analyses revealed that the hydrophobic phosphorylation motif was dispensable for the physical interaction between PKCvar epsilon and 3-phosphoinositide-dependent kinase-1 (PDK-1) as the PKCvar epsilon mutant lacking both the turn and the hydrophobic motifs could still be co-immunoprecipitated with PDK-1. These results provide fresh insights into the biochemical and structural basis underlying the isozyme-specific regulation of PKC and suggest that the very C-termini of PKCs constitute a promising new target for the development of novel isozyme-specific inhibitors of PKC.

The myeloproliferative disorder-associated JAK2 V617F mutant escapes negative regulation by suppressor of cytokine signaling 3

The somatic JAK2 valine-to-phenylalanine (V617F) mutation has been detected in up to 90% of patients with polycythemia and in a sizeable proportion of patients with other myeloproliferative disorders such as essential thrombocythemia and idiopathic myelofibrosis. Suppressor of cytokine signaling 3 (SOCS3) is known to be a strong negative regulator of erythropoietin (EPO) signaling through interaction with both the EPO receptor (EPOR) and JAK2. We report here that JAK2 V617F cannot be regulated and that its activation is actually potentiated in the presence of SOCS3. Instead of acting as a suppressor, SOCS3 enhanced the proliferation of cells expressing both JAK2 V617F and EPOR. Additionally, although SOCS1 and SOCS2 are degraded in the presence of JAK2 V617F, turnover of SOCS3 is inhibited by the JAK2 mutant kinase and this correlated with marked tyrosine phosphorylation of SOCS3 protein. We also observed constitutive tyrosine phosphorylation of SOCS3 in peripheral blood mononuclear cells (PBMCs) derived from patients homozygous for the JAK2 V617F mutant. These findings suggest that the JAK2 V617F has overcome normal SOCS regulation by hyperphosphorylating SOCS3, rendering it unable to inhibit the mutant kinase. Thus, JAK2 V617F may even exploit SOCS3 to potentiate its myeloproliferative capacity.

Functional studies on the Wilson copper P-Type ATPase and toxic milk mouse mutant

The Wilson protein (WND; ATP7B) is an essential component of copper homeostasis. Mutations in the ATP7B gene result in Wilson disease, which is characterised by hepatotoxicity and neurological disturbances. In this paper, we provide the first direct biochemical evidence that the WND protein functions as a copper-translocating P-type ATPase in mammalian cells. Importantly, we have shown that the mutation of the conserved Met1386 to Val, in the Atp7B for the mouse model of Wilson disease, toxic milk (tx), caused a loss of Cu-translocating activity. These investigations provide strong evidence that the toxic milk mouse is a valid model for Wilson disease and demonstrate a link between the loss of catalytic function of WND and the Wilson disease phenotype.

Requirement for the cell division protein DivIB in polar cell division and engulfment during sporulation in Bacillus subtilis

During spore formation in Bacillus subtilis, cell division occurs at the cell pole and is believed to require essentially the same division machinery as vegetative division. Intriguingly, although the cell division protein DivIB is not required for vegetative division at low temperatures, it is essential for efficient sporulation under these conditions. We show here that at low temperatures in the absence of DivIB, formation of the polar septum during sporulation is delayed and less efficient. Furthermore, the polar septa that are complete are abnormally thick, containing more peptidoglycan than a normal polar septum. These results show that DivIB is specifically required for the efficient and correct formation of a polar septum. This suggests that DivIB is required for the modification of sporulation septal peptidoglycan, raising the possibility that DivIB either regulates hydrolysis of polar septal peptidoglycan or is a hydrolase itself. We also show that, despite the significant number of completed polar septa that form in this mutant, it is unable to undergo engulfment. Instead, hydrolysis of the peptidoglycan within the polar septum, which occurs during the early stages of engulfment, is incomplete, producing a similar phenotype to that of mutants defective in the production of sporulation-specific septal peptidoglycan hydrolases. We propose a role for DivIB in sporulation-specific peptidoglycan remodelling or its regulation during polar septation and engulfment.

Generation of mutant leukaemia inhibitory factor (LIF)-IgG heavy chain fusion proteins as bivalent antagonists of LIF

Two leukaemia inhibitory factor (LIF) mutants, designated MH35-BD and LIF05, have been shown to have a capacity to inhibit the biological activities of not only human LIF (hLIF) but also other interleukin-6 (IL-6) subfamily cytokines such as human oncostatin M (hOSM). These cytokines share the same receptor complex in which the glycoprotein 130 (gp130) subunit is a common constituent. However, at low concentrations and in their monomeric forms, such molecules have a relatively short plasma half-life due to rapid clearance from the kidneys. Here, to prolong their serum half-lives, we have used a multi-step polymerase chain reaction (PCR) to fuse each of the LIF05 and MH35-BD cDNA fragments to a sequence encoding the Fc portion, and the hinge region, of the human immunoglobulin G (hIgG) heavy chain. The linking was achieved through an oligomer encoding a thrombin-sensitive peptide linker thus generating MH35-BD:Fc and LIF05:Fc, respectively. Both Fc fusion constructs were expressed in insect cell Sf21 and the proteins were purified by two successive affinity chromatography steps using nickel–nitrilotriacetic acid (Ni–NTA) agarose and protein A beads. The Ba/F3 cell-based proliferation assay was used to confirm that the proteins were biologically active. In addition, preliminary pharmacokinetics indicates that the Fc fusion constructs have a longer serum half-life compared to their non-fusion counterparts.

Protective and proliferative effects of survivin mutant on differentiated neuroblastoma

This research focused on the neuro-proliferation and protective effects of a survivin mutant. The results showed that this protein binds to microtubules and induces the division and proliferation of neural cells. Furthermore, it protects the neural cells against retinoic acid induced neurotoxicity, activated T-cell apoptosis and oxidative stress.

A critical role for the short intracellular C terminus in receptor activity-modifying protein function

Receptor activity-modifying proteins (RAMPs) interact with and modify the behavior of the calcitonin receptor (CTR) and calcitonin receptor-like receptor (CLR). We have examined the contribution of the short intracellular C terminus, using constructs that delete the last eight amino acids of each RAMP. C-Terminal deletion of individual RAMPs had little effect on the signaling profile induced when complexed with CLR in COS-7 or human embryonic kidney (HEK)293 cells. Likewise, confocal microscopy revealed each of the mutant RAMPs translocated hemagglutinin-tagged CLR to the cell surface. In contrast, a pronounced effect of RAMP C-terminal truncation was seen for RAMP/CTRa complexes, studied in COS-7 cells, with significant attenuation of amylin receptor phenotype induction that was stronger for RAMP1 and -2 than RAMP3. The loss of amylin binding upon C-terminal deletion could be partially recovered with overexpression of Gαs, suggesting an impact of the RAMP C terminus on coupling of G proteins to the receptor complex. In HEK293 cells the c-Myc-RAMP1 C-terminal deletion mutant showed high receptor-independent cell surface expression; however, this construct showed low cell surface expression when expressed alone in COS-7 cells, indicating interaction of RAMPs with other cellular components via the C terminus. This mutant also had reduced cell surface expression when coexpressed with CTR. Thus, this study reveals important functionality of the RAMP C-terminal domain and identifies key differences in the role of the RAMP C terminus for CTR versus CLR-based receptors.