Caterina Alfano

The Ball and Chain of Polyubiquitin Structures

Ubiquitylation is a post-translational modification implicated in several different cellular pathways. The possibility of forming chains through covalent crosslinking between any of the seven lysines, or the initial methionine, and the C terminus of another moiety provides ubiquitin (Ub) with special flexibility in its function in signalling. Here, we review the knowledge accumulated over the past several years about the functions and structural features of polyUb chains. This analysis reveals the need to understand further the functional role of some of the linkages and the structural code that determines recognition of polyUbs by protein partners.

Solution Structure of the SGTA Dimerisation Domain and Investigation of Its Interactions with the Ubiquitin-Like Domains of BAG6 and UBL4A

Background The BAG6 complex resides in the cytosol and acts as a sorting point to target diverse hydrophobic protein substrates along their appropriate paths, including proteasomal degradation and ER membrane insertion. Composed of a trimeric complex of BAG6, TRC35 and UBL4A, the BAG6 complex is closely associated with SGTA, a co-chaperone from which it can obtain hydrophobic substrates. Methodology and Principal Findings SGTA consists of an N-terminal dimerisation domain (SGTA_NT), a central tetratricopeptide repeat (TPR) domain, and a glutamine rich region towards the C-terminus. Here we solve a solution structure of the SGTA dimerisation domain and use biophysical techniques to investigate its interaction with two different UBL domains from the BAG6 complex. The SGTA_NT structure is a dimer with a tight hydrophobic interface connecting two sets of four alpha helices. Using a combination of NMR chemical shift perturbation, isothermal titration calorimetry (ITC) and microscale thermophoresis (MST) experiments we have biochemically characterised the interactions of SGTA with components of the BAG6 complex, the ubiquitin-like domain (UBL) containing proteins UBL4A and BAG6. We demonstrate that the UBL domains from UBL4A and BAG6 directly compete for binding to SGTA at the same site. Using a combination of structural and interaction data we have implemented the HADDOCK protein-protein interaction docking tool to generate models of the SGTA-UBL complexes. Significance This atomic level information contributes to our understanding of the way in which hydrophobic proteins have their fate decided by the collaboration between SGTA and the BAG6 complex.

Synergic interplay of the La motif, RRM1 and the interdomain linker of LARP6 in the recognition of collagen mRNA expands the RNA binding repertoire of the La module

The La-related proteins (LARPs) form a diverse group of RNA-binding proteins characterized by the possession of a composite RNA binding unit, the La module. The La module comprises two domains, the La motif (LaM) and the RRM1, which together recognize and bind to a wide array of RNA substrates. Structural information regarding the La module is at present restricted to the prototypic La protein, which acts as an RNA chaperone binding to 3′ UUUOH sequences of nascent RNA polymerase III transcripts. In contrast, LARP6 is implicated in the regulation of collagen synthesis and interacts with a specific stem-loop within the 5′ UTR of the collagen mRNA. Here, we present the structure of the LaM and RRM1 of human LARP6 uncovering in both cases considerable structural variation in comparison to the equivalent domains in La and revealing an unprecedented fold for the RRM1. A mutagenic study guided by the structures revealed that RNA recognition requires synergy between the LaM and RRM1 as well as the participation of the interdomain linker, probably in realizing tandem domain configurations and dynamics required for substrate selectivity. Our study highlights a considerable complexity and plasticity in the architecture of the La module within LARPs.

An optimized strategy to measure protein stability highlights differences between cold and hot unfolded states

Macromolecular crowding ought to stabilize folded forms of proteins, through an excluded volume effect. This explanation has been questioned and observed effects attributed to weak interactions with other cell components. Here we show conclusively that protein stability is affected by volume exclusion and that the effect is more pronounced when the crowders size is closer to that of the protein under study. Accurate evaluation of the volume exclusion effect is made possible by the choice of yeast frataxin, a protein that undergoes cold denaturation above zero degrees, because the unfolded form at low temperature is more expanded than the corresponding one at high temperature. To achieve optimum sensitivity to changes in stability we introduce an empirical parameter derived from the stability curve. The large effect of PEG 20 on cold denaturation can be explained by a change in water activity, according to Privalovs interpretation of cold denaturation.

Gray platelet syndrome: Novel mutations of the NBEAL2 gene

case that did not belong to groups (i) and (ii), ie those with equivocal results on one or both tests and those with a positive result on one test and negative result on the other. Groups (i) and (ii), collectively referred to as “concordant,” were used to determine the optimal Ct cutoff that maximized the agreement between tissue PCR results and double positivity/negativity. Biopsies were obtained from colon, duodenum, stomach, esophagus, and lung in 59 (39%), 44 (29%), 37 (24%), 7 (5%), and 4 (3%) cases, respectively. One hundred twenty-two (80%) cases were concordant (105 double-negative and 17 double-positive) and the remainder were equivocal. Viremic cases were marginally significantly more likely than nonviremic cases to be equivocal (28% vs. 14%, respectively; P5 0.055). Pathological evidence of GVHD was present in 57% vs. 52% of concordant vs. equivocal cases, respectively (P5 0.68). Similarly, these groups were not different with regards to the presence of clinical evidence of GVHD (82% vs. 90%, respectively). The optimal PCR Ct value for classification of concordant cases was 40, with good overall performance (AUC 0.91, P< 0.001), sensitivity 94%, specificity 79%, positive predictive value (PPV) 42%, and negative predictive value (NPV) 99%. Using this cutoff, 45% of equivocal cases were classified as negative. Table 1 shows the distribution of patients across H&E/IHC and PCR subgroups. Among viremic H&E/IHC-concordant cases, tissue PCR had a sensitivity of 100%, specificity of 50%, PPV of 44%, and NPV of 100%. Among non-viremic H&E/IHC-concordant cases, these numbers were 80%, 91%, 36%, and 99%, respectively. In this analysis on viremic and non-viremic cases, 31% and 62% of equivocal cases were classified as negative, respectively. One of the challenges in the diagnosis of CMV disease is H&E/IHC-equivocal cases. Avoiding potentially toxic anti-CMV treatment can be beneficial especially in nonviremic patients with negative tissue PCR. We demonstrate that a negative tissue PCR can be used to rule out CMV disease in H&E/IHC-equivocal cases. Although minimal spatial heterogeneity in the CMV target organ is assumed, we cannot rule out this possibility. Furthermore, we performed tissue PCR on FFPE specimens. The results may change if PCR were on fresh tissue, as it would be in the real-life setting. We expect the sensitivity of fresh tissue PCR to increase and specificity to decline. Until replicated in a controlled prospective study using fresh tissue samples, our results should not be used to guide treatment decisions. Our goal in this innovative, but preliminary, study was to assess the value of tissue PCR as an adjunct to H&E/IHC in the diagnosis of CMV disease, especially in equivocal cases. Future research could focus on clinical outcomes of non-viremic equivocal cases and whether anti-CMV treatment can be safely withheld in this group of patients. We could not address this question in the present work because all of our equivocal cases received anti-CMV treatment. Given the possibility of sampling error when using PCR on tissue biopsies, applying the same technique to fluid samples such as bronchioalveolar lavage or stool samples may be of value in future research.

A Novel RNA Polymerase-binding Protein that interacts with a Sigma-Factor Docking Site

Sporulation in Bacillus subtilis is governed by a cascade of alternative RNA polymerase sigma factors. We previously identified a small protein Fin that is produced under the control of the sporulation sigma factor σF to create a negative feedback loop that inhibits σF‐directed gene transcription. Cells deleted for fin are defective for spore formation and exhibit increased levels of σF‐directed gene transcription. Based on pull‐down experiments, chemical crosslinking, bacterial two‐hybrid experiments and nuclear magnetic resonance chemical shift analysis, we now report that Fin binds to RNA polymerase and specifically to the coiled‐coil region of the β′ subunit. The coiled‐coil is a docking site for sigma factors on RNA polymerase, and evidence is presented that the binding of Fin and σF to RNA polymerase is mutually exclusive. We propose that Fin functions by a mechanism distinct from that of classic sigma factor antagonists (anti‐σ factors), which bind directly to a target sigma factor to prevent its association with RNA polymerase, and instead functions to inhibit σF by competing for binding to the β′ coiled‐coil.

Expression and Purification of ZASP Subdomains and Clinically Important Isoforms: High-Affinity Binding to G-Actin

Z-disc-associated, alternatively spliced, PDZ motif-containing protein (ZASP) is a principal component of the sarcomere. The three prevalent isoforms of ZASP in skeletal muscle are generated by alternative splicing of exons 9 and 10. The long isoforms, either having (ZASP-L) or lacking exon 10 (ZASP-LΔex10), include an N-terminal PDZ domain, an actin-binding region (ABR) with a conserved motif (ZM), and three C-terminal LIM domains. The short isoform (ZASP-S) lacks the LIM domains. Mutations, A147T and A165V, within the ZM of ZASP-LΔex10 cause myofibrillar myopathy, but the mechanism is unknown. We have prepared these proteins, their ABR, and the respective mutant variants in recombinant form, characterized them biophysically, and analyzed their actin-binding properties by surface plasmon resonance and electron microscopy. All the proteins were physically homogeneous and monomeric and had circular dichroic spectra consistent with partially folded conformations. Comparison of the NMR HSQC spectra of ZASP-S and the PDZ domain showed that the ABR is unstructured. ZASP-S and its mutant variants and ZASP-LΔex10 all bound to immobilized G-actin with high affinity (Kd ≈ 10-8 to 10-9 M). Constructs of the isolated actin-binding region missing exon 10 (ABRΔ10) bound with lower affinity (Kd ≈ 10-7 M), but those retaining exon 10 (ABR+10) did so only weakly (Kd ≈ 10-5 M). ZASP-S, and the ABRΔ10, also induced F-actin and array formation, even in conditions of low ionic strength and in the absence of KCl and Mg2+ ions. Interestingly, the ZM mutations A147T and A165V did not affect any of the results described above.

Structural and Functional Insights into Bacillus subtilis Sigma Factor Inhibitor, CsfB

Summary Global changes in bacterial gene expression can be orchestrated by the coordinated activation/deactivation of alternative sigma (σ) factor subunits of RNA polymerase. Sigma factors themselves are regulated in myriad ways, including via anti-sigma factors. Here, we have determined the solution structure of anti-sigma factor CsfB, responsible for inhibition of two alternative sigma factors, σG and σE, during spore formation by Bacillus subtilis. CsfB assembles into a symmetrical homodimer, with each monomer bound to a single Zn2+ ion via a treble-clef zinc finger fold. Directed mutagenesis indicates that dimer formation is critical for CsfB-mediated inhibition of both σG and σE, and we have characterized these interactions in vitro. This work represents an advance in our understanding of how CsfB mediates inhibition of two alternative sigma factors to drive developmental gene expression in a bacterium.

Gray platelet syndrome

case that did not belong to groups (i) and (ii), ie those with equivocal results on one or both tests and those with a positive result on one test and negative result on the other. Groups (i) and (ii), collectively referred to as “concordant,” were used to determine the optimal Ct cutoff that maximized the agreement between tissue PCR results and double positivity/negativity. Biopsies were obtained from colon, duodenum, stomach, esophagus, and lung in 59 (39%), 44 (29%), 37 (24%), 7 (5%), and 4 (3%) cases, respectively. One hundred twenty-two (80%) cases were concordant (105 double-negative and 17 double-positive) and the remainder were equivocal. Viremic cases were marginally significantly more likely than nonviremic cases to be equivocal (28% vs. 14%, respectively; P5 0.055). Pathological evidence of GVHD was present in 57% vs. 52% of concordant vs. equivocal cases, respectively (P5 0.68). Similarly, these groups were not different with regards to the presence of clinical evidence of GVHD (82% vs. 90%, respectively). The optimal PCR Ct value for classification of concordant cases was 40, with good overall performance (AUC 0.91, P< 0.001), sensitivity 94%, specificity 79%, positive predictive value (PPV) 42%, and negative predictive value (NPV) 99%. Using this cutoff, 45% of equivocal cases were classified as negative. Table 1 shows the distribution of patients across H&E/IHC and PCR subgroups. Among viremic H&E/IHC-concordant cases, tissue PCR had a sensitivity of 100%, specificity of 50%, PPV of 44%, and NPV of 100%. Among non-viremic H&E/IHC-concordant cases, these numbers were 80%, 91%, 36%, and 99%, respectively. In this analysis on viremic and non-viremic cases, 31% and 62% of equivocal cases were classified as negative, respectively. One of the challenges in the diagnosis of CMV disease is H&E/IHC-equivocal cases. Avoiding potentially toxic anti-CMV treatment can be beneficial especially in nonviremic patients with negative tissue PCR. We demonstrate that a negative tissue PCR can be used to rule out CMV disease in H&E/IHC-equivocal cases. Although minimal spatial heterogeneity in the CMV target organ is assumed, we cannot rule out this possibility. Furthermore, we performed tissue PCR on FFPE specimens. The results may change if PCR were on fresh tissue, as it would be in the real-life setting. We expect the sensitivity of fresh tissue PCR to increase and specificity to decline. Until replicated in a controlled prospective study using fresh tissue samples, our results should not be used to guide treatment decisions. Our goal in this innovative, but preliminary, study was to assess the value of tissue PCR as an adjunct to H&E/IHC in the diagnosis of CMV disease, especially in equivocal cases. Future research could focus on clinical outcomes of non-viremic equivocal cases and whether anti-CMV treatment can be safely withheld in this group of patients. We could not address this question in the present work because all of our equivocal cases received anti-CMV treatment. Given the possibility of sampling error when using PCR on tissue biopsies, applying the same technique to fluid samples such as bronchioalveolar lavage or stool samples may be of value in future research.

Gray platelet syndrome: Novel mutations of the NBEAL2 gene: Novel NBEAL2 mutations

case that did not belong to groups (i) and (ii), ie those with equivocal results on one or both tests and those with a positive result on one test and negative result on the other. Groups (i) and (ii), collectively referred to as “concordant,” were used to determine the optimal Ct cutoff that maximized the agreement between tissue PCR results and double positivity/negativity. Biopsies were obtained from colon, duodenum, stomach, esophagus, and lung in 59 (39%), 44 (29%), 37 (24%), 7 (5%), and 4 (3%) cases, respectively. One hundred twenty-two (80%) cases were concordant (105 double-negative and 17 double-positive) and the remainder were equivocal. Viremic cases were marginally significantly more likely than nonviremic cases to be equivocal (28% vs. 14%, respectively; P5 0.055). Pathological evidence of GVHD was present in 57% vs. 52% of concordant vs. equivocal cases, respectively (P5 0.68). Similarly, these groups were not different with regards to the presence of clinical evidence of GVHD (82% vs. 90%, respectively). The optimal PCR Ct value for classification of concordant cases was 40, with good overall performance (AUC 0.91, P< 0.001), sensitivity 94%, specificity 79%, positive predictive value (PPV) 42%, and negative predictive value (NPV) 99%. Using this cutoff, 45% of equivocal cases were classified as negative. Table 1 shows the distribution of patients across H&E/IHC and PCR subgroups. Among viremic H&E/IHC-concordant cases, tissue PCR had a sensitivity of 100%, specificity of 50%, PPV of 44%, and NPV of 100%. Among non-viremic H&E/IHC-concordant cases, these numbers were 80%, 91%, 36%, and 99%, respectively. In this analysis on viremic and non-viremic cases, 31% and 62% of equivocal cases were classified as negative, respectively. One of the challenges in the diagnosis of CMV disease is H&E/IHC-equivocal cases. Avoiding potentially toxic anti-CMV treatment can be beneficial especially in nonviremic patients with negative tissue PCR. We demonstrate that a negative tissue PCR can be used to rule out CMV disease in H&E/IHC-equivocal cases. Although minimal spatial heterogeneity in the CMV target organ is assumed, we cannot rule out this possibility. Furthermore, we performed tissue PCR on FFPE specimens. The results may change if PCR were on fresh tissue, as it would be in the real-life setting. We expect the sensitivity of fresh tissue PCR to increase and specificity to decline. Until replicated in a controlled prospective study using fresh tissue samples, our results should not be used to guide treatment decisions. Our goal in this innovative, but preliminary, study was to assess the value of tissue PCR as an adjunct to H&E/IHC in the diagnosis of CMV disease, especially in equivocal cases. Future research could focus on clinical outcomes of non-viremic equivocal cases and whether anti-CMV treatment can be safely withheld in this group of patients. We could not address this question in the present work because all of our equivocal cases received anti-CMV treatment. Given the possibility of sampling error when using PCR on tissue biopsies, applying the same technique to fluid samples such as bronchioalveolar lavage or stool samples may be of value in future research.